Split (1)

train · 5.09k rows

pmcid stringlengths 6 6	title stringlengths 9 374	abstract stringlengths 2 4.62k ⌀	fulltext stringlengths 167 106k	file_path stringlengths 64 64
549206	Comparative analysis of protein coding sequences from human, mouse and the domesticated pig	Background The availability of abundant sequence data from key model organisms has made large scale studies of molecular evolution an exciting possibility. Here we use full length cDNA alignments comprising more than 700,000 nucleotides from human, mouse, pig and the Japanese pufferfish Fugu rubrices in order to investigate 1) the relationships between three major lineages of mammals: rodents, artiodactyls and primates, and 2) the rate of evolution and the occurrence of positive Darwinian selection using codon based models of sequence evolution. Results We provide evidence that the evolutionary splits among primates, rodents and artiodactyls happened shortly after each other, with most gene trees favouring a topology with rodents as outgroup to primates and artiodactyls. Using an unrooted topology of the three mammalian species we show that since their diversification, the pig and mouse lineages have on average experienced 1.44 and 2.86 times as many synonymous substitutions as humans, respectively, whereas the rates of non-synonymous substitutions are more similar. The analysis shows the highest average dN/dS ratio in the human lineage, followed by the pig and then the mouse lineages. Using codon based models we detect signals of positive Darwinian selection in approximately 5.3%, 4.9% and 6.0% of the genes on the human, pig and mouse lineages respectively. Approximately 16.8% of all the genes studied here are not currently annotated as functional genes in humans. Our analyses indicate that a large fraction of these genes may have lost their function quite recently or may still be functional genes in some or all of the three mammalian species. Conclusions We present a comparative analysis of protein coding genes from three major mammalian lineages. Our study demonstrates the usefulness of codon-based likelihood models in detecting selection and it illustrates the value of sequencing organisms at different phylogenetic distances for comparative studies.	Background Large scale sequencing projects of many different species allow us to investigate phylogenetic issues in much more detail and to identify whether certain genes have had an extraordinary evolution in one or more species and thus gain insight into the actions of natural selection. Despite the sequencing of an increasing number of mammalian genomes and the implementation of more sophisticated evolutionary models using maximum likelihood and Bayesian methodology, the branching order within the mammalian phylum is still not completely resolved. The main reason for this uncertainty is that the diversification of these orders occurred over a short period of time, making the inference of branching order a difficult problem. One of the highly debated issues concerns the relative order of branching among primates, artiodactyls and rodents [ 1 - 9 ]. Here, the Japanese pufferfish Fugu rubrices is used as an outgroup to estimate the branching order of the three species relative to each other. Codon based models [ 10 , 11 ] allow for powerful analysis of protein coding nucleotide sequences. Evolutionary hypotheses may be tested using likelihood ratio tests between nested models. For an introduction to the practical use of these models see [ 12 ], for a more thorough review of the methodology see [ 13 ]. The parameter of primary interest is the ratio of nonsynonymous to synonymous substitutions (ω), also known as the dN/dS ratio. The dN/dS ratio measures the relative importance of evolutionary forces that have shaped a particular protein. A dN/dS ratio significantly larger than one strongly suggests that positive Darwinian selection has acted on the protein. Different extensions to the basic codon model exist, and these can be divided into three main categories: (1) Lineage-specific models that average ω over sites but differentiate between lineages [ 14 ]; (2) site-specific models that average ω over lineages but differentiate over sites [ 15 ]; (3) branch-site specific models that combine the two previous extensions by allowing ω to vary over sites in all background lineages, but allow for a different value of ω in one or more pre-specified lineages [ 16 ]. The models we use here and their relationships are shown in Table 1 . Numerous studies have shown the ability of the site-specific and the branch-site specific models to detect positive selection in cases where the branch-specific models did not, indicating that averaging over sites is generally a more serious problem than averaging over lineages and that in many cases using a branch-site specific model increases the power to detect positive selection [ 17 - 22 ]. In a recent study of cDNA trios of human, mouse and chimpanzee a codon based branch-site specific model was used to search for human genes that have undergone positive selection since our divergence from other primates [ 23 ]. Here, a similar search is done on a different phylogenetic level using a collection of porcine genes. While the study by Clark and colleagues concentrates on the divergence between humans and chimpanzees (branch a in Figure 1 ) our study searches for genes that have undergone positive selection since the divergence of primates, artiodactyls and rodents. Several recent studies have shown that some of the branch-site specific models under certain conditions might have a high false positive rate when used to detect positively selected sites [ 24 , 25 ]. This problem has recently been addressed by Yang and colleagues with the implementation of a new Bayes empirical Bayes (BEB) method for predicting positively selected sites. This new method is much better at avoiding false positives while still retaining a high sensitivity (Z. Yang, pers. comm.). Here we use the new and improved BEB version of the branch-site specific model originally presented in [ 23 ] to detect genes that may have been influenced by positive selection. Results The distribution of sequence lengths of the 1120 three-species alignments is shown in Figure 2 . Since the full length cDNAs were assembled from random ESTs, there is a bias towards assembling relatively short genes. Therefore the subset of genes used in this analysis is not a random sample from the pig genome. This decreases the power of our evolutionary tests, since short alignments have less power when testing for positive selection, but we do not anticipate any other systematic bias in our results. Mammalian phylogeny The relative branching order of the three mammalian species was investigated with the individual genes as well as with a concatenated super gene. Using the empirical amino acid substitution model of Whelan and Goldman [ 26 ] we maximized the likelihood under the three conflicting topologies shown in Figure 3a–c . In 123 of the 988 alignments all amino acids are identical in the three mammalian species giving us no information to discriminate between the three topologies. Of the remaining 865 alignments 245 favour topology A, while 440 and 180 favour topology B and topology C respectively. A concatenated super gene of all 988 alignments clearly favoured topology B over topology A, which again has a higher likelihood than topology C, consistent with the results from the individual gene comparisons (Table 2 .). We used the baseml program of PAML to compare the three topologies in a nucleotide based framework. Different nucleotide based substitution models were used to maximize the likelihood on the three topologies for each of the three codon positions separately. The results of using different models of nucleotide evolution were highly similar so here we only discuss the results obtained with the HKY85 model [ 27 ]. The results based on the third codon position shows that Fugu is too distantly related to the three mammals to be informative in placement of the root of the mammals (results not shown). The first and second codon positions do not show such saturation and should therefore be useful in comparing the three topologies. Consistent with the results based on the amino acid substitution model we see that topology B is favoured in most genes, followed by topology A and topology C, respectively. The actual numbers from the second codon position are 215, 386 and 179 in favour of topology A, topology B and topology C respectively and 208 alignments are uninformative. The corresponding numbers for the first codon position are 215, 545, 175 and 53 (Table 2 .). The internal branch is rather short in all cases. Therefore in the remaining analyses we treat the mouse, human, pig split as a trifurcation. Depending on which topology is actually the right one, the only bias introduced by treating the topology as a star tree, as shown in Figure 3d , is a minor overestimation of the branch length of the species that actually roots the other two. The rates of evolution The three-species alignments were used to estimate the synonymous and nonsynonymous substitution rates of the three branches under the free ratio model, see Table 3 . Figure 4a–f shows the distribution of the synonymous and nonsynonymous branch lengths for each gene in all three species. The synonymous rates are significantly different between the three species. The average synonymous substitution rate, estimated using the concatenated super gene, is approximately 2.86 times larger in mouse compared to pig, and approximately 1.44 times larger in pig than in human. The nonsynonymous rates are more similar among the three species. The corresponding values for the nonsynonymous rates are 2.08 and 1.17 respectively. Table 3 shows the mean, median and variance of both the synonymous and nonsynonymous rate distributions as well as the values obtained from the concatenated super gene. The average values from the individual genes are highly similar to the results obtained from the concatenated super gene. Positive Darwinian selection The dN/dS ratios on the three different lineages were estimated under the free ratio model (Figure 4g–i ). Most genes in all three species have an average dN/dS ratio very close to zero with the average dN/dS ratio higher in human than in pig, which again is higher than in the mouse lineage. The one ratio model averages over sites and lineages, which makes this an extremely conservative method of detecting positive selection. Only four of the 1120 three-species alignments have an average dN/dS ratio larger than one, see Table 4 , and of those only one is significantly larger than one (XM_165930). The free ratio model allows each lineage to have its own dN/dS ratio. This model has slightly more power than the one ratio model due to its ability to find lineage specific signals. The likelihood ratio test (LRT) of these two models should not be considered as a stringent test for positive selection, but more as a test for different selective forces among lineages. The LRT shows that 154 genes have significantly different dN/dS ratios among lineages at the 5% significance level, 73 at 1% and 41 at the 0.1% level of significance. Table 5 shows the 24 genes that have a dN/dS ratio larger than one in one or more lineages as well as the result from each gene of a LRT that tests whether the estimated value of ω is significantly larger than one. As with the one ratio model only one gene shows a result significantly larger than one. The gene is the same one as reported with the one ratio model (XM_165930) and the lineage with a dN/dS ratio significantly larger than one is the lineage leading to pig. Several studies have shown that averaging over sites is more conservative when searching for positive selection than is averaging over lineages. The branch-site specific model A and model B [ 16 ] were originally designed to search for genes where only a small fraction of codons in a specific foreground lineage has evolved under positive selection. Several studies have shown that the original models are prone to predicting false positives under certain conditions, and one should therefore be very careful drawing conclusions from studies based on those models. Here we use a new and improved version of a branch-site model developed for the analyses of human, chimpanzee and mouse gene trios [ 23 ]. The new model we use here is implemented in PAML v. 3.14 and uses the new and improved Bayes empirical Bayes approach to predict which sites have evolved under positive selection in the foreground lineage. Likelihood ratio tests were done separately with human, pig and mouse as the predefined foreground lineage. The LRT when contrasting the neutral model with the branch-site model has two degrees of freedom. By using the human lineage as foreground lineage we find 288 genes that show signals of positive selection (dN/dS in the foreground lineage is larger than one). In 58 of those genes the branch-site model fits the data significantly better than the neutral model at the 5% significance level. We find 34 and 15 genes at the 0.01 and 0.001 levels of significance respectively. The corresponding numbers of genes using pig as foreground lineage are 314, 55(0.05), 23(0.01) and 5(0.001). Using mouse as foreground lineage results in 352, 67(0.05), 25(0.01) and 4(0.001). The genes found to be under positive selection in any of the three species with a LRT significance level of 0.001 are shown in Table 6 . The molecular function of the genes predicted to be under positive selection was determined using the Panther server [ 28 ] and the NCBI server using the newest build of the human genome. Both annotation servers are updated on a regular basis when new information becomes available. During the course of this study the annotation of several genes changed. Of our 1120 alignments 188 are currently not annotated as functional genes indicating that they might possibly be pseudogenes in human; see the Discussion for more details on this subject. The proportion of genes that we report to have undergone positive selection in the human lineage at the 5% level of significance can therefore be viewed as either 58/1120 ~5.2% or 43/931 ~4.6%, indicating that possible pseudogenes are only slightly overrepresented in the genes predicted to have undergone adaptive evolution. The genes predicted to have been under positive selection in the pig and mouse lineage show a similar trend. Several different models have been developed that allow for heterogeneity of ω over sites in an alignment. We used the M4 model [ 15 ] which allows each codon to fall into one of 5 categories corresponding to ω equal to 0, 1/3, 2/3, 1 and 3. The first category represents the fraction of codons that have evolved under strong purifying selection allowing no nonsynonymous changes to occur. The next two categories represent different intensities of purifying selection. The category with ω = 1 represents neutrally evolving sites, while the last category with ω = 3 represents codons that have evolved under positive selection. The results of this analysis on the concatenated super gene can be seen in Table 7 . Only 1.6 % of all codons appear to have evolved under positive selection, and approximately 69 % have been under strong functional constraints. Codon usage bias The concatenated super gene was also used to investigate the patterns of codon usage in the three species; the results of this investigation are summarized in Table 8 . A test for equal codon distributions is rejected in all three pair wise comparisons (P < 0.0001, 60 d.f.). Using nucleotide frequencies to estimate the codon equilibrium frequencies fits the data poorly, so does the equal frequency model (Table 9 ). For a description of the codon equilibrium frequency models, see the Methods. The F3 × 4 model was extended with one extra parameter that accounts for CpG avoidance at the second and third codon position. Since all changes in the second position of a codon are nonsynonymous, the frequency of NCG codons is expected to be lower than under the F3 × 4 model. The extra parameter introduced improves the log likelihood by approximately 1236 units (~44%). This can be compared to the approximately 321 units per extra parameter introduced when going from the F3 × 4 model to the codon table model. When analysing the super gene it is still better to use the actual codon frequencies, but with individual genes the number of codons can sometimes be so small that the use of actual codon counts can be problematic. We also implemented a similar model that incorporated the avoidance of CG in first and second position by introducing an additional parameter but this does not improve the fit of the model significantly (results not shown). This is probably caused by the fact that all four codons with CG in the first and second position code for the same amino acid, Arginine. Arginine has six different codons and the two codons without a CG pair (AGA and AGG) are generally favoured over the other four (Table 8 ), but this tendency is apparently accounted for when modelling nucleotide frequencies at the three codon positions, so here we only present the model that accounts for CpG avoidance at the second and third codon position. Table 9 shows that the choice of codon equilibrium frequency model has detectable effects on the parameter estimates. Most striking is the apparent overestimation of the transition/transversion ratio and the dN/dS ratio when the model is less parameter-rich. Discussion The phylogeny of the early mammalian radiation has been extensively debated over the last two decades. The classical view based on fossil evidence states that all major orders of placental mammals first appear right after the Cretaceous-Tertiary (KT) boundary approximately 65 million years ago [ 29 ]. This sudden appearance of all major placental orders is known as the mammalian radiation. With the use of molecular data this late radiation has been challenged and it is now widely accepted that the radiation of the placental orders probably occurred many million years before the KT boundary [ 29 - 31 ]. Molecular data have also been used to investigate the relative branching orders of many of the larger clades of placental mammals [ 1 - 7 , 9 , 30 ]. One of the issues that have been debated extensively is the placement of Rodentia in the placental tree. Some studies favour a basal placement of the rodents [ 1 , 3 - 5 , 32 , 33 ] while other studies favour a sister relationship between primates and rodents [ 6 - 8 ]. Recently strong evidence based on insertions, deletions and ancient transposable elements in favour of a sister relationship of primates and rodents has been reported [ 2 , 34 ]. The incongruence of single gene phylogenies was investigated in a recent study of eight yeast species [ 35 ]. The phylogeny commonly believed to be correct is completely resolved when concatenating 20 or more randomly chosen genes to form a super gene. A concatenated multi gene approach was also shown to resolve single gene incongruences in a recent study on green algae [ 36 ]. Here we use 988 full cDNA alignments comprising 672,918 nucleotides to investigate the branching order of the three mammalian species. We present results based on both single gene phylogenies and a concatenated super gene. All genes including the concatenated super gene were analysed with both nucleotide and amino acid based substitution models. All methods favour a primate-artiodactyls clade with rodents as an outgroup but with a relatively short internal mammalian branch, indicating that the mammalian radiation happened within a short period of time. The different methods used in this study have very different assumptions but they all show the same general results. The HKY85 model takes into account differences in nucleotide frequencies and transition/transversion biases and allows for differences in substitution rates among the lineages. However, it is still possible that complexities unaccounted for such as non-stationarity and irreversibility of the substitution process have created biases that lead to long-branch attraction of Fugu and Mouse and an erroneous conclusion. Furthermore, the incongruence between our analysis and many recent studies is also affected by the following. (1) The choice of outgroup; bony fishes are believed to have diverged approximately 450 million years ago [ 31 ], making saturation effects in synonymous sites a real problem. We are therefore forced to only consider nonsynonymous sites or amino acid replacements in the phylogenetic analyses. The recently completed genome sequence of the chicken ( Gallus gallus ) shows that the average value of dS between human and chicken genes is approximately 1.66 [ 37 ], which indicates that many genes may still be too distantly related for synonymous sites to avoid problems with saturation. A marsupial species would provide a much better outgroup when available [ 3 , 32 ]. (2) Taxon sampling; by only using three species the variance of the parameter estimates can be quite high and the power to discriminate between two conflicting topologies quite low. The sequencing of more species will lessen this problem. (3) Overly simplistic evolutionary models; here we use only nucleotide and amino acid based models. If a more closely related outgroup was available the use of more complex codon based models could be beneficial in resolving the apparent conflict. Several extensions have been made to the codon models during the past few years. One obvious extension to the codon models is a model that incorporates CG avoidance within and over codon boundaries. This will clearly improve the fit of the data to the model and therefore give more accurate parameter estimates. Including context dependencies over codon boundaries and information about protein structure have also been shown to increase the fit of the models to protein coding data and therefore should result in better parameter estimates [ 38 , 39 ]. (4) Gene trees and species trees can be different; the split between the three groups probably occurred within a very short period of time, allowing for the possibility that different genes actually have different phylogenies due to ancient polymorphisms at the time of the speciation. Using even larger number of genes and a sufficiently sophisticated model should lessen this problem [ 35 , 36 ]. The rate of synonymous substitution was estimated to be almost three times higher in rodents than in other mammals, in agreement with previous investigations that also showed an elevated rate in rodents [ 40 - 42 ]. This has historically often been explained by a generation time effect. Species that have short generation times experience more generations in the time span we consider and consequently they will experience more neutral substitutions over time. The fact that the pig, which has a generation time intermediate between mouse and humans, has an intermediate rate of synonymous substitutions, seems to agree with this theory. For a more thorough discussion of the generation time hypothesis in mammals see [ 43 ]. The nearly neutral theory of molecular evolution predicts that the generation time effect should be smaller for non-synonymous substitutions [ 42 , 44 , 45 ]. The simple argument is that animals with short generation times such as rodents often have a very large effective population size. In a population with a large effective population size slightly deleterious mutations will be removed from the gene pool more effectively than in a population with a small effective population size, where genetic drift will reduce the efficiency of natural selection. Figure 4g–h shows the distribution of the dN/dS ratio in the three lineages. The average dN/dS ratio is highest in humans suggesting a small effective population size, while it is smallest in mouse suggesting a larger effective population size. Previous studies of the occurrence of positive selection based on pair wise comparisons have revealed a very low occurrence of positive selection. In a study of 3595 alignments only 17 genes showed evidence of positive selection [ 46 ]. The branch specific models used here only find one gene where the dN/dS ratio is significantly larger than one. The gene reported is XM_165930. XM_165930 was originally annotated as being similar to cold shock domain protein A, but it has recently been removed from Genbank as a result of standard genome annotation processes. Codon based branch-site models similar to the ones used here were used in a paper based on a three way comparison among chimpanzees, humans and mice [ 23 ]. They report that approximately 1.6 % of all the genes studied have been undergoing positive selection in the lineage leading to modern humans. Using a similar criterion our study indicates that approximately 3.0 % of the genes studied have been undergoing positive selection on the lineage leading to humans; the corresponding numbers for pig and mouse are 2.0 % and 2.2 % respectively. When comparing these two studies it is important to consider the following three things: (1) the relatively short average length of the genes studied here decreases the power of the models to detect positive selection; (2) the use of the new BEB method for detecting positively selected sites should reduce the number of false positives, making our estimates more conservative and more accurate; (3) our study deals with a completely different phylogenetic level, covering a much longer time span than the study by Clark and colleagues. The multiple testing and the small number of taxa used in a study like this imply that the results presented should not be taken as conclusive evidence for positive selection, but more as an approach to searching among the thousands of genes to look for genes that may have evolved in a biologically interesting manner. Comparative approaches such as the one we use here can only be a first step towards showing that positive Darwinian selection may be a key part in the evolution of many different gene families. Further experimental and computational analyses must then be used to investigate the suggested candidates more thoroughly. During the course of our investigation a large fraction of the genes were re-annotated as putative pseudogenes: 188/1120 ~16.8%. However, all these genes have uninterrupted reading frames in all three species; only a tiny fraction of all codons seems to have evolved in a neutral-like fashion (ω~1), and the distributions of the synonymous as well as the nonsynonymous rates of these putative pseudogenes are almost identical to the distributions of the remaining genes (results not shown). The only difference is a slight increase in the dN/dS ratio in the human lineage, which is actually due to a few genes that experience an unusually high dN/dS ratio. Omitting these genes from the analysis removes the observed differences completely. Thus, if all these genes are indeed pseudogenes in human, the loss of function must have occurred quite recently and they may not be pseudogenes in pig and mouse. Conclusions The collection of a large set of pig cDNA sequences has enabled us to study long term evolutionary trends in mammalian genes. Our results indicate that the codon models are able to detect evolutionary signals indicating adaptive evolution in several genes. Our phylogenetic investigation of the primate, rodent, artiodactyl split disagree with most recent findings in favouring a primate, artiodactyl clade with rodents as an outgroup. Our study indicates that several genes that are not classified as genes in the most recent human annotation might after all be real genes; or at least they have become pseudogenes very recently, and the orthologous genes in mouse and pig might still be functional. This shows the potential of comparative methods in identifying functional regions of the genome. Methods cDNA alignment Complete cDNA from the domesticated pig Sus scrofa was assembled at the Danish Institute of Agricultural Sciences (DIAS) from cDNA libraries from 100 different tissues constructed at DIAS and the Royal Veterinary and Agricultural University in the following way. Total RNA was purified from selected tissues using Rneasy (Qiagen) or Tri ReagentR and poly(A+) mRNA was selected using Oligotex (Qiagene) or PolyATract (Promega). Directional cloneable cDNA was synthezised from Poly(A+) mRNA using the cDNA Synthesis Kit (Stratagene) and was ligated into Eco RI/Xho I digested pTrueBlue (GenomicsOne) or pBluescript (Stratagene) followed by electrotransformation into E. coli XL1-Blue MRF' (Stratagene). 5'-EST sequencing was performed using standard protocols (Applied Biosystem). The sequences were trimmed to the longest open reading frame and the termination codons were removed. Homologues sequences from human, mouse and the Japanese pufferfish Fugu rubrices were obtained with the blastall program with default parameters; the E-score was set to 10 -8 . We constructed two different datasets, one with and one without Fugu rubrices . Individual alignments were made using ClustalW version 1.83 with default parameters [ 47 ]. We kept the pig reading frame intact in the alignments by removing any columns where the alignment gave rise to gaps in the pig sequence. Alignments that resulted in premature stop codons, or were shorter than 30 codons, were removed. We used the one ratio model to estimate the total branch length of the tree as well as the synonymous branch lengths. These distributions were used to detect peculiar genes where one or more sequences might not be a true orthologue, and all outliers were thereafter removed from the dataset. This analysis gave 1120 alignments of mouse, human and pig, and of these 988 also included Fugu. The 1120 original cDNAs from Sus scrofa have been deposited in Genbank with the following accession numbers: AY609387-AY610506. Phylogeny and rates of evolution Nine hundred and eighty-eight four-species alignments were concatenated into a super gene. The three topologies were compared using the super gene as well as each individual gene. Both nonsynonymous nucleotide substitutions and amino acid substitutions were investigated with PAML v. 3.14 [ 48 ]. The nonsynonymous substitutions were represented by the first and second codon positions of all codons, and the three different topologies were investigated with baseml using the HKY85[ 27 ] model (model = 4) of nucleotide substitutions. The likelihood was then maximized under the three different topologies using all the individual genes as well the concatenated super gene. The codeml program with the codons translated to amino acids (seqtype = 3) were also used to investigate the three topologies. We used different models of amino acid evolution to maximize the likelihood under the three topologies and since the results were highly similar we only present the results from the empirical method of Whelan and Goldman (model = 2, aaratefile = wag.dat)[ 26 ]. Using the 1120 three species alignments, the synonymous and nonsynonymous rates of evolution were estimated with the codeml program (seqtype = 1) using the free ratio model (model = 2) with the transition/transversion ratio estimated from the data (fix_kappa = 0). Investigation of selection The different tests for positive Darwinian selection are all based on extensions of the basic codon based likelihood model [ 11 ]. Likelihood ratio tests (LRTs) were used to compare nested models where one allows for positive selection and the other does not. The probability that a codon i substitutes into another codon j during the time interval t is determined by the rate matrix Q = ( q ij ) with entries for i ≠ j , with corresponding substitution probability matrix given by exp(Qt). Here π j is the equilibrium codon frequency of codon j , κ is the transition/transversion ratio and ω is the dN/dS ratio. All parameters are estimated independently for each gene. The star topology of the three species is used to estimate the branch lengths (τ human , τ pig , τ mouse ) for synonymous and non-synonymous substitutions. Positive selection was tested in two different ways. Test 1 averages over sites but differentiates among lineages. The LRT compares the free ratio model where all three lineages have a different value of ω estimated from the data with the one ratio model where all three lineages share a common value of ω [ 14 ]. We note that this test is more a test of variable dN/dS ratios among lineages than a test for positive selection. The free ratio model has three parameters for ω and the one ratio model only one. The LRT statistic is calculated as 2 times the differences in maximum log likelihood and is asymptotically distributed as a χ 2 distribution with 2 degrees of freedom. The genes found in one or more lineages evolving with a dN/dS ratio > 1 are compared to a nested model where the dN/dS ratio is fixed at 1 in the lineages shown to have a dN/dS ratio larger than one to see whether the result can be attributed to natural selection or just relaxation of selective pressures. Test 2 is based on a new and improved version of the branch-site method presented in [ 23 ]. We will refer to this model as model A. The LRT is based on a comparison of the neutral model and model A. The neutral model assumes two categories of sites, a proportion p 1 of sites where ω 1 are estimated from the data and is forced to lie between 0 and 1, and a proportion p 2 of neutrally evolving sites where ω 1 = 1 ( p 1 + p 2 = 1). Model A furthermore allows a pre-specified branch to have a proportion of sites that evolve with a different value of ω estimated from the data. This value cannot be smaller than 1. The LRT follows a χ 2 distribution with 2 degrees of freedom. If the value of ω in the foreground lineage is estimated to be equal to one the model collapses to the neutral model. PAML v. 3.14 [ 48 ] was used to estimate likelihood and parameters under each model. Codon equilibrium frequencies can be estimated from data using either simple proportions in the full data set (the CT model with 60 parameters), assuming equal frequencies (Fequal model), multiplying overall counts of nucleotide frequencies (F1 × 4 model, 3 parameters) or counts of nucleotide frequencies for each codon position (F3 × 4 model, 9 parameters). The codon table (CT) was used for analysis of the concatenated super gene and the F3 × 4 model was used on the individual genes. CpG Extension of the codon models A simple extension of the F3 × 4 codon equilibrium frequency model can incorporate CpG avoidance by adding an extra parameter that penalizes a C followed by a G in the second and third codon position. The new model is parameterised as follows Here π i 1 1 represents the frequency of nucleotide i 1 , at codon position 1, and ψ(0 < ψ < 1) is a CpG penalizing parameter. The scaling factor c ψ ensures that the codon frequencies sum to one. Authors' contributions FGJ carried out the analyses and was the primary writer of the text. AH and FGJ together implemented some of the analysis tools. FGJ, AH and MHS together developed the ideas and discussed the interpretation of the results. MF and CB gathered the EST data used. HHJ assembled the cDNAs and carried out Blast searches. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549206.xml
514897	Factors associated with psychotropic drug use among community-dwelling older persons: A review of empirical studies	Background In the many descriptive studies on prescribed psychotropic drug use by community-dwelling older persons, several sociodemographic and other factors associated with drug use receive inconsistent support. Method Empirical reports with data on at least benzodiazepine or antidepressant drug use in samples of older persons published between 1990 and 2001 (n = 32) were identified from major databases and analyzed to determine which factors are most frequently associated with psychotropic drug use in multivariate analyses. Methodological aspects were also examined. Results Most reports used probability samples of users and non-users and employed cross-sectional designs. Among variables considered in 5 or more reports, race, proximity to health centers, medical consultations, sleep complaints, and health perception were virtually always associated to drug use. Gender, mental health, and physical health status were associated in about two-thirds of reports. Associations with age, marital status, medication coverage, socioeconomic status, and social support were usually not observed. Conclusions The large variety of methods to operationalize drug use, mental health status, and social support probably affected the magnitude of observed relationships. Employing longitudinal designs and distinguishing short-term from long-term use, focusing on samples of drug users exclusively, defining drug use and drug classes more uniformly, and utilizing measures of psychological well-being rather than only of distress, might clarify the nature of observed associations and the direction of causality. Few studies tested specific hypotheses. Most studies focused on individual characteristics of respondents, neglecting the potential contribution of health care professionals to the phenomenon of psychotropic drug use among seniors.	Background The use of prescribed psychotropic drugs by older persons has been a subject of interest for several decades [ 1 , 2 ]. Psychotropic drugs are defined as substances that act directly on the central nervous system, affecting mood, cognition, and behavior, and usually include anxiolytics, sedatives and hypnotics, antidepressants, neuroleptics, anticonvulsants, and stimulants. The topic continues to draw researchers' attention for several reasons, including (1) the high prevalence of older users (especially of benzodiazepines) and their typically long-term consumption, (2) their special vulnerability to drug induced iatrogenesis, (3) the discrepancy between rates of mental disorder and rates of drug use among older people, and (4) inappropriate prescribing. The prevalence of psychotropic drug use among community-dwelling older persons (usually defined as those 65 years and older) varies from about 20% to 48% [ 3 - 5 ]. More than half of them take psychotropics for six months or longer, and are therefore considered long-term users [ 4 , 6 , 7 ]. For example, 69% of Canadian elders using benzodiazepines have done so for at least a year [ 3 ]. Long-term use is contraindicated because benzodiazepines lack effectiveness beyond a few weeks or months of sustained use for their principal indications, the relief of insomnia and anxiety [ 8 - 17 ]. Since aging increases the likelihood of drug accumulation and intoxication, older persons are particularly vulnerable to adverse effects from psychotropic drugs [ 18 ]. Notable harmful consequences of psychotropic drug use include memory impairment, psychomotor slowing, delirium, falls with a risk of hip fracture, automobile accidents, and psychiatric hospitalizations [ 19 - 21 ]. Although more psychotropic drug users are found among older persons than any other age group, the prevalence of mental disorders appears to be lower among older than younger adults. This has been shown for major depression, anxiety disorders and sleep disorders [ 6 , 22 - 30 ]. Other observations, such as the small rate of hospital admissions for psychiatric reasons among older persons (1.1%) [ 4 ] compared to their high rate of psychotropic drug use relative to younger adults' rate, confirm these findings. The inappropriate prescription of psychotropic drugs may account partially for the discrepancy between low levels of distress and high levels of drug use among older persons. Inappropriate prescriptions include questionable drug combinations (such as two benzodiazepines), excessive treatment duration, and drugs contraindicated for use by older people (such as long half-life benzodiazepines). Authors estimate that one-fifth to one-half of psychotropic drug prescriptions to the elderly are "inappropriate" [ 4 , 16 , 31 , 32 ]. These findings suggest that older persons' psychological well-being is not the principal determinant of their psychotropic drug use. In the present article, we review recent empirical studies in order to identify other factors that may account for psychotropic drug use among older persons. A large body of work bears on this age group and their use of psychotropic and other drugs [ 33 , 34 ]. Earlier reviews examined prevalence rates [ 35 - 39 ], the issues of long-term use [ 40 ] and dependence [ 41 , 42 ], as well as various models of drug use [ 43 , 44 ]. However, to our knowledge none focuses on community-dwelling psychotropic drug users. Consequently, the present review critically examines various factors associated with such drug use among this population. Each factor is discussed in terms of the empirical support it has received, of hypotheses put forth to explain its association with drug use, and of suggestions for future research. Methods Reports were selected on April 20, 2001, from the following bibliographic databases: MedLine, Cumulative Index to Nursing & Allied Health Literature , Psychlit , Eric and Sociological Abstracts for the years 1990 to 2001. Various keywords were used. Selected reports had to: (1)constitute either a peer-reviewed journal article or a government publication published in English or French; (2) present specific retrievable empirical results concerning older persons, regardless of other age groups studied (some reports [ 16 , 39 , 45 ] were rejected because of this criterion); (3) report on non-institutionalized, community-dwelling participants at the time of the study; and (4) provide specific results on at least benzodiazepine or antidepressant drug use, regardless of other drug classes studied (some reports [ 29 , 46 , 47 ] were rejected because of this criterion). A total of 61citations met these criteria in the databases. Eliminating overlaps left only 32 separate reports, all of which were retrieved and are included in this review. Their reference lists were also consulted, but no other study was identified by this mean. The 32 publications report on 30 different studies conducted in ten different countries. Unless indicated otherwise, each report is treated as a separate study. Most reports originated from the United States (31%) and Canada (28%), followed by France (12.5%), Sweden (9.5%), and 3% each (one report) from Australia, Austria, Ireland, the Netherlands, Spain, and the United Kingdom (Table 1 [see additional file 1 ]). Of 26 reports that disclosed a source of funding, 16 (61.5%) identified government agencies, 6 (23%) government-industry or government-foundation partnerships, and 4 (15.5%) industry. Most studies (62%) collected their data during the 1990s, but several did so during the 1980s (34%) and 1970s (3%). As far as could be determined, the median time from end of data collection to publication was 6 years (range: 1 – 12 years). Most reports (24, 75%) presented multivariate data analyses, usually multivariate logistic regression, where the independent contribution of various variables to the variance in psychotropic drug use could be ascertained while controlling for the contribution of other variables. Although variables with statistically significant associations (p < .05) to drug use that remained in the regression equation were often considered "predictors," strictly speaking only an association is demonstrated in this fashion, and the direction of causality can rarely be established. This is especially so when a theoretical model is not specified prior to the data analysis, as was the case in the vast majority of reports. The eight remaining studies conducted bivariate analyses solely, and most examined three or less variables. In the following review, associations of sociodemographic factors and life conditions with psychotropic drug use are examined first. A factor was deemed well supported by the review studies when 70% of the results from all studies that considered this factor were statistically significant and pointed in the same direction. Second, methodological and conceptual issues relevant to the study of psychotropic drug use among the elderly, and salient in the reviewed studies, are discussed. Results Psychotropic drug use and sociodemographic characteristics Prevalence of psychotropic drug use Weighted average prevalence rates of psychotropic drug use were estimated from all reports having collected data from probability samples or entire populations (n = 22). In longitudinal studies, data from the last year of the study were used. Average prevalence of any psychotropic drug use from 9 studies was 29.0% (range 11.8% – 42.5%). For drugs identified in the reports as benzodiazepines, minor tranquilizers, anxiolytics, or sedative-hypnotics, the average prevalence in 13 studies was 21.5% (range 6% – 43.8%). In 11 studies with data on antidepressants, the average prevalence was 6.9% (range 2.3% – 14%). Finally, for neuroleptics, it was 3.1% (6 studies, range 1% to 6%). Age Older people are more likely than any other age group to use any type of medication [ 29 ]. However, it has been observed that medication use decreases in those over 75 years [ 4 ]. Some suggest that this occurs because: doctors exercise more caution when prescribing to the oldest old [ 48 , 49 ], survivors into advanced age are healthier and thus use fewer medications [ 4 , 50 ], the elderly face fewer stressful events [ 50 ]. The observation of a decline in psychotropic drug use with very advancing age is not universal [ 51 ]: Blazer et al. [ 6 ] and Mamdani et al.[ 49 ] found that it reaches its peak prevalence among those older than 85 years. As Table 2 [see additional file 2 ] shows, 22 studies carried out 23 tests of the association between age and drug use, but only 8 (35%) of the results showed an age-specific trend (5 found an increase and 3 a decrease). This low percentage prevents firm conclusions about any age trend in regard to psychotropic drug use among older people. Gender Over the past three decades, numerous hypotheses have been proposed to explain the higher prevalence of psychotropic drug use among women than men: women are more inclined to reveal their emotional problems to their doctor[ 35 , 36 , 52 - 55 ], to request prescriptions explicitly [ 36 , 54 , 55 ], to hold more positive views of psychotropic drugs [ 50 ]. Some authors have suggested that men prefer to use alcohol rather than prescribed drugs to deal with emotional problems [ 56 , 57 ]. Graham and colleagues [ 42 ] failed to support this last hypothesis in a sample of 826 older persons. Other authors suggest that since women live longer than men, they experience more effects of aging, losses and health problems, all of which increase their likelihood of using a psychotropic [ 37 , 49 , 55 , 58 ]. Physicians might be more willing to prescribe a psychotropic drug to a woman than to a man [ 49 , 54 , 55 , 59 ]. Also, women visit physicians more often, increasing their chance of receiving a prescription [ 36 , 55 , 57 ]. However, in studies using population-wide databases, Brown et al.[ 60 ], Jorm et al.[ 55 ], and Weyerer and Dilling [ 45 ] controlled both the number of health problems and physician visits and showed that women still used more psychotropic drugs than men. Kirby et al. [ 53 ] found the same result when controlling for mental health status. Contradictory results are reported by Mayer-Oakes et al., [ 61 ] and Swartz et al., [ 62 ] who showed the use of benzodiazepines to be associated not to gender but to physical health status, poorer in women than in men. In this review, 73% of the relevant studies support the established finding that women are more likely to use psychotropic drugs than men. In two studies, the relationship with gender holds only among those aged between 65 and 74 years, and in a third, the results are opposite. However, the studies shed little light on reasons for this disparity. Only two studies identified as a secondary objective to look at the gender issue in relation to psychotropic drug use [ 42 , 49 ]. A few studies tested hypotheses to examine the role of physical health status and mental health status relative to gender among seniors, but none received consistent support [ 42 , 45 , 53 , 55 , 60 - 62 ]. No other hypothesis was directly tested in this body of studies. Some authors borrowed hypotheses from studies with middle-aged adults to discuss their results [ 49 , 53 , 55 , 63 ]. Authors in other studies which found a statistically significant relationship between gender and psychotropic drug use did not discuss or interpret the association [ 6 , 48 , 55 , 60 , 64 - 74 ]. In sum, many studies confirm that more women than men use psychotropic drugs, but no single compelling explanation for this difference among the aged emerges from this body of studies. Race The role of culture, ethnicity, and race has received little attention so far in the literature on older persons' psychotropic drug use. Explaining cultural, ethnic, or racial disparities in use of health services is a complex endeavor, requiring analysis of predisposing, structural, access, and other variables in interaction [ 75 - 78 ]. In this review, two studies from Canada compared likelihood of use among French and English speakers, one finding an association with French speakers, albeit in a non-probability sample [ 79 ]. In addition, six studies from the United States compared the prevalence rate of psychotropic drug use between Whites and African-Americans. All studies but one controlled for income or education, and all found that Whites are significantly more likely to use psychotropic drugs. These results concur with many findings showing differential use of health services along racial and ethnic lines in the United States [ 75 ]. Interpretations of the race-specific findings from these six studies focused mostly on professional-and individual-level determinants. Brown et al. [ 60 ] suggested that doctors prescribe fewer psychotropic drugs to older African-Americans by prudence, since the former are more sensitive to some drug effects. Other authors proposed that different prescription patterns result from differences in the expression of psychological distress [ 62 , 80 ] or from the lower rate of depression among African-Americans [ 6 ]. The smaller proportion of African-Americans and other minorities in the United States with private health insurance also may play a role, especially with regard to newer, more expensive drugs [ 81 ]. In that country, more studies are needed to test these hypotheses and others involving structural and access variables, also with other significant minority groups such as Latinos. Beyond racial or ethnic status, studies are needed to understand whether distinct cultural attitudes independently predict psychotropic drug use among older people after various sociodemographic and structural variables are controlled. Marital status It has been suggested that older widows would be more likely to use psychotropic drugs because of the distress of bereavement [ 82 ]. Eleven of 32 studies examined psychotropic drug use in relation to marital status, but only 4 (36%) confirmed an association between drug use and not being married (single, widowed, divorced, or separated). A spouse's death, especially if preceded by long illness, is likely to represent a significant stressful event accompanied by insomnia, anxiety or depression for any person and this can lead to psychotropic drug use. However, strictly speaking, stressful events or psychological distress rather than marital status as such would be implicated, and perhaps only on a relatively short-term basis. In summary, marital status so far is not revealed as a significant factor accounting for psychotropic drug use in older people. Socioeconomic status Living in deprived environments and having an unskilled occupation, low income and little formal education are variables associated with psychological distress among adults, and it might be assumed that the same would hold among older people. However, the studies reviewed fail to support this association. Socioeconomic status (SES) is typically operationalized by education, income, and occupation. As Table 2 [see additional file 1 ] shows, 13 studies examined one of more of these variables. Lower education was significantly associated with drug use in only 3 of 11 results (27%). Lower income was significantly associated with drug use in 3 of 7 results (43%). Two studies examining occupational status before retirement found an association: one found that the self-employed were less likely to use psychotropics [ 83 ], the other, using a non-probability sample, found more blue-collar workers among users [ 79 ]. In sum, most studies did not support the impact of lower educational level or lower income on psychotropic drug use among older persons. The sparse findings regarding occupation are difficult to interpret. While the importance of SES as a predisposing or mediating influence on drug use might be more established in general population studies, it may be less relevant in the older age group because of a lower rate of psychological distress and greater access to income security plans and retirement pensions that prevent extreme poverty. Insurance status In the United States and until recently in Canada, older persons benefited from Medicare or provincial health insurance plans without medication coverage. Insurance coverage has been shown to influence psychotropic drug use by the elderly [ 84 ]. Five studies in this review (four from the USA and one from Canada) examined this issue in six different tests but only two (33%) supported a relationship between having insurance coverage for medication and being more likely to use psychotropic drugs. Besides the fact that the most frequent psychotropic drugs used by older persons during the time period covered by the 32 studies in this review (for example, benzodiazepines like lorazepam or tricyclic antidepressants) were available in inexpensive generic versions, no compelling hypothesis exists to account for these results. However, newer psychotropics, such as selective serotonin reuptake inhibitors which reached peak usage during the late 1990s, have been marketed at much higher prices, and the impact of insurance coverage on their use might be shown to differ in future studies. Proximity to health centers No study examined meso-level variables related to SES, such as census-tract or neighborhood poverty, except proximity to health centers. The use of health services, including medications, may be increased by proximity to such services [ 85 ]. Close or easy availability of health services augments medical consultations (and consequently the request of medication or its prescription). This hypothesis is supported in six of seven studies (85%) in the present review. Life conditions Stressful events Stressful events may increase people's likelihood of using psychotropic drugs. Only three studies examined this relationship, each reporting a different result: a positive association [ 65 ], a negative association [ 50 ], and no association [ 79 ]. The precise role of stressful events within a model of psychotropic drug use by older persons clearly needs to be elucidated. To explain the mixed results, future research might consider the transformation of stressors over time: stressful events may impact significantly on the initiation of psychotropic drug use, but may recede to a negligible level in long-term consumption. A longitudinal design is needed to examine this relationship more adequately, but unfortunately none of the 12 longitudinal studies in this review explored stressful events in relation to psychotropic drug use. Illnesses and other medications A recent qualitative synthesis of the literature concludes that the pathway to psychotropic drug use among older people typically includes the presence of organic disease [ 86 ] (and loneliness, see ahead). Since elderly people suffer from more diseases than younger people, they might use more psychotropic and of course non-psychotropic medications. In another vein, some anxious elderly users of psychotropic drugs might worry excessively about their health and be more likely to consult physicians and receive other medications [ 85 ]. In this review, nine of 16 studies (56%) found a significant association between the presence or the number of physical illnesses and psychotropic drug use. In their conceptual model, Gustaffson et al. [ 69 ] suggest that affliction by a new disease constitutes a stressful event that worsens an older person's mental health status, leading to the need (personally or professionally perceived) for a psychotropic drug. Nonetheless, the results overall are equivocal, perhaps because researchers neglected to consider both the type and the duration of health problems. For example, living with high blood pressure and being struck with congestive heart failure are dissimilar experiences. Typically, researchers counted only the presence or number of illnesses and did not distinguish between individuals who have been living with a disease for a long period of time and those recently experiencing it. As actually measured, the illness factor did not clearly discriminate between older users and non-users of psychotropic drugs. Taking into account both the nature and the duration of illnesses in future studies might better elucidate the relationship between health status and psychotropic drug use. Four of six studies (60%) examining the relationship between psychotropic drug use and use of other medications found a significantly positive association, but the direction of causality remains unclear. Health perception Health perception–the self-evaluation of one own's health–has been shown to be more strongly associated with psychotropic drug use than actual diagnosis of disease [ 33 ]. Some researchers see here an indirect relationship, where poor health perception negatively influences the mental health status of a person, which in turn leads to the request for psychotropic drugs [ 65 , 69 ]. Previous studies support the correlation between mental health status and health perception [ 87 - 90 ]. In this review, seven of eight studies (87.5%) examining the association between health perception and drug use found a positive relationship. However, two thirds of the reviewed studies were cross-sectional, preventing conclusions about cause-effect relationship, and typically more than 80% of the psychotropic drugs used by their subjects were benzodiazepines. This raises the possibility that some subjects had poor health perception as a result of benzodiazepine consumption. Iatrogenic effects of long term benzodiazepine use may worsen health and functional capacity, hence health perception [ 73 , 91 ]. The present findings confirm that health perception has a place within a model of psychotropic drug use among the elderly, yet leave its precise role unclear. A longitudinal study would help to elucidate this role. Social support It has previously been proposed that low social support is associated with psychotropic drug use among older people [ 92 , 93 ]. In 10 studies here reviewed, various scales operationalized constructs identified as social support, social network, social relationships, family relationships, and social isolation. A variable of living alone or with others was included in two of these studies, and subjective reports of loneliness in two studies. However, in 13 tests of the association between some of these variables and drug use, 9 results (69%) failed to observe any significant relationship. All five tests of association with "social support" and all three tests of association with "social relationships" found no relationship with drug use. The single association of "social relationships" was positive, as was one of two of "living alone." Overall, results are counter-intuitive since one would expect that without significant social support to aid in time of difficulties, a person might be more likely to seek medical help, increasing the chances of receiving a drug prescription. Either social support is a minor strand in the tapestry of psychotropic drug use, or the concept of social support may be inadequately operationalized by standard scales. These may need to measure, beyond frequency of social contacts and residential status, subjective judgments of loneliness and of opportunities for social intimacy. Whereas a summed scale score indicating "social isolation" was not associated with benzodiazepine use in one study [ 61 ], in both studies where the older person's feeling of "loneliness" as such was elicited, its association with psychotropic drug use was found to be significant after controlling for other variables, including "social support" scores [ 69 , 70 ]. Rather than the presence or extent of social support, a subjective feeling of loneliness may influence, directly or indirectly, psychotropic drug use [ 86 ]. Mental health status The association between a diagnosis or rating of mental disorder or psychological distress and the use of psychotropic drugs is evidently anticipated. In 18 studies, 22 tests of this association were carried out, and it was significant in 17 (77%). An intriguing observation is that two of the four studies that failed to support the association included only the antidepressant drug class [ 67 , 71 ]. While the association between mental health and psychotropic drug consumption is well supported, it is worth noting that the magnitude of the association varies considerably across studies. The following two pairs of longitudinal and cross-sectional large-sample studies using multivariate logistic regression analysis illustrate this variability: benzodiazepine users were 2.13 times more likely than non-users to report depressive symptoms at 10-year follow-up [ 6 ], and 6.7 more likely to report emotional or nervous problems [ 63 ]. Psychotropic drug users were 2.78 times more likely to report depressive symptoms at 6-year follow-up [ 68 ], and 4 times more likely to report anxious or depressive symptoms [ 94 ] (Table 3 ). We discuss ahead how this variability may stem from methodological differences, especially in the measurement of the key variables in the associations. Table 3 The association between mental health variables and psychotropic drug use Study Results 65 Allard et al. (1995), n = 500 In bivariate analysis, drug use negatively correlated with morale (r = -0.32, p < 0.001) 6 Blazer et al. (2000), n = 4,162 In multivariate logistic regression, benzodiazepine use significantly associated with depressive symptoms at 10-year follow-up (OR: 2.13, p < 0.05) 68 Dealberto et al. (1997), n = 2,812 In multivariate logistic regression, drug use significantly associated with depressive symptoms at 6-year follow-up (OR: 2.78, p < 0.001) 69 Gustafsson et al. (1996), n = 421 In LISREL model, drug use positively correlated (0.63) with poor mental health (chi 2 = 3.38, p = 0.33) 94 Paterniti et al. (1998), n = 1,389 In multivariate logistic regression, drug users 4 times more likely to report anxious and depressive symptoms than non-users (OR: 4.0, CI: 2.5–6.5) 63 Gleason et al. (1998), n = 5,181 In multivariate logistic regression, benzodiazepine users 7 times more likely to report emotional or nervous problems than non-users (OR: 6.66, CI 5.1, 8.7) 74 Taylor et al. (1998), n = 5,222 Depressive symptoms multiply by 3 the likelihood of using hypnotics and by 5.1 the likelihood of using anxiolytics. Anxious symptoms multiply by 4.2 the likelihood of using hypnotics and by 3 of using anxiolytics. Sleep In the general population, Ohayon and Caulet [ 28 ] have clearly shown that a sleep disorder, such as primary insomnia diagnosed according to DSM-III-R criteria [ 95 ], is associated with the use of psychotropic drugs. These authors also noted that complaints of poor or inadequate sleep were associated with drug use. In the present review, all five studies (100%) that evaluated this latter association among older persons supported it. Although as mentioned earlier they do not report more sleep problems than younger persons, the presence of sleep complaints appears to play an important role in their psychotropic drug use. Medical consultations The number of annual visits to a doctor's office has an obvious influence on the number of prescriptions since it has been shown that up to 75% of visits by older people end up with a prescription [ 96 , 97 ]. All seven studies (100%) having examined this factor in this review supported its association with psychotropic drug use. Because no drug can be prescribed without the active participation of a physician, the physician's role is undoubtedly critical. However, it might differ over time. It has been previously suggested [ 98 ] that the first prescription of a benzodiazepine drug and the renewal prescription are two separate phenomena: the doctor might be more directive during the visit leading to the first prescription, whereas the older patient might be more directive to ensure its renewal. Thus, the physician's role in psychotropic drug use among community-dwelling older persons might best be modelled according to the duration of use. In previous research, several characteristics of physicians, notably a high number of consultations, have been significantly associated with the likelihood of prescribing psychotropics to older patients [ 99 ]. Summary of findings on sociodemographic factors and life conditions Among the 16 factors identified in this review of 32 empirical reports, only the variables of language and stressful events were examined in less than five different studies. Gender, age, and mental health status were most frequently examined. The variables of race, medical consultations, proximity to health centers, sleep complaints and health perception are significantly associated with drug use in all or almost all studies incorporating them. Gender and mental health status are associated with drug use in over 70% of studies, while physical illness and number of medications are associated in slightly over half of relevant studies. However, significant associations with age, marital status, socioeconomic status, and social support are observed in only 27% to 36% of studies in which these factors are examined. Methodological issues in the study of psychotropic drug use by the elderly Conflicting results could be explained partially by methodological and conceptual characteristics of many of the studies reviewed. The following discussion focuses on study design, sample, concept definitions and measurements (particularly of mental health status and psychotropic drug use), as well as distinction between short-and long-term psychotropic drug use. Table 1 [see additional file 1 ] lists these and other methodological characteristics of the studies reviewed, as well as summaries of findings on prevalence of psychotropic drug use. Design Descriptive methods (100%) using cross-sectional data (62%) predominate in this body of recent reports of psychotropic drug use in the elderly. Cross-sectional data are vulnerable to sample bias, especially when respondents are not randomly selected, as was the case in 9 of 30 (30%) studies. Given the greater costs involved, the proportion of longitudinal research (38%) seems respectable. Possibly because of cost factors, these studies examined fewer variables in relation to psychotropic drug use. However, besides highlighting the phenomenon of long-term use of psychotropic drugs by the elderly [ 55 ], they provided clear demonstrations of the deleterious effect of psychotropic drug consumption on cognitive capacities [ 19 ] and of how nursing home admission increases psychotropic drug prescriptions [ 5 ], for example. Despite their greater cost and demanding logistics, longitudinal designs are essential to understand more accurately how and why an older person begins, ceases, continues, and modulates psychotropic drug use. Finally, the absence of qualitative studies, which usually allow for a better grasp of elderly users' own perspectives on their use, is noteworthy; most studies so far have reflected only the researchers' points of view. Samples Virtually all samples (29 in the 30 distinct studies) were composed of older consumers and non-consumers of psychotropic drugs. Comparisons between these two groups allow researchers to identify what differentiates users from non-users of psychotropic drugs. However, one notices from this review that researchers do not always succeed. One possible reason may be that short-and long-term users are combined in one group. Indeed, in almost every study it appears that investigators are comparing long-term users with non users. About 20% to 30% of community-dwelling older persons use psychotropic drugs [ 3 , 84 ], with over two-thirds having been users for more than a year, and over one half for more than one year. Among the very old, up to 93% have consumed for more than a year [ 100 ]. Thus, the characteristics of users with less than six months of use are not well known. In one longitudinal study, the one factor that most accurately distinguished long-term users of psychotropic drugs from non-users was having been a user of the drug at the first measurement three years earlier [ 68 ]. The researchers found that older users were 15 times more likely to be users 3 years later – 71 times more likely in the case of antidepressant drugs – whereas the fact of being depressed increased the likelihood of drug use at follow-up by 4.7 times. Thus, if most psychotropic drug use among the elderly transforms into long-term use, the process of transformation itself requires more observation and explanation, again by means of longitudinal research. Immediate problems leading to the first prescription of a psychotropic drug, such as psychological distress or insomnia, might have little relevance among long-term users. Possibly, drug dependence has developed among some long-term users and sustains long-term consumption [ 41 ]. The six-month cut-off period habitually used to distinguish short-from long-term consumption in studies may be too long if dependence is taken into account, as patterns of physiological and psychological dependence may already have become established within such a time frame. However, current studies have not taken into account the dependence issue. For instance, one can wonder how withdrawal symptoms are influencing beginning and long-term users of psychotropic drugs. Fear of withdrawal symptoms might explain long-term use of a certain percentage of elderly users. In one previous study, 71% of middle-aged psychotropic drug users wanted to stop consuming these medications, but half of them feared stopping because of withdrawal symptoms [ 47 ]. It seems desirable to increase knowledge on this aspect of the phenomenon among older people, given the well documented potential of benzodiazepines to provoke dependence. It also appears that over the past decade, the use of mixed samples of users and non-users of psychotropic drugs has provided little information about older consumers themselves. Although comparison is essential for understanding, perhaps more studies should target older drug users exclusively. For instance, we know of no study examining mental health outcomes of older psychotropic drugs users over time. One might deem it illogical if no studies had been conducted on the long-term effects of antihypertensive medication on blood pressure. Grad [ 101 ] made the same observation when commenting on the few empirical studies of the impact of long-term use of benzodiazepines on the quality of sleep of community-dwelling older persons. Mental health status, distress, and psychological well-being The concepts of mental health, psychological distress, depression, and social support are often studied in relation to psychotropic drug use, but operationalized differently across studies. For instance, in the body of studies here reviewed, mental or emotional state is generally measured by standardized, self-rated scales that focus on the manifestation or experience of various symptoms of psychological or bodily distress, or by structured diagnostic interviews aiming to identify mental disorders according to official diagnostic criteria. In all, at least 12 different instruments or scales, 4 different systems of diagnostic criteria (ICD-9 and ICD-10, DSM-III-R and DSM-IV), and 3 unstructured open questions were used in 18 studies to measure concepts identified as depression (12 studies), anxiety (6 studies), psychological distress, mental disorders, life events (3 studies each), psychiatric syndromes (2 studies each), and morale, nervous or emotional disorders, melancholy, nerves, emotional condition, dementia, and sleep problems (1 study each). This variability in concepts and instruments surely impacts the variability of observed relationships between drug use and mental health status (Table 3 ). In addition, it is relevant to ask whether the sole use of scales measuring psychiatric symptoms is appropriate to understand psychotropic drugs use among older people. As discussed, the rate of mental disorders, including sleep problems, is lower among older than middle-aged adults, and largely outpaces their use of psychotropic medications. This suggests that many older people are prescribed psychotropic drugs for mild to moderate psychological difficulties that would not qualify as DSM mental disorders, and that might not produce significant impairment in daily functioning. For example, in two studies [ 61 , 79 ], while drug users displayed more depressive symptoms than non-users according to the Center for Epidemiologic Studies Depression scale (CES-D), their scores did not reach the standard threshold of 16 points necessary for a diagnosis of depression. Results such as these suggest that scales less oriented to symptoms, distress, or disorder, such as measures of psychological well being that focus on so-called "positive" psychological dimensions such as self-acceptance, autonomy, relationships, and purpose in life [ 102 - 104 ], should supplement traditional-type scales. Psychological well-being is not merely the reverse of psychological distress, and the relationship of psychological well-being to symptoms of distress is complex [ 105 ]. No scale of psychological well-being was used in any of the reviewed reports. However, in one recent study of older persons, Guerette [ 106 ] found that among several measures of mental status, measures of psychological well-being produced the strongest associations with benzodiazepine use. Measures of psychotropic drug use Some conflicting or ambiguous results across studies can be attributed to how psychotropic drug use should be measured. Two issues are discussed here: what drug classes to include, and what time period of use to consider. Depending on the study, one or more different classes of drugs are included among the substances measured as psychotropic medications, although, for example, indications for the prescription of benzodiazepines differ from those for the prescription of neuroleptics. Using a composite variable of any psychotropic drug use when examining the association with relevant sociodemographic factors or life conditions probably affects the observed association and consequently our understanding of the phenomenon. It would seem better to determine separately what variables are associated with benzodiazepine, antidepressant, and other drug use. Benzodiazepines warrant careful attention, as these drugs may be classified as anxiolytics, sedatives, hypnotics, and anticonvulsants–and we would not expect logically the correlates of anticonvulsant use in the elderly to resemble those of hypnotic use. In addition, researchers rarely specify which drugs they classify as "minor tranquilizers," or how they distinguish between "anxiolytics" and "sedatives." These definitional issues have long existed in pharmacoepidemiology and we should not expect them to be solved in these studies, but without assurances that drug categories do not overlap, measures of the dependent variable occasionally remain imprecise. The average prevalence of neuroleptic drug use among community-dwelling older persons, estimated at 3.1% from six studies with probability samples, deserves comment in this respect. It has been common wisdom that such drug use has been rare except in institutionalized samples. The studies also confirm that the main psychotropic drugs prescribed to seniors are benzodiazepines and antidepressants. However, most of these studies, conducted in the early 1990s, probably missed the increased popularity of atypical neuroleptics. These drugs have so far enjoyed a reputation as less toxic drugs than conventional neuroleptics, encouraging their prescription among the older age group [ 98 ]. Several problems associated with benzodiazepine use among seniors are not associated with neuroleptics. However, all neuroleptics carry their own substantial risks of adverse effects, such as tardive dyskinesia and cognitive dysfunction (conventionals) and weight gain and diabetes (atypicals) [ 107 , 108 ]. In future studies, investigators will probably need to examine the prevalence of these drugs' use among the elderly. The category of reversible cholinesterase inhibitors, also known as anti-dementia, anti-Alzheimer's, or "cognition enhancers," is also unmentioned in any of the reviewed studies. Introduced in the mid-1990s, drugs such as donazepil and rivastigmine are increasingly prescribed to community-dwelling elderly showing subtle signs of dementia and simple forgetfulness (often termed "mild cognitive decline") according to a preventive ethos [ 109 ]. The second measurement issue to consider relates to the period of time for which data are collected, a source of confound noted by other reviewers [ 41 , 42 , 110 , 111 ]. In the 30 studies, temporal windows varied widely, from "current use" (4 studies), "current and past use" (1 study), "regular use" (2 studies), two days (2 studies), one week (1 study) two weeks (3 studies), one month (3 studies), three months (5 studies), and one year (7 studies). In two studies this period is undefined. When drug utilization is measured based on more than a week, one risks inflating the prevalence rate by including consumers who no longer use the drugs. Conversely, one risks omitting occasional users when referring to the last two days only [ 45 ]. Graham and Vidal-Zeballos [ 42 ] suggested asking respondents about short-term consumption (past 2 days and "recent use") and about long-term use (past year). This suggestion does not however solve problems of inaccurate self-report. Studies have found that older persons frequently hide or deny their use of psychotropic drugs [ 112 - 114 ]. To avoid these pitfalls, researchers in six studies determined psychotropic drug use according to municipal, provincial, state or national prescription databases held by government agencies or health maintenance organizations. The number of prescriptions, however, can greatly differ from actual drug consumption [ 16 ]. For example, about 50% of the elderly do not comply with their prescription regimen [ 115 , 116 ]. and an unknown proportion share their pills with relatives or friends [ 85 ]. Finally, researchers in at least 10 studies measured drug use by inspecting medication containers at the home of the respondent [ 83 ]. This method confirms that drugs have been obtained but leaves the compliance problem unresolved. A good rationale exists nonetheless to encourage this method, probably the most reliable way to get as close as possible to actual consumption [ 55 , 63 , 83 , 111 ]. Given that one third of studies in this review used this method suggests that its cost may be relatively acceptable. Interestingly, only two studies used telephone surveys. Owing to their relatively lesser cost than personal interviews, and since the development of reliable technologies, telephone surveys using random-digit dialing and random-select dialing are increasingly used to contact respondents in health related surveys. Conclusions Without being exhaustive, the present literature review is comprehensive and the range of reports, methods, samples, and geographical locations provides a reasonably solid base to support most recommendations. This review was limited to empirical studies. To better understand the phenomenon of psychotropic drug use among community-dwelling elderly, it is desirable that historical, psychological, sociological, political and regulatory, as well as medical aspects of the phenomenon be considered. While some factors are clearly associated with psychotropic drug use in the reviewed studies (such as race, proximity to health centers, sleep complaints, and health perception), few investigators test specific hypotheses to account for the associations. For example, sparse work focuses on cultural factors that might explain drug use disparities between Whites and African-Americans. Similarly, with most drug users being long-term and most drug treatments for insomnia losing their effectiveness with long-term use, the strong association between sleep complaints and drug use needs thorough examination. One of the least studied aspects of the phenomena in the reviewed studies concerns the role of health care professionals. Physicians, nurses, and pharmacists all interact significantly with community-dwelling older persons, especially with those who take psychotropic medications. Only through a health care professional such as a physician and a pharmacist would the vast majority of older people obtain a psychotropic drug to relieve a sleep problem. Recently, the Internet and mail-order pharmacies without face-to-face interaction have facilitated individuals' access to prescription drugs. Each professional's role, as mentioned, is also likely to alter as the individual's phase of consumption transforms from short-to long-term. Researchers must face the challenge to incorporate variables related to health care professionals' attitudes and behaviors, as well as to new modes of distribution of psychotropic drugs to consumers, in their study of the phenomenon. Approximately one third of community-dwelling older persons use psychotropic medications. If the rate of psychiatric disorders among a population serves as a guideline, then obviously older persons' use of psychiatric drugs far outpaces these drugs' standard indications and has extended into areas where drugs have little documented effectiveness. Viewed in this light, the ubiquitous phenomenon of long-term psychotropic drug use should evoke concern and caution. The discipline of nursing can definitely contribute to the rational use of these agents among older people. As suggested in this review, researchers do not still fully grasp the dynamics of psychotropic drug use among this population and creative and rigorous research from several disciplines, and from interdisciplinary perspectives, is needed. However, nurses concerned by the problem of the overuse of medication and its adverse consequences can already implement and evaluate programs to educate older people and allied health care and social service professionals about the risks of psychotropic drugs and alternatives to drugs for the management of everyday anxiety, loneliness, depression, and especially insomnia. Nurses can also actively implement and evaluate drug withdrawal programs aimed at long-term users who have had difficulty in withdrawing, and especially at short-term users who might soon be trapped into dependency and thus long-term use. Conversely, diverse patterns of psychotropic drug use undoubtedly exist among older persons, and positive patterns of use, emanating from users' own experiences and discoveries, need to be documented and disseminated. Competing interests None declared. Authors' contributions PV and DC conducted the literature review and drafted the manuscript. SL and JC revised the literature review and subsequent drafts. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Characteristics of 32 empirical reports on psychotropic drug use among community-dwelling older persons, 1990–2001. Reports on psychotropic drug use among community-dwelling older persons. Click here for file Additional File 2 Factors associated with psychotropic drug use among community-dwelling older persons in 32 empirical reports, 1990–2001. Factors associated with psychotropic drug use among community-dwelling older persons. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514897.xml
546041	Rice Genome Approaches Completion	null	In April 2002, Science published draft genome sequences for the two major subspecies of cultivated rice, Oryza sativa . The release of the rice genome—the first plant crop to be sequenced—was big news. Rice is a staple crop for more than half of the world's population, and it was hoped that the availability of the genome sequence might enable scientists to develop more productive rice strains or strains that are more environmentally friendly. Furthermore, the rice genome might provide the key to understanding the genetics of other major cereal crops, all of which have much larger genomes. But the sequences published in 2002 were only draft genomes, containing many gaps and errors—works-in-progress rather than finished products. Now, a large group of scientists led by the Beijing Institute of Genomics is publishing a much improved, near-complete genome analysis of the indica and japonica subspecies of O. sativa , which are eaten in India and China, and Japan, respectively. Their analysis team, led by Gane Ka-Shu Wong, provides important insights into the evolution of rice. First of all, the team improved their original whole-genome shotgun sequencing of indica by generating significantly more sequence data, and then they used better methods to assemble these data. In whole-genome shotgun sequencing, the entire genome is chopped into random fragments, each fragment is sequenced, and then powerful computer programs search for overlaps and put all these data in order. It's like putting a fiendishly difficult jigsaw puzzle together by looking for patches of matching color. The key to the improvement in the genome sequence analysis is that the researchers have used the combined DNA sequence data from the two subspecies to facilitate the sequence assembly. The result is a nearly 1,000-fold increase in contiguity for the two genome sequences. In other words, while the original draft was very fragmented, in the new version, 97.7% of the genes can be found, in either the indica or the japonica dataset, on one piece of DNA whose position along the chromosomes is well defined. The researchers have also used their improved genome sequence to investigate the evolutionary history of rice. Central to evolution is the development of new functions through mutation of existing genes. But when mutations occur in functional genes, the result is rarely beneficial, so it is thought that evolution is more likely to proceed first by duplicating existing genes and then experimenting on the “backup” copy of the gene. Wong and colleagues report that there is evidence in the rice DNA sequences for a whole-genome duplication event just before the grasses diverged from other flowering plants, about 55–70 million years ago. This genome duplication may have played a role in the origin of the grasses, which then spread rapidly across the world to provide important sources of food that, among other things, possibly influenced human evolution. A bowl of indica (white, long grains) and japonica (brown, short grains) rice Analysis of the rice genomes also indicates that a small chromosomal segment was duplicated about 21 million years ago and that there is massive ongoing duplication of individual genes. These individual gene duplications provide a continuous source of raw material for gene genesis and very likely contribute to the differences between members of the grass family. Now the challenge is to use the rice sequences as a basis for detailed genetic analyses of additional cereal crops and for the development of improved strains of not only rice, but wheat, maize, and other important food crops.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546041.xml
529257	Adjuvant interferon gamma in patients with drug – resistant pulmonary tuberculosis: a pilot study	Background Tuberculosis (TB) is increasing in the world and drug-resistant (DR) disease beckons new treatments. Methods To evaluate the action of interferon (IFN) gamma as immunoadjuvant to chemotherapy on pulmonary DR-TB patients, a pilot, open label clinical trial was carried out in the Cuban reference ward for the management of this disease. The eight subjects existing in the country at the moment received, as in-patients, 1 × 10 6 IU of recombinant human IFN gamma intramuscularly, daily for one month and then three times per week up to 6 months as adjuvant to the indicated chemotherapy, according to their antibiograms and WHO guidelines. Sputum samples collection for direct smear observation and culture as well as routine clinical and thorax radiography assessments were done monthly. Results Sputum smears and cultures became negative for acid-fast-bacilli before three months of treatment in all patients. Lesion size was reduced at the end of 6 months treatment; the lesions disappeared in one case. Clinical improvement was also evident; body mass index increased in general. Interferon gamma was well tolerated. Few adverse events were registered, mostly mild; fever and arthralgias prevailed. Conclusions These data suggest that IFN gamma is useful and well tolerated as adjunctive therapy in patients with DR-TB. Further controlled clinical trials are encouraged.	Background Tuberculosis (TB) is not yet a defeated affection. Although controllable at a community level and curable in individuals, its eradication seems distant. At present, at least one third of the World's population, more than 1 500 million individuals are infected with the Mycobacterium tuberculosis. Every year, around 8 – 10 million new cases occur [ 1 ]. Two million people die annually due to non-AIDS related TB, which is the highest number of deaths attributable to a single infectious agent [ 2 ], and corresponds to the 7 th cause of death worldwide [ 3 ]. TB represents 26 % of avoidable deaths in developing countries [ 4 ]. The emergency of drug resistant (DR) or multidrug-resistant (MDR) strains has increased this global problem, leading to a high morbidity and mortality [ 5 ]. MDR-TB affected patients' mean survival ranges from 2 to 14 months [ 6 ]. According to the World Health Organization's (WHO) data, MDR patients global proportion is 2.2 % [ 7 ]. The infection is mainly transmitted by inhalation of the bacilli coming from respiratory airways infected secretions. Once inhaled, the bacilli are subjected to phagocytosis within the alveolar macrophages, where they can be destroyed. Nevertheless, Mycobacteria have developed mechanisms to adapt to the noxious intracellular environment. Thus, it can persist, replicate and disseminate, leading to new infectious foci. Resistance emergence depends on several factors such as initial bacillary load, inadequate or incomplete chemotherapy administration, and the patient's immune condition. Chemotherapy is successful in most cases, given that the treatment schedule is thoroughly followed. It is prolonged, costly, and needs to be directly observed. Otherwise it is inadequate to kill all the bacilli and drug resistance emerges. Toxicities are frequent as well [ 8 ]. The immunologic approach to TB treatment can be promising since only 10 – 20% of infected people develop the disease and many of them have spontaneous remission [ 9 ]. Interferon (IFN) gamma plays a main role in the immunity to TB. It is a glycoprotein, secreted by CD4+, CD8+ and NK cells. Nevertheless, CD4+ Th1 lymphocytes are the main producers in response to a stimulus [ 10 ]. Enough evidences exist related to IFN gamma action on the macrophages immunoregulatory activity [ 11 - 14 ]]. Lack of production of this cytokine [ 15 ] or expression of its receptor [ 16 , 17 ] is associated to the infection's most lethal forms. Interferon gamma has also a potent antifibrotic effect [ 18 - 20 ]. Therefore a pilot clinical study was done with the aim to evaluate IFN gamma effect on drug resistant pulmonary TB patients regarding their clinical, bacteriological and radiological evolutions. The results show that the use of this protein in eight DR-TB patients (four of them MDR) as adjuvant to antibiotics had short and middle-term beneficial effects. Methods An open-label, non-randomized, non-controlled, pilot trial was carried out at the "Benéfico -Jurídico" Hospital, Havana, which is the national reference unit for TB and other respiratory diseases. According to the national TB program, all patients with unfavorable response to treatment are remitted to this center. The study population was constituted by eight Cuban patients, both sexes, more than eighteen years old, with diagnosis of TB without a favorable response to the usual therapy, who gave their written, informed consent to participate. The diagnosis comprised clinical findings such as cough and expectoration, pulmonary lesions at thorax radiography, and positive DR-TB sputum-smear and culture. To confirm drug resistance the Canetti's multiple proportions method [ 21 ] was used as antibiogram. Exclusion criteria were another chronic disease, pregnancy or nursing, severe psychiatric dysfunction, multiple sclerosis or another autoimmune disorder, other pulmonary infections, HIV co-infection, and treatment with glucocorticoids or any other immunosuppressor medication. The previous drug therapy received by the patients included 4 drugs (isoniazid, rifampin, streptomycin, and pyrazinamide) daily during 2 months, then isoniazid and rifampin twice per week for 2 additional months. Since their sputum tests had not become negative at this point they were returned to the 4 drugs regime plus ethambutol daily for 3 months, and finally isoniazid, rifampin, and ethambutol three times per week for 5 months. The trial was done in compliance with the Helsinki Declaration. The protocol was approved by the hospital's Ethics Committee and by the Cuban Regulatory Authority. Data from nineteen historical control cases were obtained from the hospital's archives. Patients received 1 000 000 IU of human recombinant IFN gamma (Heberon Gamma R ® , Heber Biotec, Havana), intramuscularly, daily during 4 weeks and then 3 times per week for the next 20 weeks. Participants stayed as in-patients during the study period. They received anti-TB drugs (WHO schemes) [ 22 ], according to the resistance detected in each case by the antibiogram (Table 1 ). Drugs were given as follows: rifampin 10 mg/Kg (maximum 600 mg), ethambutol 20 mg/Kg (max. 1500 mg), ethionamide 10 mg/Kg (max. 750 mg), pyrazinamide 15–30 mg/Kg (max. 2000 mg), ciprofloxacin 15–20 mg/Kg (max. 1500 mg), amikacin 15 mg/Kg (max. 1000 mg), and kanamycin 15 mg/Kg (max. 1000 mg) daily. After the end of the 6-months IFN gamma treatment period, chemotherapy continued up to 9 months if the scheme included rifampin and 18 months otherwise. Evaluations were carried out at entry and monthly during IFN gamma treatment. A complete physical examination was done. Sputum samples were taken for acid-fast-bacilli smear and culture, as well as blood samples for hematological counts, globular sedimentation rate, alanine aminotransferase, and creatinin determinations. Thorax radiographies were also recorded. Afterwards, patients were followed up with half-yearly evaluations during one year. Treatment efficacy evaluation included clinical, bacteriological and radiological outcomes. Complete response was defined as total disappearance of all signs and symptoms, negative sputum acid-fast-bacilli smear and culture, and pulmonary lesions improvement at X-ray. Partial response included signs and symptoms decrease, negative sputum smear and culture and stable X-ray lesions. No response consisted in signs and symptoms persistence, positive bacteriological examinations, and lesions stabilization or progression. Safety and tolerability of the IFN gamma treatment were monitored by means of a rigorous control of the adverse events that could be presented. Results Eight patients were enrolled in the study. Those were all the pulmonary DR-TB cases in the country during the inclusion period, from December 1999 to February 2002. They had not responded to the usual Directly Observed Treatment Short-course (DOTS) chemotherapy regime. Strain resistance was acquired in all cases. This was well determined since in Cuba all detected cases are screened for resistance on their first isolate. The patients did not have any extra-pulmonary manifestation of the disease. Their demographic and baseline characteristics are shown in Table 1 . Five of them were men, six of them non-white. The age ranged between 23 and 54 years old, and body mass index (BMI) between 13.2 and 22.0 Kg/m 2 . Their main symptoms were cough, expectorations, dyspnea, stertors, distal cyanosis, and finger clubbing. Bacteriological tests codification was mostly high and all patients showed active lesions at thorax radiography. Most of them had accelerated globular sedimentation rates (GSR). Anemia or other hematological alterations were not recorded. A rapid favorable evolution was obtained after treatment with IFN gamma (Table 2 ). Clinical improvement was evident since the first month of treatment, when all signs and symptoms (except for finger clubbing) had disappeared in all patients and BMI increased in all but one of them. Sputum acid-fast-bacilli smears and cultures were negative since the 1 – 3 months of treatment. The eight patients had radiological improvement, with lesions size reduction (total disappearance in one case) (Figure 1 ). GSR decreased in five out of 6 patients who had abnormal values at inclusion. After six additional months follow-up, patients # 3 and 4 normalized it to 10 and 15 mm/h, respectively. At the end of the IFN gamma treatment all the patients were evaluated as complete responders. The treatment with IFN gamma was safe and well tolerated. Four patients presented at least one adverse event. These events were arthralgias, fever, headache and asthenia. All adverse events were mild, except for one moderate fever, which was efficiently controlled with acetaminophen. Significant differences were not detected in other clinical laboratory tests. After completion or the IFN gamma 6-months treatment the patients continued with the corresponding chemotherapy schedule. Seven of the eight patients remained bacteriologically, clinically and radiologically negative at least twelve months after the treatment with IFN gamma concluded. However, patient number five relapsed six months after the end of IFN therapy. He developed additional resistance to rifampin and ethionamide and a chronic obstructive respiratory disease that contributed negatively to his evolution. Table 3 shows the results obtained at the same hospital with the 19 DR-TB cases during the five years prior to the present study. These patients had also failed to the standard DOTS regime. They were all resistant to isoniazid and rifampin, 13 were resistant to streptomycin, 7 to kanamycin, 4 to ethambutol, and one to pyrazinamide. Resistance was primary in 2 cases and acquired in the rest. Their average age was 58 years. Management was essentially the same as for the patients included in the study, except for IFN gamma treatment. None of these DR-TB cases reached culture conversion at three months of treatment with chemotherapy and less than half had converted at six months. Their clinical outcome was also worse. Discussion In spite of the reduced size of the population studied, the results suggest the efficacy of IFN gamma on DR-TB, when used as adjuvant to chemotherapy. All eight patients were considered as complete responders at the end of IFN treatment with disappearance of the disease signs and symptoms, sputum tests conversion, and pulmonary lesions improvement. Bacteriological and radiological improvement correlated with the clinical evolution; BMI increased and manifestations as cough and expectoration did not recur after IFN gamma treatment. Clinical practice demonstrates that these results are very difficult to obtain in such a short period of time with the chemotherapy alone. Any conclusion from this study is also limited by the fact that it was not a controlled trial. This was not possible due to the low incidence of TB (7.6/100,000 inhabitants in 2002) [ 23 ] and DR-TB in Cuba [ 7 ]. Therefore a historical control with the results obtained at the same hospital by the same investigators in patients treated only with chemotherapy is used for comparison. Despite the fact that this kind of control is not fully comparable since they were not in the original design of the trial, a clear difference is shown regarding patients' performance. Literature reports on chemotherapy-treated DR-TB patients show similar unfavorable outcome [ 6 ]. The fact that four patients received rifampin, according to the strain sensitivity, which is one of the election drugs for the treatment of TB with excellent results in patients treated with DOTS regime, does not diminish the consideration regarding the possible benefit exerted by IFN gamma since these same patients had already not responded to this antibiotic as part of the DOTS. Moreover, one patient recurred after he had finished IFN gamma treatment for 6 months, despite still being under chemotherapy regime. This suggests that in his case a new IFN gamma cycle combined to the antibiotics would have been necessary, but this was not previewed in the protocol. However, only a controlled trial can definitely clarify the role of IFN gamma and second line antibiotics in this kind of patient's improvement. The radiological results demonstrated IFN gamma antifibrotic properties as well. All patients had a reduction in the pulmonary lesions size, while one showed a complete resolution. This effect cannot be attributable to the antibiotics, since it is well known that DR-TB patients only develop radiological improvement long time after sputum smears and culture become negative. In many cases extensive fibrotic lesions never improve, and stay stable for life. This antifibrotic action agrees with that obtained with IFN gamma in idiopathic lung fibrosis [ 24 ] and suggests that IFN gamma can have future indications in other pulmonary diseases where fibrosis is present. Therapy was well tolerated. It was not necessary to suspend the combined treatment due to adverse events; mostly mild. Adverse events such as arthralgias, fever and headache coincide with those reported for interferons [ 25 ]. Immunity to TB depends on the development of CD4+ cells- and macrophages-mediated Th1 response. The role of IFN gamma as the main macrophage-activator Th1 cytokine has been clearly established in animal models infected with M. tuberculosis [ 15 , 26 , 27 ]. IFN gamma action on the macrophages leads to kill intracellular Mycobacteria . It stimulates macrophages to produce tumor necrosis factor alpha (TNF α), oxygen free radicals and nitric oxide, increase surface display of MHC antigens and Fc receptors, decrease lysosomal pH, and increase the intracellular concentration of some antibiotics [ 11 - 14 , 28 ]. Regarding its antifibrotic effect, IFN gamma inhibits lung fibroblast proliferation and chemotaxis in a dose dependent manner, and reduces collagen synthesis [ 18 , 19 , 29 ]. Furthermore, this protein is a potent inhibitor of the transforming growth factor β (TGF-β)[ 20 ], involved in the pathogenesis of many fibrotic lung diseases [ 30 - 32 ]. On the other hand, mutations in the IFN gamma gene [ 15 ], or in the IFN gamma receptor alpha chain gene [ 16 , 17 ], increase susceptibility to develop the disease. Patients with disseminated BCG and other infections present defects in IFN gamma and other cytokines secretion and action [ 33 - 35 ]. IFN gamma therapy has been shown effective for cerebral tuberculosis caused by a multidrug-resistant strain [ 36 ]. The aerosol route of administration has been proposed as organ specific delivery method, obtaining a high release to infected alveoli [ 37 ]. Condos et al. reported clinical and bacteriological improvement and tolerability with aerosolized IFN gamma in five patients with MDR-TB [ 38 ]. In addition, other previous trials demonstrated promising results in patients infected with other Mycobacteria [ 10 , 39 - 42 ]. Conclusions These results can suggest a beneficial effect of IFN gamma when it is used as adjuvant in the treatment of tuberculosis patients that have resistance to standard chemotherapy, and encourage carrying out more extensive, controlled studies. Combination with second-line drugs can reduce the time of treatment, diminishing toxicities and possible relapses; in many cases could reduce the application of recessional surgery. Further controlled clinical trials are needed to confirm these results. Competing interests Authors IGG, CMVS, and PALS are employees of the Center for Biological Research, which is part of the Center for Genetic Engineering and Biotechnology, Havana network, where IFN gamma is produced. The rest of the authors have no competing interests at all. Authors' contributions RSM conceived the study and carried out the bacteriological determinations. IGG participated in the study design and coordinating, and wrote the manuscript draft. NFO, MVQ, MTM, DC, and DMM took care of patient recruitment, management, and follow-up. CMVS participated in the study design and result analysis. PALS took part in the design, results analysis and manuscript writing. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529257.xml
554096	Theories of schizophrenia: a genetic-inflammatory-vascular synthesis	Background Schizophrenia, a relatively common psychiatric syndrome, affects virtually all brain functions yet has eluded explanation for more than 100 years. Whether by developmental and/or degenerative processes, abnormalities of neurons and their synaptic connections have been the recent focus of attention. However, our inability to fathom the pathophysiology of schizophrenia forces us to challenge our theoretical models and beliefs. A search for a more satisfying model to explain aspects of schizophrenia uncovers clues pointing to genetically mediated CNS microvascular inflammatory disease. Discussion A vascular component to a theory of schizophrenia posits that the physiologic abnormalities leading to illness involve disruption of the exquisitely precise regulation of the delivery of energy and oxygen required for normal brain function. The theory further proposes that abnormalities of CNS metabolism arise because genetically modulated inflammatory reactions damage the microvascular system of the brain in reaction to environmental agents, including infections, hypoxia, and physical trauma. Damage may accumulate with repeated exposure to triggering agents resulting in exacerbation and deterioration, or healing with their removal. There are clear examples of genetic polymorphisms in inflammatory regulators leading to exaggerated inflammatory responses. There is also ample evidence that inflammatory vascular disease of the brain can lead to psychosis, often waxing and waning, and exhibiting a fluctuating course, as seen in schizophrenia. Disturbances of CNS blood flow have repeatedly been observed in people with schizophrenia using old and new technologies. To account for the myriad of behavioral and other curious findings in schizophrenia such as minor physical anomalies, or reported decreased rates of rheumatoid arthritis and highly visible nail fold capillaries, we would have to evoke a process that is systemic such as the vascular and immune/inflammatory systems. Summary A vascular-inflammatory theory of schizophrenia brings together environmental and genetic factors in a way that can explain the diversity of symptoms and outcomes observed. If these ideas are confirmed, they would lead in new directions for treatments or preventions by avoiding inducers of inflammation or by way of inflammatory modulating agents, thus preventing exaggerated inflammation and consequent triggering of a psychotic episode in genetically predisposed persons.	Background When the solution to a clinical or scientific puzzle eludes us for more than a century, as with schizophrenia (formerly dementia praecox), we need new ways of thinking about the problem [ 1 , 2 ]. Efforts to understand schizophrenia have focused on neurons and, especially, the role of presumed excess dopamine neurotransmission. We believe that genetic, environmental, and stochastic factors combine with epigenetic factors to create episodes of the illness [ 3 - 5 ]. Thus, the syndrome of schizophrenia is viewed as an endpoint in a dynamic process variously conceptualized as degenerative or developmental or alternating at different points in the process [ 6 - 10 ]. Degenerative models imply that after a period of normal development, the organism, or one of its parts, takes a wrongful turn in its trajectory and begins to malfunction. This describes the eventual outcome for all life forms and is a biological restatement of the second law of thermodynamics. Since degeneration is universal, stating that an illness is degenerative is not particularly helpful. What would be helpful is to determine when in the life course the degeneration begins and how the degeneration is initiated and proceeds. Answers to the "when?" and "how?" questions would then describe the degenerative process in developmental terms. Developmental models of schizophrenia implicate abnormalities of early brain development predisposing to future schizophrenia. The proponents of the model further argue that the perturbations of development are limited to the early times of development and are discontinuous. Without this qualifier, developmental models are indistinguishable from degenerative models where the degeneration commences early in the life span. The early abnormalities are not necessarily the cause of schizophrenia, but, instead, create a state of risk for a future episode of schizophrenia. That is, a diathesis or predisposition is not a disease. Consequently, there must be factors later in life that convert the vulnerability to an illness. These additional factors are presumed to damage development in such a way that a predisposition becomes actualized. To gain a complete understanding of the syndrome, we must again return to the question of " what happens?" Following this line of reasoning, the distinction between degenerative and developmental models blurs. In fact, a medical-behavioral condition can be both developmental and degenerative as exemplified by Down syndrome [ 11 - 13 ]. Individuals born with trisomy 21 exhibit a number of developmental anomalies including cardiac malformations, abnormal dermatoglyphics, skeletal changes, and muscular hypotonia, to name a few. As trisomy 21 infants mature, most exhibit degrees of mental retardation. By about age 50, these individuals invariably develop Alzheimer-like CNS degenerative changes that can be seen at autopsy [ 13 ]. Schizophrenia involves both developmental and degenerative features. From the time of Bleuler [ 14 ] and Kraepelin[ 15 ], "It is certain that many a schizophrenia can be traced back into the early years of the patient's lives..." [ 14 ] p. 252. The 'follow back' studies of schizophrenia support these views [ 16 ]. Likewise, prospective studies of children at high risk for schizophrenia report developmental anomalies in motor skills, cognition, and attention long before the onset of overt illness [ 17 - 19 ]. Overt psychotic symptoms for some individuals usually start in the late teenage years or early twenties, but the illness can start as early as middle childhood [ 20 ] and may, more rarely, start in old age [ 21 ] p 73]. The evidence suggesting early developmental perturbations in schizophrenia is compelling. At the same time, there certainly are examples of deterioration reminiscent of Kraepelin's suggestion for some people with schizophrenia. However, deterioration in clinical course may not indicate CNS deterioration. Instead, the decline could be a secondary consequence of an illness that disrupts education, economic achievement, and social functioning leading to a downward spiral in all aspects of adult life. Consistent with an early degenerative process, there are reports of declining cognitive function preceding onset of psychosis [ 22 ]. Proponents of neurodevelopmental models suggest that the premorbid cognitive abnormalities are developmental risk factors for future schizophrenia (c.f [ 23 ]) and argue that such abnormalities show little evidence of decline after onset [ 6 , 24 ]. Whether developmental or degenerative, the premorbid cognitive deficits seen in schizophrenia are also seen in other disorders [ 25 ] and lack specificity and sensitivity thus detracting from the concept that the cognitive abnormalities seen in schizophrenia are useful endophenotypes [ 26 ]. The strongest evidence for a neurodegenerative phenomenon comes from imaging studies showing progressive loss of brain volumes [ 27 - 29 ]. Neuropathological studies fail to find widespread classic signs of neurodegeneration such as gliosis though there are exceptions to this generalization [ 30 ]. Observations of abnormal dendritic arborization [ 31 , 32 ] are consistent with the neuroimaging evidence suggesting abnormal connectivity between brain regions [ 29 ]. As a cautionary note, most of the neuroimaging and neuropathology results are subject to confounds from the effects of medications and various other treatments, post-mortem intervals, possible effects of diet, smoking habits, as well as a myriad of other potential confounds associated with glucocorticoid mediated stress following chronic illness and associated life's limitations [ 33 , 34 ]. The symptoms of schizophrenia are highly variable. Within families (and thus presuming relative homogeneity of genetic and environmental factors) symptoms can vary widely over time, as illustrated by identical quadruplets concordant for schizophrenia [ 35 ]. Even within affected individuals, symptoms will wax and wane and may even remit [ 36 ] suggesting a life long process. The major behavioral symptoms of schizophrenia include alterations in cognition, memory, perception, thought (inferred from language), motor functions, and affect. People with schizophrenia may show abnormal dermatoglyphics and other minor physical anomalies [ 37 - 42 ]. Other oddities to be incorporated in a comprehensive explanation of schizophrenia include highly visible nail fold capillaries [ 43 , 44 ] and the rarity of rheumatoid arthritis among schizophrenic persons [ 45 ]. These physical characteristics suggest the need to look beyond the nervous system per se to have a comprehensive view of the illness. The fact that the schizophrenia syndrome, as currently defined, is relatively common provides important information about the frequency of causal factors. About 1% of the population will experience schizophrenia during the lifespan. Except for a few rare exceptions, this 1% risk is remarkably constant around the globe regardless of culture, geography, or ethnicity. Men and women are affected equally. These facts mean that the risk factors for schizophrenia must also be common and ubiquitous. Given that the concordance rate for schizophrenia in identical twins [ 46 ] is only about 50%, there must be at least two global risk-increasing categories for schizophrenia, i.e., something(s) genetic and something(s) environmental. Assuming these risk factors are independent of each other, the joint probability of acquiring both risk factors is the product of their population frequencies that, for schizophrenia, equals about .01. To make a simplifying assumption to allow easy calculations, let us say that the two risk factors are present with about equal frequency in the population. With this simplification, straightforward mathematics indicates that the individual frequencies of these factors are close to the square root of the population frequency of 1%. That would mean that about 10% of the population would encounter at least one risk factor. The math indicates that the greater the number of independent risk factors, the more common they are. [See [ 47 ] for further elaboration]. Our challenge is to develop a theory of schizophrenia that can plausibly explain an illness that affects all domains of behavior (thought, affect, motor performance, etc), that has elements of developmental perturbations early in life leaving clues such as minor physical abnormalities, and also has elements of degenerative changes. At the same time, the defect is so subtle that we can't find the cause(s) with our best modern technology. Furthermore, in spite of brain-wide dysfunctions, many individuals with schizophrenia remain sufficiently intact that, with good treatment and a bit of luck, can maintain jobs and function usefully in society. Thus, we need to find frequent and ubiquitous factors that can affect virtually all brain functions as well as creating somatic signs, but they operate in ways that leave these functions only slightly "off kilter" as compared to the complete disruption seen in strokes, or classical degenerative disorders such as Alzheimer, or as seen in Down syndrome where the behavioral pathology is apparent from earliest stages. As we try to explain schizophrenia, we must account for most all of the developmental and degenerative features of schizophrenia. To account for the panoply of signs and symptoms seen in schizophrenia, any complete theory of schizophrenia must include organism wide systems. In addition to the nervous system, the immune system and the vascular system are defensible candidates. Both are invoked in the following theory: Some schizophrenia psychoses are the result of damage to the micro-vascular system in the brain initiated by genetically influenced abnormal inflammatory processes acting in response to ubiquitous environmental factors that trigger inflammatory responses, including infection, trauma, or hypoxia. It is the relative infrequency of the vulnerable genotypes in the population [ 48 ] that results in only a small proportion developing overt psychosis. We wish to emphasize that our hypothesis specifically identifies the microvascular system as the critical site of inflammation. We postulate that the inflamed micro-vessels lose their coupling with astrocytes, leading to disrupted regulation of cerebral blood flow and damage to the blood brain barrier. These disruptions in homeostatic mechanisms then lead to abnormal signal processing. Our focus on inflammation of the vessels differentiates our hypothesis from models of widespread parenchymal inflammation such as seen in psychotic syndromes following, for example, encephalitis lethargica, or paraneoplastic syndromes. Many acute inflammatory disorders of the brain involve inflammation of both the parenchyma and the vasculature. By contrast, we are proposing a chronic, smoldering, inflammation of the vessels alone. And, finally, we distinguish our hypothesis from the theories of schizophrenia implicating direct parenchymal infection of the brain (c.f. [ 49 ]) and also differentiates our hypothesis from speculations about schizophrenia that invoke infectious agents altering DNA [ 50 ]. Many prior debates about inflammation in the brains of people with schizophrenia have focused on the presence of absence of gliosis (see [ 51 ] for review). The consensus opinion is that gliosis, though present in some cases, is not a consistent feature of the neuropathology of schizophrenia. However, as Harrison [ 51 ] points out, evaluating gliosis is fraught with a multitude of problems and is not a definitive indicator of degenerative/inflammatory changes in the brain. More recent efforts have demonstrated activation of microglia in the brains of some individuals with schizophrenia implying an ongoing immunopathological process in addition to what ever happened early in development [ 52 ]. Ongoing neurodegenerative processes are suggested by increased levels of S100B, a small calcium binding astrocytic protein that is involved in inducing apoptosis and modulating proinflammatory cytokines [ 53 - 55 ]. It is likely that the current clinical syndrome of schizophrenia is etiologically heterogeneous. We do not pretend to explain all (DSM or ICD) cases of syndromal schizophrenia. Instead, we put forward our hypothesis as an attempt to define a psychiatric syndrome in terms of a particular pathophysiology. Following this course may then help refine our nosology (see also section on 'specificity' below) and cause us to recalculate basics 'facts' such as prevalence rates. Discussion A primer on CNS blood supply Neurons derive their energy from oxygen and glucose delivered by the vascular system, plus lactate and glycogen derived from astroglia [ 56 ]. The combination of neurons, astroglia, and micro-vessels form a metabolic trio [ 56 ] whereby the glia extend processes interacting with neurons on the one hand and, on the other, form endplates interdigitated into capillary walls. Rather than being passive conduits, the CNS vascular system is the most precisely managed and the most complex fluid dynamic system known. Regulation of cerebral blood flow (CBF) is managed primarily by a coupling between astrocytic glial cells [ 56 - 59 ] and capillary endothelium [ 60 - 65 ]. Astrocytes sense local neuronal metabolic activity and adjust blood flow as needed. Cerebral vessels change caliber in response to vasoactive substances released by astrocytes activated by glutamate receptors [ 56 , 66 , 67 ]. Serotonin [ 68 ], acetylcholine [ 69 ] and dopamine [ 66 , 70 , 71 ] transmission between astrocytes and micro vessels also play roles. When the neuronal activation of discrete areas is sustained over longer periods, vasoactive substances stimulate angiogenesis resulting in increased capillary density [ 67 ] thus enhancing local neuronal circuitry. Conversely, decrease in capillary density is likely to reduce the functional capacity of brain areas so affected [ 67 ]. Consequently, capillary beds in the cortex are not distributed in uniform fashion [ 72 ]. There are close relationships among local neuronal activity, density of capillary bed, and the distribution of valve-like flow control structures [ 73 ]. Developmentally, the CNS vascular system originates from capillary endothelial cells that migrate into developing neuro-ectoderm under the influence of trophic factors such as vascular endothelial growth factor (VEGF) [ 74 ] and erythropoietin [ 75 ] both produced by astroglia. The developing micro-vasculature, although comprising only 0.1% of the entire brain, and operating under the influence of genetic directives, has a key role in the development, maintenance and repair of the brain [ 76 ]. In turn, VEGF has trophic effects on neurons and glial cells, and the activity of VEGF influenced angiogenesis is directly proportional to the high metabolic activity of neocortical development [ 77 ]. Thus, angiogenesis and neurogenesis occur simultaneously and synergistically [ 78 - 80 ]. In addition to formation of capillaries themselves, intricate anastomoses between micro-vessels further 'fine tune' the metabolic support of developing glia and neurons [ 81 ] The genetics of infectious & inflammatory diseases When infectious agents give rise to inflammatory vascular disease, the nature of the infectious agent may be less important that an individual's genetically influenced inflammatory response. The concept that infectious disease may have a genetic component is, of course, not new. Many agricultural geneticists make their livings by breeding disease resistance into both plants and animals [ 82 , 83 ]. One of the founders of behavioral genetics, Franz Kallmann [ 84 ], showed genetic factors influenced acquiring tuberculosis (DZ concordance = 26%, MZ concordance = 87%), an observation that was confirmed in modern times [ 85 , 86 ]. Many other infectious diseases appear to have genetic factors influencing susceptibility or resistance to the infection [ 87 - 97 ]. Mechanisms for genetically mediated responses to infection occur through genetic variations in immune mediators such as cytokines[ 96 ] and HLA factors [ 98 , 99 ]. Familial Mediterranean Fever (FMF) [ 100 , 101 ] provides a heuristic Mendelian example. The gene for FMF is located on the short arm of chromosome 16 and produces pyrin (marenostrin) that functions in a negative feed back loop to suppress inflammation. Absence of pyrin leads to exaggerated inflammatory responses. Vasculitis is one of the consequences [ 102 ]. Additionally, very high rates of rheumatic fever (RF) or rheumatic heart disease (RHD) are found in relatives of patients with FMF[ 103 ]. Having even one mutant gene appears to lead to immune hyperactivity to streptococcal antigens. We also know that antibody [ 104 ] production and cytokine activity [ 105 ] in RF patients is more marked than non-rheumatics. It is clear that genes influence the host's response to infection. A similar line of reasoning applies to other inducers of inflammation such as traumatic injury [ 106 ] or hypoxia [ 107 , 108 ]. Just as the CNS blood supply is highly regulated, the inflammatory systems in the brain require 'fine tuning.' Given the limited ability for adult brain to regenerate, and assuming there is little tissue to spare, it would make sense that the brain should be protected from overabundant inflammatory reactions [ 109 ]. Astrocytes play a key role in the expression of inflammatory cytokines, chemokines, and growth factors involving the modulation of gene expression for these factors [ 109 - 111 ]. Let us suppose that schizophrenia develops following an infection (or trauma or anoxia – the environmental contributors) but the host's response is determined by genetic factors regulating the nature and degree of inflammation. That infectious agents may be operative in schizophrenia is supported by several of lines of evidence. Summaries can be found in numerous sources [ 49 , 50 , 112 - 116 ]. The same concept applies to trauma [ 106 ] or anoxia [ 79 , 107 ] that may also stimulate inflammatory processes. Vascular disease and psychopathology The syndrome of schizophrenia is likely to be etiologically heterogeneous and a multitude of CNS disorders can give rise to schizophrenic-like psychoses [ 117 ]. The idea that CNS micro-vascular diseases, in particular, are factors in psychotic disorders is also an old idea [ 118 , 119 ] that deserves a second look in light of new perspectives offered by developments in the genetics of inflammatory diseases. There are many examples of psychoses resulting from micro-vascular CNS disease including lupus and Sjögren syndrome [ 120 ]. Neuroimaging and neurocognitive deficits in these disorders are similar to those seen in schizophrenia [ 121 ]. Psychoses associated with substance abuse are also associated with CNS vasculitis [ 122 ]. Furthermore, infectious agents such as syphilis [ 123 ] and rheumatic fever (RF – see below), lead to micro-vascular disorders of the CNS that are associated with psychiatric symptoms including psychoses. Thomas, et. al. [ 124 ] also demonstrated small vessel abnormalities in the depressed elderly. At the same time, there is growing interest in cytokines and other inflammatory agents in psychoses[ 125 ] as well as growing awareness that inflammatory reactions are modulated by neuropeptides [ 126 ]. Inflammatory processes often damage the precise regulation of cerebral blood flow. The wide spectrum of clinical conditions thought to be created, in part, by inflammatory CNS micro-vessel disease include Alzheimer disease where it is thought that inflammatory processed damage the micro-vascular endothelium causing insufficient blood flow leading to oxidative stress, a build up of amyloid, and eventual cell death [ 127 - 135 ]. Cerebral palsy is also conceptualized as an infectious-inflammatory-vascular disorder where the vascular lesion is complete thrombosis [ 136 ]. Neurotoxic effects of methamphetamine and cocaine appear to be due to induction of inflammatory genes in small vessel endothelial cells [ 122 , 137 ], thus explaining the vascular damage seen in amphetamine and cocaine abuse that was previously attributed to contaminants of injected drugs [ 122 , 138 - 140 ]. Returning to the early stages of life, we have seen that the development of the neurons and glia are intimately associated with, and dependent on, the parallel development of the CNS vasculature. If the stated theory is correct, and given the developmental perspective of schizophrenia ---early developmental perturbations of the CNS set the stage for later schizophrenia--- we would expect to find support for the idea that inflammatory events early in life affect CNS vascular function. Such is the case. Whether the early insults are traumatic, infectious, or hypoxic; inflammatory process are involved in the attempts to protect and repair by modulating angiogenesis [ 141 - 148 ]. Thus, the reports implicating pregnancy and birth complications (anoxia, trauma or maternal infections) in the development of some cases of schizophrenia [ 149 , 150 ] could all be mediated by the common pathway of inflammatory-vascular mechanisms. Individuals who's genes created perturbations in inflammatory-vascular regulation would continue to experience abnormalities of protection and repair in response to subsequent CNS insults. Over time, the accumulation of 'hits' could lead to brain dysfunction to the extent seen in psychoses. The greater the number and duration of 'hits,' the greater the risk for a deteriorating /degenerative course. That neuroleptics may alter the permeability of the blood brain barrier and modify immunoregulation in the CNS [ 151 ] strengthens the argument for early treatment as a strategy to prevent deterioration. Alterations of cerebral blood flow in schizophrenia Since the time of Seymour Kety's pioneering efforts [ 152 , 153 ], there has been interest in altered cerebral blood flow in people with schizophrenia. An in-depth review of this large literature is beyond the scope of this paper. The interested reader is referred to discussions of reduced anterior cerebral perfusion leading to the concept of 'hypofrontality' in schizophrenia [ 154 , 155 ] and to more recent reviews [ 156 - 158 ]. Bachneff's [ 159 ] review and theory about defects in regulation of CNS microvascular systems is particularly relevant. These reviews summarize a consistent body of evidence showing reduced cerebral blood flow in brains of people with schizophrenia especially to anterior regions. Flow deficits are seen in medication-naive new onset cases [ 160 , 161 ] and more established cases free of neuroleptics [ 162 ] suggesting that flow perturbations are neither the consequence of duration of illness nor treatment. Neuroleptics can alter cerebral blood flow [ 163 , 164 ] although the effects may be regionally and drug specific [ 165 , 166 ]. Decreased frontal flow is often associated with negative symptoms [ 167 , 168 ]. In addition to the frontal cortex, flow abnormalities in people with schizophrenia have been noted in the cingulate cortex [ 169 , 170 ], thalamus [ 171 ], basal ganglia [ 172 ], parietal cortex [ 167 , 170 ] and cerebellum [ 171 ]. Furthermore, in some instances, flow rates are increased [ 160 , 170 ]. Rather than a simple hypothesis of hypofrontality in schizophrenia, theorizing is evolving toward a concept of "dysfunctional circuits"[ 160 ] or "inefficient dynamic modulation" [ 173 ] of cerebral metabolism which is supported by other examples of abnormal modulation of cerebral blood flow in response to activation tasks [ 171 , 174 ]. Disturbances of blood flow in schizophrenia are well documented but are not limited to schizophrenia. Disturbed cerebral blood flow is also reported in obsessive compulsive disorder [ 175 ] and depression [ 176 , 177 ] as well as in Alzheimer disease (cited earlier). The usual interpretation is that alterations of blood flow arise as a consequence of abnormal neuronal metabolism. The theory proposed by this paper turns the causal arrow around to suggest that abnormalities of blood flow lead to altered neuronal-glial function that, in turn, leads to psychopathology. There has been scant direct visualization of the vascular system in schizophrenia, but at least one laboratory has found evidence of atypically simplified angioarchitecture and failure of normal arborization of small vessels [ 32 ]. Post- streptococcal behavioral syndromes as a model Post-streptococcal neuropsychiatric syndromes include Syndenham chorea, the PANDAS/obsessive compulsive syndrome, tics including Tourette syndrome, and possibly, ADHD [ 178 - 184 ]. Psychotic disorders are also implicated [ 183 , 185 ] and see citations below. Sydenham chorea is the best-known neuropsychiatric complication following streptococcal pharyngitis. The association of psychoses and Sydenham chorea as well as with RF even in the absence of chorea, was discussed in the 17 th and 18 th centuries starting with Sydenham himself (see [ 186 ]). The interest in psychoses associated with RF continued throughout the 1900's [ 187 - 197 ]. People with a history of Sydenham chorea and/or rheumatic fever are at high risk for developing psychopathology later in life [ 198 , 199 ] with a relative risk for schizophrenia as high as 8.9 in a 10 year follow-up of 29 Sydenham patients [ 200 ]. There is a suggestion that the family members of Sydenham patients are also at higher risk for psychosis [ 201 ]. During the 1940's-1960's when RF was still quite prevalent, people with psychoses appeared to have higher than expected rates of histories of RHD or RF)[ 195 , 202 , 203 ] or rheumatic chorea [ 204 ]. Psychotic patients with RHD more often had early (<age 19) onset, movement disorders, progressively insidious courses and poor long-term outcomes [ 203 ]. Preliminary data from a Minnesota study also finds increased rates of RHD in psychotic patients, a pattern of increased psychiatric hospitalization following an epidemic of RF, and a clinical course for "rheumatic psychoses" that disproportionately led to a severe and continuous decline in function [ 205 ]. Although schizophrenia-like psychoses were the most common psychopathology related to rheumatic syndromes, manic-depressive, involutional, and senile psychoses were also observed [ 183 , 197 ]. An inflammatory reaction of the CNS vascular endothelium (vasculitis) is a common denominator in the both acute and chronic cerebral consequences of rheumatic fever. [ 186 , 187 , 190 , 195 , 197 , 206 - 209 ]. The microvascular lesions suggest both an obliterating process likely due to micro-emboli from rheumatic cardiac valves and an inflammatory process involving irregular proliferative changes in the vascular endothelium, dilatation of the lymphatic spaces surrounding the capillaries suggesting increased permeability of the capillary endothelium, and inflammatory cell infiltrates. Disruption of the blood brain barrier suggested by the evidence of increased permeability of the small vessels could compromise the immunological protection of the brain leading to the formation of the anti-neuronal antibodies seen in post-streptococcal CNS syndromes. In parallel fashion, people with schizophrenia show evidence of altered blood brain barrier and consequent alterations in immunological markers [ 210 ] The post-strep psychopathologies provide a precedent for the hypothesis of this paper by demonstrating that an infectious process can trigger a series of inflammatory reactions that lead to a variety of somatic and psychiatric syndromes, including psychoses where vascular pathology is implicated. The pathogenicity of a strep infection is a function of the strain (genotype) of the bacterium and the genetically mediated inflammatory mechanisms of the host [ 211 ] and illustrates how a ubiquitous and often relatively benign environmental factor can create more serious sequelae in a limited number of genetically predisposed individuals-true genotype by environment interaction. Summary The ideas here are not completely new. Eugen Bleuler [ 14 ] remarked: "The fragility of the blood vessels which appears in many schizophrenics, both acute and chronic, seems to indicate a real vascular pathology (p.167)." We bring old ideas forward into the light of new understandings offered by molecular genetics and inflammatory diseases. Since the late 1800's there has been evidence of inflammatory neuro-vascular abnormalities in psychiatric illness that were initiated by infectious agents. CNS lues (syphilis) is the best-known example. This paper expands the concept to suggest that a variety of environmental insults (infection, trauma, anoxia) may follow a common final pathway to psychopathology by stimulating inflammatory processes that damage the capillary-glial-neuron triad as illustrated in Figure 1 . Abnormal behaviors develop as a result of disruptions in astroglial mediated coupling of cerebral blood flow to neuronal metabolic needs. These subtle disruptions are hard to find, as the microvasculature comprises only about 0.1% of the brain and are of a scale more appropriate for electron microscopy. None-the-less, the hemodynamic perturbations have sufficient impact to cause subtle but widespread disruption of the normally harmonious coordination of CNS function leading to a condition variously conceived as a "neurointegrative defect"[ 212 ], "synaptic slippage" [ 213 ], "abnormal signal transduction" [ 4 ], "inefficient dynamic modulation" [ 173 ] or "synaptic destabilization" [ 214 ]. The ultimate impact would lead to psychopathology including psychoses as the vascular-glial-neuron triad is progressively damaged over time after repeated inflammatory episodes. The resultant failure to regulate the delivery of oxygen and energy adequately would lead to oxidative stress [ 215 - 217 ]. Oxidative stress, in turn, can further damage the microvasculature and the blood brain barrier [ 218 - 220 ]. The astroglial-capillary partnership that protects the integrity of the blood brain barrier would be compromised, thus exposing neural tissue to damage from immunological attack [ 221 ]. Known precedents of such processes are found in the behavioral changes seen in CNS vascular inflammatory diseases such as lupus and the post-strep syndromes described above. This theory could explain how developmental events such as prenatal infections [ 150 , 222 ], and other birth and pregnancy complications [ 149 ] including anoxia [ 223 ] are linked to later schizophrenia – infection, trauma, or anoxia all stimulate inflammatory processes [ 224 ]. The data suggesting an (statistical) influence of season of birth [ 116 ] is also consistent with the hypothesis as infectious epidemics often follow seasonal patterns. Some of the minor physical anomalies such as unusual scalp hair patterns and dermatoglyphic changes are explained because the development of these phenomena are linked to each other [ 225 ], to the development of the central nervous system [ 226 ], and are developmentally modulated by the pleiotropic effects of the same substances that modulate brain vascular development (e.g., vascular endothelial growth factor/vascular permeability factor [ 227 ] and epidermal growth factor [ 228 ]). The waxing and waning of symptoms would correspond to waxing and waning of inflammations as individuals are exposed, recover, and then re-exposed in conjunction with other physiological and hormonal influences, as seen in lupus [ 229 ]. The nature and severity of symptoms would depend on where in the brain the inflammation takes place and this may be stochastic. As the micro- vascular system is everywhere in the brain, lesions could produce the variety of symptoms seen in schizophrenia including dysfunctions of thought, emotion, memory, motor skills and autonomic regulation. The developmental age of the individual will also make a difference. Inflammatory processes that alter angiogenesis during prenatal development will likely have more dire consequences than inflammatory reactions that start after CNS maturation although even the adult brain remains susceptible [ 230 ]. We have attempted to schematically illustrate this dynamic process in Figure 2 . This theory also captures many of the little oddities observed in schizophrenia. For example, the reported abnormalities of the nail fold capillary beds seen in some people with schizophrenia [ 44 ] are also seen in people with inflammatory disorders such as FMF [ 231 ] and rheumatoid arthritis [ 232 ]. Another oddity is the negative association between schizophrenia and rheumatoid arthritis [ 45 ]. There are parallels in the post-streptococcal syndromes where RF and acute post-streptococcal glomerulonephritis very rarely occur in the same patient [ 233 ]. Some strains of group-A-streptococci identified by their M-protein serotypes are rheumatogenic while others are nephritogenic [ 233 , 234 ]. Phage or phage-like elements inserted into the streptococcal DNA are a major source of variation between streptococcal strains and these elements determine pathogenicity [ 235 ]. Additionally, host variation in humoral and cellular immune response shape the outcome of infection[ 211 ] By analogy, individuals with vascular/CNS involvement following, for example, streptococcal infections may be systematically spared from joint involvement as a function of both the invading strain and the individuals susceptibilities. Alternatively, as postulated for Alzheimer disease (cited earlier) that is also less common in people treated for arthritis, the anti-inflammatory treatments for arthritis might reduce the risk of inflammatory brain disease. Another line of evidence compatible with this theory is the observation that genetic linkages for schizophrenia coincide with sites for glial growth factor cell regulators [ 214 ] and, as we have seen, the glia are key intermediaries of CNS inflammation and vascular regulation. More specifically, emerging data demonstrate associations between schizophrenia and genetic polymorphisms in regulators of inflammation such as tumor necrosis factor alpha genes [ 236 , 237 ] and interleukin-1 genes [ 238 ]. Another piece that fits into the puzzle is the fact that neuroleptics have inflammatory modulating properties [ 239 - 244 ] and neuroleptic treatment may be synergized by addition of anti-inflammatory drugs [ 245 ]. It may well be that the environmental components of psychiatric illness such as schizophrenia are relatively minor, ubiquitous, or chance events [ 246 , 247 ] that have the potential to stimulate the inflammatory systems. However, the nature of the insults may be less important than individuals' genetically influenced and idiosyncratic responses to the insults, similar to individuals with FMF who have an exaggerated inflammatory response. Thus, the genetic components of the inherited predisposition to mental illness may lie "upstream" in the immune system rather than in the CNS per se. The possibility that the environmental agents may be nearly universal (e.g. who has not had a strep throat or viral syndrome?), will mean that the prevalence of the etiological factor will be similar in control and experimental groups thus making it too easy to dismiss key environmental factors in null hypothesis designs [ 47 , 248 ]. Rather than focus on the environmental contributors that could be non-specific and ubiquitous, it will be more productive to look for genotypes that respond abnormally to triggers of inflammation and microvascular dysfunction (cf[ 48 ]). These individuals would be the ones who are at high risk for psychiatric illness. However, the inflammatory processes involve a cascade of steps involving many genes. But this, too, fits with the polygenic features of schizophrenia [ 249 ]. Identification of high-risk individuals, combined with such tools as immunizations or anti-inflammatory agents may promote prevention of much psychiatric morbidity. Already, the cytokine regulator and vascular growth factor erythropoietin is suggested as a possible neuroprotective factor in schizophrenia [ 250 ] Future directions The speculations about psychoses developing from vascular/inflammatory processes provide direction for future research across many domains. In addition to pursuing direct evidence of altered activities in inflammatory/immune systems in people with psychoses, the inflammatory/vascular theory has implications for epidemiology, genetics, neuroimaging and neuropathology. For the epidemiologist, the challenge will be to detect relatively small signals against a very noisy background. We hypothesize that the triggers for inflammation can be many and varied and are common factors in the environment. Imagine starting with the clinical syndrome of Sydenham chorea and comparing the rates of strep throat in those affected vs. comparison sample of people without Sydenham chorea. Null hypothesis testing with small sample sizes and nearly ubiquitous etiological agents are clearly not adequate. A second epidemiological challenge is to cast a broad enough net to capture the wide variety of possible contributing factors. Rather than taking a one by one approach to exploring the etiological contributions of, say, virus titers, anoxia, physical trauma, the epidemiologist should look for any and all. It would be predicted that individuals with multiple "hits" (e.g. in utero exposure to virus and low Apgar scores and childhood head trauma) would be at greater risk than those exposed to just one event. If in utero inflammatory processes are active in the genesis of schizophrenia we would also predict an increased rate of fetal deaths in families of schizophrenic probands. A third epidemiological opportunity lies in the search for non-psychiatric inflammatory-related disease or traits in people with psychosis. If something is askew in the inflammatory process in schizophrenia, the effects will show up in other parts of the body. Though requiring replication, the association of psychosis with hemolytic anemia in lupus [ 251 ] provides an illustrative example. In addition to rheumatoid arthritis, the associations of diabetes and cancer have been explored in schizophrenia; one of is exploring rheumatic heart disease [ 205 ]. Population-based health registries should be used in a search for co-morbid physical illness. For geneticists, the proposed theory obviously points to linkage/association studies using inflammation genes; a few examples were cited previously [ 236 - 238 ]. A simple step with extant data might start with a meta analysis defining chromosomal "hot spots" for linkage with schizophrenia and search the gnome maps for immune regulators at these sites as Moises, et al [ 214 ] have done for glial growth regulators. Family, twin, and adoption methodologies can all be applied to the issue of co-morbid or co-segregating physical conditions. The inflammatory/vascular theory has much to suggest to neuroimaging research especially in the realm of reinterpreting regional perturbations in metabolic activity as primary disturbances of flow regulation rather than intrinsic neuronal metabolic abnormalities. It would be interesting to assess the impact of vasoactive compounds and inflammatory modulators on neuroimaging studies of regional blood flow. Likewise, further pursuit of neuroimaging evidence of disrupted blood brain barrier, as initiated by Dysken, et al [ 252 ], and with manipulation of inflammatory systems as suggested by Mueller and Ackenheil [ 253 ] would test our hypothesis. The neuropathology of schizophrenia, focused mostly on the neurons, is notable for inconsistencies in findings (see [ 51 , 254 ] for reviews). Such inconsistency is exactly what would be predicted by an inflammatory/vascular theory where the lesions are truly functional in the sense that the function of the brain alters in relation to perturbations in blood flow regulation. Only the more prolonged and serious inflammation will leave visible traces of neuronal damage and such damage may be patchy and inconsistent from one patient to another. However, over the early years of CNS development, alterations in cellular organization or migration may result from disrupted angiogensis that must go hand in hand with neuronal and glial development. The location and extent of CNS change will be a function of severity of inflammation and timing during development. Such consequences will be hard to demonstrate in human post-mortem tissues and animal or in vitro models may be more fruitful areas for study the effects of inflammation on neurogenesis and blood flow regulation. To our knowledge, human post mortem studies have not utilized vascular cast methodology and this should be considered, perhaps casting one half of a specimen brain while subjecting the other half to cellular analysis. Specificity Because of our interests and expertise, we have focused our attention on schizophrenia as the behavioral phenotype resulting from inflammatory-vascular pathology but the theory presented here is likely to be more general. Indeed, our use of examples of psychoses associated with known inflammatory- vascular pathologies (e.g. autoimmune CNS vascular disease or infectious CNS vascular disease as seen in syphilis) makes it clear that a vascular-inflammatory theory may apply to a wide range of psychotic conditions that may also include psychoses associated with mood disorders. Whereas, the classical genetic studies support the separateness of schizophrenia and mood disorders [ 255 ], there are modern molecular signs that schizophrenia and mood disorders share genetic elements in common [ 256 , 257 ]. Furthermore, mood disorders, like schizophrenia, show evidence of frontal lobe pathology, enlarged ventricles, abnormal cerebral blood flow [ 33 , 258 ] and vascular abnormalities [ 124 ]. To what extent all of these changes are epiphenomena of being psychotic (treatment effects or stress, etc) remain debatable [ 259 ]. However, finding similar brain changes in a variety of psychotic conditions does not necessarily mean these changes are epiphenomena. Examples from neuropsychiatry teach us that the underlying pathology does not necessarily define the behavioral symptoms. Thus, psychoses with Huntington disease may be affective-like or schizophreniform. Similar pathophysiological mechanisms may underlie a variety of psychotic phenotypes. The evolution of behavioral symptoms for any given pathophysiology may depend on a variety of moderating variables such as an individual's developmental age when the disease process begins, gender, hormones, genetic 'landscape' upon which the disease process unfolds, along with the nature, frequency, and intensity of successive triggers of inflammatory response. Reprise A broad spectrum of observations leads to a working hypothesis that schizophrenia and, possibly, other psychiatric syndromes are the result of genetically mediated inflammatory reactions that damage the neuron-glial-capillary triad with resultant loss of ability to fine tune regional brain metabolism. This hypothesis incorporates genetic, epigenetic [ 260 ], and environmental factors. Furthermore, an inflammatory/vascular theory can explain the variety of behavioral symptoms seen in schizophrenia, the variable course of the illness, and the numerous other puzzling observations such as an excess of minor physical anomalies. Should this theory prove heuristic, it would point to the use of inflammatory modulators in treating the illness. Perhaps more importantly, identifying individuals who were at high risk for the disorder in high genetic risk families as well as the general population, because of abnormalities of their inflammatory systems, holds hope for prevention through early intervention using inflammatory modulators. List of abbreviations ADHD attention deficit hyperactivity disorder BDNF brain derived neurotropic factor CBF cerebral blood flow CNS central nervous system DZ dizygotic FMF familial Mediterranean fever MZ monozygotic NGF nerve growth factor NO nitric oxide PANDAS pediatric autoimmune neurological disorder associated with strep. RF rheumatic fever RHD rheumatic heart disease VEGF vascular endothelial growth factor Competing interests The author(s) declare that they have no competing interests. Authors' contributions This article was the joint effort of both authors with input as noted below. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554096.xml
548681	Use of email for patient communication in student health care: a cross-sectional study	Background Citizens increasingly use email in personal communication. It is not however clear to what extent physicians utilize it for patient communication. Our study was designed to examine physicians' activity in using email and to estimate the proportion of email messages missing from documentation in electronic patient records (EPR). Methods All physicians (n = 76; 48 general practitioners and 28 specialists) at the Finnish Student Health Service received a questionnaire by email, and were asked to print it and keep a daily tally of visits, phone calls and email messages over the study period of one working week (5.5. – 9.5.2003). The response rate was 70%. The data originating from the questionnaire were compared with statistical data from the EPR during the study period. Results The majority (79%, 41/52) of doctors reported using email with patients, averaging 8.6 (range: 0–96) email contacts and a percentage rate of "email / visit" 20% (range: 0–185%) in one working week. Doctors in the capital city region and those doctors who had a positive attitude toward email for patient communication were most active in email use. Up to 73% of email contacts were not documented in the EPR. Conclusion The activity in using email with patients verified among Finnish physicians is compatible with recent study results elsewhere. The notable proportion of un-recorded email messages establishes the need for an electric communication system built into the EPR to improve the quality of patient care and to limit medico-legal risks.	Background Citizens increasingly use email in personal communication [ 1 ]. It is not however clear to what extent physicians utilize it for patient communication. In reports from the 1990s 1–14% of doctors in the USA and Norway used email in patient work [ 2 , 3 ]. In recent studies up to 73% of physicians had used email for patient communication [ 4 - 6 ]. International and national recommendations and guidelines have been published on email use between doctor and patient; the contents of the Finnish guidelines are well in line with European guidelines [ 7 , 8 ]. These guidelines emphasize the suitability of email for only certain limited purposes and stress the risks to information security. Use of email between doctor and patient has been studied in one controlled and randomized and three cross-sectional studies [ 4 , 5 , 9 , 10 ]. In a study by Katz and colleagues the number of email contacts of physicians in their study group (46 email messages/100 scheduled visits) was greater than that in the control group (9/100) [ 5 ]. Houston and associates found that the majority of doctors received daily 1–5 email messages from their patients [ 9 ]. According to Sittig, physicians received daily in average 2.6 messages, and monthly an average of 40 per 140 visits [ 10 ]. Gaster and colleagues noted that physicians on average received 7.7 email messages in a month from their patients. Physicians in university clinics were most active in email use, while those in municipal primary health care were least active. Of physicians 58% reported in the questionnaire that the email contacts with patients were for the most part not registered in patient records [ 4 ]. Among Finnish citizens of working age young adults are the most active users of email and Internet [ 1 , 11 ]. University students use these electronic net services even more actively than the young adult population as a whole. In a study from 2002 99% of students reported using email and Internet at least weekly [ 12 ]. All students have an email address at the university and their health providers at the FSHS can be reached by email. The student health care system can be seen as an appropriate setting to use email for patient communication [ 13 ]. The students represent a young, well educated, relatively healthy part of population which has been identified to be the most active to use email in patient-doctor communication [ 2 , 5 , 14 ]. The Finnish Student Health Service The Finnish Student Health Service (FSHS) provides primary health care services to approximately 140.000 university students in Finland. The FSHS has health stations in 16 university cities. Services include health promotion, consultations with general practitioners and with other clinical specialists, mental health care, and dental care. Since 1993 FSHS has provided health counseling in Internet. Since 1999 all physicians have had an email account at their disposal in health stations and an email address of type: firstname.surname@yths.fi . Principles of communication by email with FSHS' employees and of other forms of electronic services (email service for cancellation of appointments, health counseling service on the Internet, and email service for feedback) are available at the FSHS' website. The Social Insurance Institution, the university cities, the State of Finland, and the students themselves finance FSHS services. Students pay an annual obligatory health care fee as a part of the Student Union's membership fee. There is no other fee for preventive services, visits to general practitioner or public health nurse, and laboratory or X-ray examinations prescribed during these visits. Use of Internet services is also free of charge. The FSHS employs 560 persons and 63% of the physicians are general practitioners. In this paper general practitioners also include specialists of general practice/family medicine, whereas "specialists" refers to clinical specialists other than psychiatrists or oral surgeons. Aims of the study The aim of the study was to seek answers to the following questions: 1. How actively did physicians at student health care use email in communication with their patients? 2. How much did they use email compared to phone calls and patient visits? 3. Who were the active doctors using email with patients? 4. What proportion of visits and phone calls could be candidates for substitution by email communication? 5. Did the volume of visits, phone calls and email messages documented in the EPR of the FSHS during the study period differ from that of visits, phone calls and email messages registered in the study? Methods All physicians (n = 82) in the FSHS' functionary register in April 2003 received a questionnaire by email. We excluded six physicians, who were not any more working for the FSHS and took exception to the two authors. The actual number of survey population was 74. The questionnaire (see Additional file 1 ) included background factors and a registration (in form of daily tally) of numbers of patient contacts, phone calls and email messages over one working week. Respondents were also asked to assess the number of visits and calls replaceable by email, and the number of email messages including a request, which could not be fulfilled without face-to-face contact. Also doctors' attitudes toward email use for patient communication were asked. The first mailing of questionnaire took place 28.4.2003 and a reminder was sent 5.5.2003. Recipients were asked to print the survey form, fill it in by hand, and return it by internal mail. Overall 52 out of 74 (70%) physicians returned a completed survey. Respondents were grouped according to age, location, speciality licence, and type of employment (Table 1 ). Facts on years of birth were collected from the register book "Finland's Doctors 2002" and other background factors from the FSHS' functionary register. Table 1 Background variables of all physicians at the FSHS and of the respondents. All physicians Respondents (n = 76) (n = 52) % % Gender Male 34 29 Female 66 71 Age (years) 45 or under 33 31 46–55 39 42 56 or over 28 27 Location Capital city region 38 42 Turku and Tampere 28 27 Other 1) 34 31 Speciality licence General practitioner 63 69 Specialist 37 31 Type of employment Permanent 68 85 Non-permanent 2) 32 15 Distribution (%) of background variables of all physicians at the Finnish Student Health Service and of the respondents of the survey. 1) Small university towns 2) Vicars and fee-based working doctors All physicians at the FSHS utilize EPR (Medicus ® ). We collected the numbers of patient contacts documented at Medicus ® for the study period using the statistical software Cognos ® . We entered data using Microsoft Excel ® software and performed statistical analysis using StatsDirect Statistical ® (3,2,7 -version) software. Statistical analyses were conducted using the proportion test for two independent groups, the χ 2 test, Fisher's exact test, the unpaired t-test, the Mann-Whitney test and the Kruskal-Wallis test. All tests were made two-sided and p-values below .05 were regarded as statistically significant. Results Respondents Of all respondents 29% were men and 71% women (Table 1 ). The respondents and all doctors at the FSHS were compared according to the background variables. The doctors who answered represented well the overall body of physicians working in the FSHS. The mean age of male respondents was 52.8 (range 34–65) and of female 48.5 years (range 29–65). This was in the same range as the mean age of all doctors at the FSHS (men 51.3 and women 48.3 years). Activity of using email In one working week 79% of doctors had used email and 98% the phone for patient communication. Respondents reported 2296 patient visits, 948 phone calls and 449 email contacts. They had on average 8.6 email contacts and 18.2 phone calls per week with their patients (Table 2 ). They reported a mean percentage for "email per visit" of 20%, and phone calls in proportion to visits ("phone call per visit") averaging 40%. Eleven doctors (21%) reported more email contacts than phone calls. Table 2 Characteristics of the use of email. Characteristic Mean Median Minimum Maximum n 1) n 1) n 1) n 1) Type of patient contact Visit 44.2 46.5 7 78 Phone call 18.2 18.0 0 59 Email 8.6 3.5 0 96 % % % % Proportion of the respective contact types Phone call / visit 40 36 0 100 Email / visit 20 12 0 185 Email / phone call 55 22 0 600 Characteristics of the use of email in one week (5.5. – 9.5.2003) among physicians (n = 52) at the Finnish Student Health Service. 1) Number of contacts We tested the variables showing email usage and phone calls with respect to background factors. There were no statistically significant differences by gender, age group, speciality licence or type of employment. Of physicians in the capital city area 41% reported more email contacts than phone calls. Among doctors working in Turku and Tampere the proportion was 7.1%, and among those working in small towns 6.3%. The difference between capital city region and other locations was statistically significant (p = .015). Respondents' general attitude toward email for patient communication was evaluated by the statement "Email contacts with patients facilitate my work." The group of "positives" was formed of the 56% of respondents who on a five step Likert scale replied: "same opinion" or "nearly same opinion." The "positives" reported more email contacts than others (email per visit: median18% versus median 3%, p < .001). All eleven who had reported more email contacts than phone calls belonged to the "positive" group. Patient visits and phone calls replaceable by email Doctors estimated that 2% (57/2296) of patient visits could have been replaced by email. Of phone calls 21% (196/948) could have been substituted with email. Respondents estimated that 10% (45/449) of patients' email messages required a personal consultation. Documentation in the EPR The number of visits registered in the survey did not differ from the presumed number documented in the EPR (Table 3 .). The difference noted between registered and presumed email contacts shows that 73% of email contacts were not entered in the EPR. Table 3 Patient contacts documented in the EPR and registered in the survey. Type of patient contact Number of patient contacts Statistical significance of the difference between expected and registered contacts 2 ) All physicians Respondents Documented data in electronic patient record (Medicus R ) Expected 1) Registered n n n p Visit 3098 2107 2296 0.237 Phone call 1115 758 948 < 0.001 Email 178 121 449 < 0.001 Patient contacts documented by all physicians at the Finnish Student Health Service (n = 76) in the electronic patient record (Medicus R ) and registered by respondents (n = 52) in a survey over one working week (5.5. – 9.5.2003), as well as the statistical significance of the difference in contact numbers. 1) Based on the proportion of respondents among all physicians 2) Proportion test for two independent groups Discussion Even if university students do not represent the whole population, they can act as "pilot population" representing adults of working age of a future information society. Our study population – doctors taking care of students – was small with only 52 respondents. Thus the results of the study cannot be indiscriminately generalized. Because of the small study population comparison of the subgroups may not be reliable. Although the study group was small, it well represented all the doctors at the FSHS, and the response rate was high. A further strength was that we compared the number of patient contacts documented in the questionnaires during the study week to the statistical data of contact numbers from the EPR at the same time. In other studies no corresponding comparison has been made. The doctors were asked to keep a daily tally of visits, phone calls and email messages, and to evaluate how many visits or phone calls could have been replaced by email. Many doctors undoubtedly did this simultaneously with patient work. Some doctors might have been in a hurry, they probably supplemented the questionnaire at the end of the day. To achieve a more accurate evaluation of visits and phone calls replaceable by email, a continuous assessment (visit by visit, phone call by phone call) could have been stressed even more in the instructions. The doctors at the FSHS do not have a specific electronic communication system focused on patient communication. They use their general, unprotected email system also to communicate with patients. A specific communication system used only for patient communication would enable an automatic collection of the patient communication data and create a more accurate database than our data collection method. Katz and colleagues have made the only controlled and randomized study concerning physicians' use of email [ 5 ]. Our own results on the average number of doctors' email contacts and email usage are of the same magnitude as those referred to above and in other recent studies in the USA [ 4 , 9 , 10 ]. In the present study 79% of respondents used email for patient communication. The proportion of those who had used email is clearly larger than in older studies, and at the same level as reported in recent international studies [ 2 - 6 ]. Our study revealed individual differences in the use of email in patient work. Differences in physicians' activity in using email have previously been reported in only one study [ 4 ]. Deriving of our small study group only especially glaring association between subgroups of respondents could be verified. Our findings still support the results published by Gaster and associates. Physicians working in the capital area were more active email users than their colleagues elsewhere in Finland. Physicians reckoned that email could replace only 2% of visits. This confirms Sittig's evaluation in 2003 that email could possibly cover a small percentage of visits [ 10 ]. Increasing the use of email can thus not considerably reduce the number of patient visits. On the other hand it could make physicians' crowded telephone hours easier [ 15 ]. When we compared contacts in the EPR with contacts registered daily on the questionnaires we found that the majority of email contacts were not registered in the EPR. This finding is supported by Gaster and colleagues who asked physicians themselves to describe how often they usually registered email contacts in patient records [ 4 ]. FSHS provides specific electronic services for focused issues (email service for cancellation of appointments, health counseling service on the Internet, and email service for feedback). Principles of recommended issues to use email between health providers and patients are available for students at FSHS' websites. We have had a presumption that email messages between FSHS' physicians and their patients mainly handle patient care. Nyström's congress report from 2004 supports our presumption. He explored 139 email messages from 103 individual patients at his GP practice at the FSHS and noticed that 77 % of email messages handled medical tests, and 16 % handled follow-ups of symptoms or illnesses [ 16 ]. Thus the information in email communication should be entered in patient records. All university students in Finland have access to Internet and email at their universities. Use of email as communication method in health care does not in their case cause inequalities in health. A general tendency in the societies to provide also health services widely in electronic form (in Internet or by email) can contribute to inequalities for those who are not able to use modern technologies [ 13 , 17 ]. Conclusions Doctors at the FSHS had an average of 8.6 email contacts with their patients during one week. The proportion of email contacts per visit was 20%. Physicians estimated that email contacts could substitute 2% of patients' visits and 21% of phone calls. Of email contacts 73% were not documented in the EPR. Our study indicates that email communication really constitutes a part of patient work. This should be taken into account in planning working time and daily timetables. Use of software not integrated with the EPR increases the physician's registering load and currently involves extra work. It is not possible to confirm the patient's identity reliably using two separate systems. A system allowing retrieval of patient's identity safely and with no need to register separately the email communication in the EPR would promote the patient's adequate treatment and reduce the physician's medico-legal risks. There is a need for a larger study on email utilization between patient and physician which better covers the medical profession. The consumer point of view should also be better taken into account. A content analysis of email messages for patient communication combined with assessments of email documentation in the EPR could have strengthened present study regarding the importance of email registration in patient records. In Finland the Ministry of Social Affairs and Health has instigated a major project to safeguard health care [ 18 ]. One part of this programme demands that the whole public health care field should be using EPR by 2007 [ 19 ]. More research data are needed on electronic communication with patients and on users' experiences. The future EPRs should include a purposeful, safe and secure means of patient communication. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JC participated in the design of the study, coordinated it, keyed data into computer, performed the statistical analysis and drafted the manuscript with co-authors. MN conceived of the study, participated in the design of the study and wrote parts of manuscript in English. IV participated in the design of study and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Questionnaire Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548681.xml
535905	Neuroanatomical organization of gonadotropin-releasing hormone neurons during the oestrus cycle in the ewe	Background During the preovulatory surge of gonadotropin-releasing hormone (GnRH), a very large amount of the peptide is released in the hypothalamo-hypophyseal portal blood for 24-36H00. To study whether this release is linked to a modification of the morphological organization of the GnRH-containing neurons, i.e. morphological plasticity, we conducted experiments in intact ewes at 4 different times of the oestrous cycle (before the expected LH surge, during the LH surge, and on day 8 and day 15 of the subsequent luteal phase). The cycle stage was verified by determination of progesterone and LH concentrations in the peripheral blood samples collected prior to euthanasia. Results The distribution of GnRH-containing neurons throughout the preoptic area around the vascular organ of the lamina terminalis was studied following visualisation using immunohistochemistry. No difference was observed in the staining intensity for GnRH between the different groups. Clusters of GnRH-containing neurons (defined as 2 or more neurons being observed in close contact) were more numerous during the late follicular phase (43 ± 7) than during the luteal phase (25 ± 6), and the percentage of clusters was higher during the beginning of the follicular phase than during the luteal phase. There was no difference in the number of labelled neurons in each group. Conclusions These results indicate that the morphological organization of the GnRH-containing neurons in ewes is modified during the follicular phase. This transitory re-organization may contribute to the putative synchronization of these neurons during the surge. The molecular signal inducing this plasticity has not yet been identified, but oestradiol might play an important role, since in sheep it is the only signal which initiates the GnRH preovulatory surge.	Background Gonadotropin-releasing hormone (GnRH), the peptide responsible for the regulation of secretory activity of the pituitary gonadotropes, is found in a diffuse neuronal system situated in the preoptic area and anterior hypothalamic area [ 1 ]. The neurons project their terminals to the median eminence where the peptide is released into the hypothalamo-hypophyseal portal vessels. The secretion of GnRH is normally pulsatile, as demonstrated by the measurement of GnRH following the direct sampling of hypothalamo-pituitary portal blood in various species such as sheep [ 2 , 3 ]. The frequency of this pulsatility is the main characteristic of this secretion and it encodes each part of the sexual cycle in the female [ 4 ]. The highest circulating concentration of GnRH is induced during the follicular phase in the female by the sequential action of the two main ovarian steroids (progesterone and oestradiol). These act within the brain to trigger a large and sustained period of GnRH release, the GnRH surge, which stimulates the preovulatory LH surge, and subsequently ovulation. The pulsatile nature of GnRH secretion, as well as the large amount of peptide released during the preovulatory surge, indicate that the activity of GnRH-containing neurons can be synchronized, and it has been demonstrated that the GnRH neurons of the preoptic area receive synaptic contacts from GnRH neurons [ 5 - 7 ]. Oestradiol, which initiates the preovulatory surge of GnRH, has been shown to be involved in the synaptic plasticity demonstrated in various neuronal populations, including those in the arcuate nucleus [ 8 , 9 ]. Moreover, in rats, an increase in the expression of the molecular markers of synaptic remodelling is observed at the time of the surge in the preoptic area where most of the GnRH neuron cell bodies are found [ 10 ]. In sheep, changes in the expression of the polysialylated form of the neural cell adhesion molecule around the periphery of the GnRH cell bodies, associated with the seasonal changes in GnRH secretion, are indicative of potential plastic changes in this neural system linked with changes in secretory activity [ 11 ]. In addition, oestradiol and progesterone have been shown to modify the morphology and staining intensity of GnRH neurons within the ovine hypothalamus [ 12 ], suggesting that ovarian steroids could elicit plastic changes in GnRH neurons during the oestrous cycle. However, very little work has been done regarding neuronal organization at the level of the GnRH cell bodies during the oestrous cycle in the ewe. The aim of this work was to study the number, distribution and potential contacts present between GnRH-containing cells in intact ewes under natural conditions, focusing particularly on the neuroanatomical organization of these neurons around the preovulatory surge when a high level of oestradiol is released by the growing follicles. Results Plasma hormones Because there are some individual variations in the response to the induction of ovulation treatment (oestrus onset, preovulatory LH surge onset, etc.), an analysis of plasma hormone concentration was carried out to confirm the allocation of animals to experimental groups according to the characteristic hormonal level previously described [ 13 ]. In group 1, as illustrated in figure 1 , progesterone and LH concentrations were low, characteristic of the late follicular phase. In group 2, progesterone remained low (less than 0.05 ng/ml) and a surge of LH was observed for 4 out 5 animals. Analysis of the LH profile concentrations showed that these animals were killed either during the ascending (n = 2) or descending (n = 2) part of the LH surge; for the remaining animal only a slight increase in LH secretion was observed at the end of the sampling period. In groups 3 and 4 a preovulatory surge of LH was clearly identified in all animals. These LH surges were followed by a clear increase in progesterone concentrations and the progesterone level was in the range of 2.5 to 4.5 at the end of the sampling period (except for one animal 1.9 ng/ml in group 4). Global analysis of plasma hormone concentrations at the last sampling time before animals were killed revealed significant differences between groups for LH (p < 0.01) and progesterone (p < 0.05). As shown in figure 1 , the LH level was higher in group 2 than for any other group (p < 0.01), and progesterone concentrations were lower in groups 1 and 2 than in groups 3 and 4 (p < 0.01). For groups 1, 2, 3 and 4, mean levels ± SEM were respectively 1.07 ± 0.12, 28.20 ± 7.08, 0.56 ± 0.03, 0.65 ± 0.06 for LH and 0.05 ± 0.0, 0.05 ± 0.0, 2.74 ± 0.10, 3.62 ± 0.26 for progesterone. However, the ewes showing no clear LH surge (group 2) or a moderate increase in progesterone concentration (group 4) were considered as slightly different from any other animal of the same group and were not used for determining the number or relative clustering of GnRH neurons. Four of the eight animals in experiment 2 were killed during the LH surge (2 during the ascending and 2 during the descending part). The 4 remaining animals were killed 4–6 hours after the end of the LH surge and were discarded from the study. GnRH-immunoreactive neurons Experiment 1 GnRH-containing neurons were distributed in the preoptic area (fig 2 ) as previously described [ 14 ]. There was some variation in the staining intensity in the same section, but it was usually strong. The black DAB/Nickel ammonium sulfate precipitate was distributed throughout the cytoplasm in the cell bodies and processes. The nucleus was never stained. GnRH-ir fibres were also observed around perikarya, presenting large varicosities. The distribution of GnRH cell bodies was homogenous between animals. GnRH-ir neurons were counted when the nucleus was clearly visible. There was no difference in the total number of GnRH-ir neurons in the experimental groups (group 1: 291 ± 38, n = 5, group 2: 347 ± 41 n = 4, group 3: 345 ± 38 n = 5, group 4: 241 ± 75 n = 4) (fig 3A ). However, the organization of the labelled neurons differed in the different experimental groups. Clusters of two or more GnRH-ir neurons in which there appeared to be close contacts between neuronal cell bodies (figs 4a,4b ) were found in the preoptic area of all groups. However, the number of clusters and the proportion of identified neurons involved in clusters varied significantly between the experimental groups. These clusters (mainly pairs of neurons) were more numerous in groups 1 (43 ± 7) and 2 (41 ± 8) at the moment of the preovulatory surge than in groups 3 (28 ± 2) and 4 (25 ± 6) in the luteal phase (p < 0.05) (fig 3B ). The percentage of clusters was higher in group 1 (15.1 ± 1.2) than in groups 2 (11.8 ± 1.0), 3 (8.3 ± 0.4) and 4 (10.7 ± 0.9) (fig 3C ). Experiment 2 In the second experiment, four additional animals were studied during the GnRH surge, and the data compared with those of the group 2 ewes in the first experiment. As shown in table 1 , there was no difference in the proportion of clusters of GnRH-ir neurons in the four animals sampled during the ascending phase of GnRH surge and the four sampled during the descending phase of the surge. Discussion Our observations indicate that the organization of GnRH-ir neurons was modified during the oestrous cycle, since more clusters were observed during the follicular phase. However, we did not observe any difference between the ascending and descending phases of LH release in group 2. Therefore, our data support the hypothesis that there are changes in communication between GnRH cell bodies prior to the initiation of the preovulatory GnRH surge. The increase in the number of clusters was not mirrored by a parallel change in the total number of GnRH cell bodies. However, as the number of clusters was relatively small compared to the GnRH cell population (constituting 10% to 15%), the putative increase in the number of GnRH-ir neurons could have been masked due to the large variations in the number of labelled neurons between animals. An alternative hypothesis is that the number of GnRH neurons remains constant, and that the variations in the number of clusters is linked to variations in the peptide level, which are consistent with the variations in the staining intensity observed in all animals. In this case, the level of GnRH would be higher in clustered neurons than in isolated neurons at the moment of the preovulatory surge, and therefore neuronal communication would be more efficient in clustered neurons. The percentage of clusters increased only in group 1, while the difference between the number of clusters in groups 2, 3 and 4 was not statistically significant. This special morphological organization may constitute an anatomical support for the synchronization of GnRH neuronal activity needed to induce the GnRH preovulatory surge. Clusters of GnRH-ir cell bodies have been previously described in sheep [ 15 ] (Lehman et al, 1986), rats and monkeys [ 16 ]. In rats they represented only 2–7% of the GnRH neuronal population [ 16 ]. In our study, the proportion of clusters was similar (10–15%) to that observed in monkeys (3–15%). However, contrary to our observation, the numbers did not change with the different hormonal conditions in the monkey [ 16 ]. This might be related to the distribution of GnRH cell bodies which differs between monkeys and sheep [ 17 ], and it is possible that the increase of clusters observed in our experiments was related to a specific GnRH neuronal population involved in the GnRH surge secretion. Indeed, Fos expression at the time of the preovulatory LH surge has been shown to be expressed in a subset of GnRH neurons in rats [ 18 ]. A neuronal re-organization of GnRH-containing neurons has also been previously described in anoestrous ewes where the neurons are ensheathed by glial processes which decrease the number of axo-somatic synaptic contacts [ 19 ]. In this latter situation, photoperiod would be the major signal to induce such a modification. Another example of the relationship between synaptic afferents and GnRH secretion has been described in the monkey where the increase of GnRH activity at puberty correlates with the decrease in synaptic afferents on GnRH-containing neurons [ 20 ]. This morphological regulation of GnRH-containing neurons, through synaptic contacts or perikaryal apposition, involves glial processes, since in both sheep and monkeys these neurons are ensheathed by numerous glial processes [ 19 , 21 ]. Steroids, mainly oestradiol, are the most potent regulators in the control of GnRH neuronal activity. Therefore, we may hypothesize that this "plasticity" results from an oestradiol effect, as has been shown in the monkey where oestradiol modulates the number of synaptic contacts on GnRH neurons [ 22 ]. In rats and monkeys, numerous studies have demonstrated that oestrogens induce synaptic plasticity in the control of gonadotropin secretion (rat: [ 23 ]; monkeys: [ 24 ]). In our study, the highest number of clusters was found during the preovulatory surge which occurred about 24 hours after the expected oestradiol increase, this delay being consistent with an oestradiol effect. There was no difference in the number of labelled neurons in the experimental groups, and this observation may indicate that the level of GnRH-immunoreactivity was stable in the neurons of the preoptic area. It has previously been demonstrated in the ewe that GnRH messenger ribonucleic acid expression changes before the onset of the oestradiol-induced luteinizing hormone surge [ 25 ], and that the staining intensity of GnRH-ir perikarya increases after oestradiol treatment, but that the number of labelled neurons does not change [ 12 , 26 ]. In addition, the variations of LH secretion induced by progesterone withdrawal are not linked to a variation in GnRH mRNA [ 27 ]. Unlike the apparent stability of the GnRH level in the perikarya, GnRH-immunoreactivity decreases in the median eminence after ovulation [ 28 ]. This information could indicate that variations in GnRH secretion arise from its release from the median eminence terminals without variations in peptides in the perikarya. Conclusions In conclusion, we have demonstrated that the morphological organization of the GnRH neurons of the preoptic area is modified during the oestrous cycle, although the overall number of GnRH-ir neurons does not change. During the preovulatory surge, the GnRH neurons are more frequently found in clusters, and the percentage of clusters is significantly higher immediately prior to the preovulatory LH surge. Actual contacts between GnRH-ir neurons cannot be determined using light microscopy, but can only be demonstrated using electron microscopy. Because oestradiol is the most powerful regulator of GnRH activity during the oestrous cycle, we can hypothesize that this plasticity may be induced by steroids. The role of this plasticity remains to be demonstrated, but it could increase interneuronal communication during the preovulatory surge. Methods All experimental procedures were carried out in accordance with authorisation A37801 of the French Ministry of Agriculture. Animals Intact adult Ile de France ewes (n = 28) from the laboratory flock, maintained under natural photoperiod were used. Two experiments were performed (one year apart) during the breeding season (between November 15 th and December 15 th ). Animals were fed daily with hay, straw and corn, and water was available ad libitum . All of the ewes had lambed at least once. Experimental design Experiment N° 1 Animals (n = 20) were killed at specific time points in the oestrous cycle. In order to synchronize oestrous cycles, animals were treated with an intravaginal progesterone-releasing device (CIDR InterAg, Hamilton, New Zealand) for 14 days. Following removal of the progesterone device, animals were treated (i.m.) with 500 UI of Pregnant Mare Serum Gonadotropin (PMSG), a treatment known to induce the LH surge and ovulation in sheep (for review see [ 29 ]). It has been shown that oestrous behaviour in this breed precedes the LH surge by 7.0 ± 1.6 hours [ 30 ]. Therefore, oestrous behaviour was recorded for each ewe every 6 hours, from 18 hours after PMSG administration until the animal was observed to be in oestrus using a ram wearing an apron. The animals were then allocated to four groups: Group 1: Animals killed 1–2 hours after oestrus, i.e. before the LH surge onset, (n = 5) Group 2: Animals killed 8–12 hours after oestrus, i.e. during the preovulatory LH surge, (n = 5) Group 3: Animals killed 8 days after the preovulatory LH surge, (n = 5) Group 4: Animals killed 15 days after the preovulatory LH surge, (n = 5) Experiment N° 2 Analysis of LH secretion (see paragraph below) revealed that animals in group 2 in experiment 1 were killed either in the ascending (n = 2) or descending (n = 2) phase of the LH surge. Therefore, in order to increase the N for each sub-group, 8 animals were synchronised as previously described (one year later, during the breeding season) and killed 12 and 20 hours (n = 4) after oestrus. For both experiments, blood samples were collected by venepuncture every 2 hours from 18 hours after PMSG administration until sacrifice (groups 1 and 2) and daily thereafter (groups 3 and 4). Hormone assays Plasma samples were assayed in duplicate for LH with 100 μl aliquots of plasma using a previously described RIA method [ 31 ]. All samples from one experiment were run in a single assay. Intra-assay coefficient of variation averaged 9% and assay sensitivity was 0.16 ± 0.05 ng/ml (4 assays) of standard 1051-CY-LH (i.e. 0.31 ng/ml of NIH-LH-S1). Progesterone was determined in a single assay after hexane extraction of 100 μl of plasma [ 32 ]. The sensitivity was 5 pg/tube and the intra-assay CV 10%. Immunohistochemistry A solution of heparin (25,000 units) was injected iv 10 min before decapitation. Both carotids were catheterised and the head was perfused with 2 litres of 1% sodium nitrite followed by 4 litres of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). After perfusion, the brain was quickly removed and a block containing the whole preoptic area was isolated and post-fixed for 2 hours at +4°C in the same fixative. The blocks were immersed in 30% sucrose containing 0.1% sodium azide at +4°C for 5 days. Serial 40 μm coronal sections were obtained using a freezing microtome and stored at +4°C in PBS containing 0.1% sodium azide. Every fifth section was stained for GnRH immunoreactivity. Sections were incubated for 2 hours at +4°C in PBS containing 0.3% triton X100 and 1% H 2 O 2 and then preincubated for 30 min at room temperature in blocking solution (PBS, 6% normal sheep serum, 0.3% triton, 0.1% sodium azide). Sections were exposed to GnRH primary antiserum at a dilution of 1/10,000 in PBS containing 0.3% triton, 0.2% HSA, 0.1% sodium azide for 4 days at +4°C. After washing in PBS, sections were incubated for 3 hours at room temperature in the secondary antibody (1/500 with PBS containing 0.2% BSA). After washing in PBS, sections were incubated in rabbit peroxidase-antiperoxidase complex diluted 1/10,000 in PBS containing 0.2% BSA overnight at +4°C. After washing twice in PBS and in Tris buffer (0.05 M pH 7.6), the sections were reacted with 3,3-diaminobenzidine tetrahydrochloride (0.02%) and nickel ammonium sulfate (0.25%) in the same Tris buffer containing 0.0025% H 2 O 2 for 20 min. The reactions were stopped by rinsing the sections with Tris buffer. Sections were mounted onto gelatine-coated slides, dried, counterstained with a solution of neutral red for 5 min and mounted with a cover-slide using Depex. The characteristics and the specificity of the GnRH antibody were as previously described [ 14 ]. Data analysis The LH surge was assumed to start when LH concentration exceeded 6 ng/ml of plasma, i.e. about twice the maximum value of a pulse during the follicular phase for this breed and for this LH assay. For hormone values, a global comparison was carried out using a non-parametric ANOVA with exact general score test, and comparison between groups was carried out using the non-parametric exact permutation test for independent samples (Stat Exact Software, Cytel Software Corporation, Cambridge, MA, USA). The first observation of the sections allowed the immunoreactive area surrounding the OVLT to be determined (fig. 2 ). We selected two sections where the size of the OVLT was maximum in the third ventricle, and the two rostral and caudal sections were studied. A total of six sections at 160 μm intervals were studied for each ewe. In each section, all immunoreactive neurons for GnRH were counted using a light microscope, and groups of closely-associated neurons were noted. Total number of cells and number of clusters were analyzed by a 2-way (group and section number) ANOVA (SuperANOVA, Abacus concept, California) followed by a student-Newman-keuls post-hoc test for two by two comparisons. The percentage of cells forming clusters was calculated for each animal. It was analyzed by a one-way (group) ANOVA after arcsin transformation followed by a student-Newman-keuls post-hoc test for two by two comparisons. The results are expressed as means ± standard error of the means. Authors' contribution MB performed the immunohistochemical reaction and counted the neurones, AC and YT conceived the study, and participated in its design, coordination and in the collection of biological samples. BM participated in the final statistical analysis of results and corrected the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535905.xml
514713	Rubia cordifolia, Fagonia cretica linn and Tinospora cordifolia exert neuroprotection by modulating the antioxidant system in rat hippocampal slices subjected to oxygen glucose deprivation	Background The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD). Methods Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NO 2 ) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O 2 .- ), hydroxyl radicals ( . OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression. Results All the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia. Conclusions RC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression level and may be an effective therapeutic tool against ischemic brain damage.	Background It is generally believed that a major portion of post-traumatic neuronal necrosis after brain injury does not result from diffuse primary injury, but rather from a secondary process. The injury appears to trigger a cascade of molecular events that lead to gradual vascular and neuronal tissue degeneration, thus destroying the anatomical substrate necessary for the neurological recovery. A large body of evidence obtained from a wide variety of experimental studies of acute CNS injury strongly suggest that various reactive oxygen species (ROS) and nitrogen species (RNS) have been implicated in the progressive secondary degeneration that follows the injury [ 1 - 3 ]. ROS and RNS have been associated with secondary injury that amplifies the magnitude of final neuronal damage. Both biochemical analyses and studies with transgenic mice has shown that ROS/RNS production persists for many hours after the initial insult. This offers a potential therapeutic window for pharmacologic intervention of clinical relevance. Several classes of pharmacologic mimetics of superoxide dismutase/catalase have been synthesized. Evaluation of these catalytic antioxidants in laboratory models of acute brain injury has shown both robust neuroprotection and a prolonged therapeutic window at doses apparently devoid of neurotoxicity [ 4 , 5 ]. Another important aspect determining extent of oxidative stress mediated post-reperfusion injury subsequent to ischemia is the antioxidant status of the affected tissue as it is of great importance for the primary endogenous defence against free radical attack. Various types of antioxidant enzymes like Cu-Zn superoxide dismutase (SOD), peroxidases such as glutathione peroxidase (GPx), catalase have been reported to be neuroprotective [ 5 , 6 ]. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) are tropical herbs and have been extensively used in the treatment of various types of haematological, hepatic, neurological and inflammatory conditions [ 7 ]. The antioxidant and anti-inflammatory and immuno-modulatory properties of RC and TC has also been well documented [ 8 - 11 ]. Although RC has been reportedly used as an Ayurvedic medication in a wide variety of conditions, reports regarding the use of FC and TC are not available. In light of the aforementioned properties of RC and TC and relatively scant studies on FC we hypothesized that these herbs may overcome the oxidative stress mediated injury during ischemic neuronal injury via modulating the antioxidant pool of the cells. In order to test this hypothesis we devised a two pronged strategy in the present study to evaluate the effect of the drugs on the status of antioxidant enzymes such as GPx and Cu-Zn SOD, the levels of reduced glutathione (GSH), the principal redox regulator of the cell, and the status of nitric oxide (NO) generation the in the hippocampal slices subjected to oxygen-glucose deprivation (OGD). Methods Reagents All reagents unless stated otherwise were obtained from Sigma Chemical Co. USA, Merck, India and Loba Chemie, India. All animals used were as per the institutional animal ethics committee approval. Preparation of hippocampal slices Method for preparation of slices was similar to those of Taylor & Weber[ 12 ]. Normal New Zealand male Wistar rats weighing between 180–220 gm. were anaesthetized with ether and decapitated, whole brain was removed and placed in ice-cold oxygenated artificial cerebrospinal fluid (aCSF) containing (in mM) NaCl – 125, KCl – 3.5, CaCl 2 – 2.0, MgSO 4 – 1.0, NaHCO 3 – 26, Na 2 HPO 4 – 1.25 and D-glucose – 10 mM. The chilled brain was removed and the hippocampal regions were dissected, slices of 0.6 mm were obtained on a modified Stadie – Riggs microtome and were immersed in oxygenated aCSF between 22–28°C for 120 min to allow the tissue to get stabilized. Induction of in vitro oxygen-glucose deprivation (OGD) hypoxic ischemia Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) [ 12 ]. (Taylor + Weber) by suspending the slices in D-Glucose deficient aCSF equilibrated with 95% Nitrogen gas and 5% CO 2 , until the partial oxygen pressure was less then 20% as that of normoxic aCSF (pO 2 was measured using a blood gas analyzer and was found to be ≤ 35 mm Hg). Slices were incubated in oxygen glucose deficient aCSF for 30 min at 37°C. Transferring the slices to reperfusion chamber containing aCSF started reperfusion. The extent of injury was estimated by assaying LDH release in the medium using an LDH kit. Study groups Hippocampal slices were divided into 3 groups of OGD for present study. a) The overall control group consisted of the slices immediately stabilized for 2 hours at 22–26°C in normoxic aCSF. b) The experimental group consisted of stabilized slices subjected to OGD without any drug treatment. c) The experimental treated group consisted of OGD slices reperfused in the medium (aCSF) containing 1) 2 mg/ml concentration of RC, TC and FC for 30 min. 2) Ascorbic acid and Reduced glutathione equivalent to their content in RC, FC and TC for 30 min. Biochemical assays In all the above study groups the following biochemical parameters were monitored. 1) Free Cu-Zn superoxide dismutase (Cu-Zn SOD) was assayed using the method of Beyer et al[ 13 ]. 2) Reduced Glutathione (GSH) was assayed by the method of Teitz [ 14 ]. 3) Glutathione peroxidase (GPx) was assayed by the method of Paglia et al. [ 15 ]. 4) Nitric oxide (NO) was measured as nitrite (NO 2 ) in the supernatant by the method of Green et al. [ 16 ]. 5) Protein was estimated by the method of Lowry et al. [ 17 ]. Electron Paramagnetic Resonance (EPR) measurement EPR (Magnettech X-band Miniscope MS-100, Berlin Germany) was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O 2 . ), hydroxyl radicals ( . OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-No and Sin-1 respectively. All the free radical donors (100 μM – 500 μM) were incubated in phosphate buffered saline (pH 7.4, 37°C) containing the spin-trap, Tempone-H (1 mM), oxidation of which generates 4-oxo-tempo with a characteristic three-line EPR signal centred at 3365 G. Development of this signal was monitored for 60 min from addition of the oxidising species and compared to parallel incubations containing RC, FC and TC (10 or 50 μM, n = 3). The amplitude of the first line of the spectrum was measured; data are expressed in arbitrary units. The EPR parameters for these experiments were as follows: Microwave frequency – 9.4 GHz; microwave power – 20 mW; modulation frequency – 100 kHz; modulation amplitude – 1500 mG; centre field 3365 G; sweep width 50 G; sweep time 20 sec; No. of passes – 1; receiver gain 3E1. Reverse Transcriptase Polymerase chain reaction (RT-PCR) GCLC (Glutamyl – cysteinyl ligase catalytic subunit), iNOS (inducible nitric oxide synthase) and Cu-Zn SOD mRNA was isolated using TriZOL reagent from the hippocampal slices and reverse transcribed to study the effect of RC, FC and TC on the expression status of the above genes in OGD untreated slices after a period of 24 and 48 hours post OGD. Gene expression in treated groups was studied after 24 hours. GAPDH served as the house-keeping gene. After an initial reverse transcription, the cDNAs obtained were amplified using the following respective primers and PCR conditions: GCLC was amplified by 32 thermal cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 mins followed by an extension at 72°C for 10 mins using primers: for ' 5'gtggtactgctcaccagagtgatcct and rev ' 5'tgatccagtaactctgggcattcaca. iNOS was amplified at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for a total of 27 cycles followed by a 10-min extension at 72°C using primers: for '5'-gtgttccaccaggagatgttg-3' and rev ' 5'-tggggcagtctccattgcca-3'. Cu-Zn SOD was amplified at 94°C for 45 s, 56°C for 30 s, and 72°C for 45 s for a total of 23 cycles followed by a 10-min extension at 72°C using primers: for '5'-tctaagaaacatggcggtcc-3' and rev'5'-cagttagcaggccagcagat-3'. GAPDH was amplified using 20 thermal cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 30 s, followed by final extension for 10 mins at 72°C, primer used were for' 5'ccacccatggcaaattccatggca and rev 5'tctagacggcaggtcaggtcaacc. Results Intracellular levels of GSH is differentially modulated by RC, FC and TC Figure 1 depicts the effect of the three herbs RC, FC and TC on the intracellular GSH levels in treated rat hippocampal slices post glutamate toxicity. A significant elevation in the GSH level was recorded in untreated controls as compared to the stabilized group (p < 0.001). Where as RC and FC did show an increase in the GSH levels as compared to TC. Figure 1 Effect of RC, FC and TC on the cytosolic GSH levels during OGD in rat hippocampal slices. * p <0.001 compared to stabilized, and + p < 0.001 compared to OGD. Results expressed are a mean of 6 independent experiments ± SEM. Effect of RC, FC and TC on GCLC gene expression Figure 2 shows the effect of RC, FC and TC on GCLC (Glutamyl – cysteinyl light chain) RT-PCR gene expression, all the three herbs were found to be inducers of the gene expression as compared to the untreated controls. Expression of housekeeping gene GAPDH was unaltered in all the lanes. Figure 2 a: Representative GCLC mRNA expression in relation to GAPDH in control, OGD and OGD + drug treated rat hippocampal slices. Lane 1 – Control, Lane 2 – OGD 24 hr, Lane 3 – OGD 48 hr, Lane 4 – OGD + FC, Lane 5 – OGD+TC, Lane 6 – OGD+RC. b: Percentage densitometric expression of GCL mRNA expression in relation to GAPDH in control, OGD and OGD + drug treated rat hippocampal slices. * p < 0.01 and ** p < 0.001 versus control, + p < 0.01 versus * & ** and ++ p < 0.001 versus * & *. Results expressed are a mean of 6 independent experiments ± SEM. RC, FC and TC directly scavenge free radicals RC, FC and TC contain polyphenols, a class of compound that can directly interact with electrophillic species and thus can act as a direct scavenger of free radicals. To test this hypothesis we employed EPR spectroscopy to study the interaction of RC, FC and TC with certain free radicals such as O 2 .- , . OH, NO and ONOO using specific donors. EPR spectroscopy revealed that addition of RC, FC or TC (10 μg/ml) to the O 2 .- generator, pyrogallol (100 μM; n = 3, Fig 3a ) or the . OH generator, menadione (500 μM; n = 3, Fig 3b ), NO generator, DETA-NO (100 μM; n = 3, Fig 3c ) and ONOO generator Sin-1 (100 μM; n = 3, Fig 3d ) in the absence of a spin trap failed to generate a spin signal over a 60 min period. This indicated, either the herbs interacted with the free radicals covalently and therefore yielded no paramagnetic signals or did not interact at all. However, 3-line spin signals characteristic of formation of the stable radical 4-oxo-tempo developed in a time-dependent manner when the same oxidant generating substances were incubated with the recognised spin trap, tempone-H (1 mM). Co-incubation of RC, FC and TC with pyrogallol (100 μM) in the presence of tempone-H caused a significant inhibition of spin signal development over a 60 min time period (p < 0.001, 2-way ANOVA – results of Bonferroni post-hoc analyses and non-linear curve fits are shown on the figure; n = 3). Figure 3 Non-linear curve fit analysis of the effect of RC, FC and TC on scavenging free radicals such as A) O 2 - , B) OH . , C) NO and D) ONOO - generated by pyrogallol, menadione, DETA-NO and SIN-1 respectively. The reference curve fit is depicted in the respective groups. Results expressed are a mean of 3 independent experiments ± SEM. Intracellular levels of GPx are increased by RC, FC and TC Figure 4 depicts the effect of the three herbs RC, FC and TC on the intracellular GPx levels in treated rat hippocampal slices post glutamate toxicity. A significant rise in the GPx level was recorded in treated slices as compared to the stabilized and control group (p < 0.001). Here too RC and FC show an increase in the GPx levels as compared to TC. Figure 4 Effect of RC, FC and TC on the glutathione peroxidase levels in rat hippocampal slices. + p < 0.01 versus stabilized and ++ p < 0.001 versus OGD. Results expressed are a mean of 4 independent experiments ± SEM. Cu-Zn SOD gene expression and enzyme level is upregulated by RC, FC and TC Figure 5 depicts the effect of the three herbs RC, FC and TC on the expression and activity levels of intracellular Cu-Zn SOD in stabilised, untreated and treated rat hippocampal slices post OGD. A significant drop in the SOD level was recorded in untreated OGD slices as compared to the stabilized group (p < 0.001). A significant rise in the levels of both SOD gene expression and the enzyme activity was observed for RC, FC and TC (p < 0.001). Figure 5 Effect of RC, FC and TC on Cu-Zn SOD gene expression and cytosolic free levels during OGD in rat hippocampal slices. RT-PCR pictograph of Cu-Zn SOD is shown aligned to the corresponding groups. p < 0.001 versus stabilized, + p < 0.001 versus OGD. Results expressed are a mean of 6 independent experiments ± SEM. RC, FC and TC and exogenous antioxidants decrease nitric oxide generation and iNOS gene expression The effect of exogenously added antioxidants namely ascorbic acid and reduced glutathione equivalent to their concentrations found in three herbs RC, FC and TC on nitric oxide generation in comparison to the three herb extracts is shown in figure 6 . All the three herbs along with the exogenously added GSH and Vit C significantly inhibited NO 2 generation in the treated OGD slices. The decrease in the level of NO 2 was found to parallel a decrease observed for the expression of the iNOS gene in the same group of the hippocampal slices (Figure 7 ). Figure 6 Effect of RC, FC and TC and amounts of ascorbic acid and GSH equivalent to that present in the herbs (RC, FC and TC* respectively) on NO 2 generation in hippocampal slices. + p <0.001 versus control (100%) and ** p < 0.001 versus OGD. Results expressed are a mean of 3 independent experiments ± SEM. Figure 7 Effect of RC, FC and TC on the iNOS gene expression in rat hippocampal slices subjected to OGD and OGD+RC/FC/TC. Lane 1 – Control, Lane 2 – OGD 24 hr, Lane 3 – OGD 48 hr, Lane 4 – OGD + FC, Lane 5 – OGD+TC, Lane 6 – OGD+RC. p < 0.05 vs control, p < 0.001 vs control, +p < 0.001 vs *. Results expressed are a mean of 6 independent experiments ± SEM. Discussion Ischemic cells are known to be under oxidative stress and hence experience oxidative injuries like membrane alterations, lipid peroxidation, increased ionic influx, especially Ca and Na etc. [ 18 - 20 ] elevated levels of O 2 - , NO, NO 2 , decreased activity of Ca 2+ Mg 2+ ATPase and Na + /K + ATPase [ 21 ]. In the present study we have reported the protective effects of RC, FC and TC during OGD insult to rat hippocampal slices. GSH was protected from depletion during the insult and this protection could be correlated to an elevation in the expression of the g-GCS gene. The peroxide scavenging enzyme, glutathione peroxidase (GPx) activity was also significantly restored by the three plant extracts. Treatment of the OGD-hippocampal slices with pure vitamin C and GSH in proportions equivalent to that found in the extracts exhibited a parallel effect on NO generation as to that found with the drugs. RC, FC and TC effectively reduced free radical levels by mechanisms involving increased expression of Cu-Zn SOD, decreased expression of iNOS and simultaneous scavenging of the free radicals such as O 2 - , OH . , NO and ONOO. Overall, RC, FC and TC exhibit potential cytoprotective ability in rat hippocampal slices subjected to OGD. In addition to its role as an antioxidant, the GSH status of a cell is critical for various other biological events that include transcriptional activation of specific genes and modulation of redox-sensitive signal transduction and hence pro-inflammatory processes during cerebral ischemia [ 22 ]. GSH also plays a crucial role in the regulation of expression of several redox-sensitive antioxidant and anti-inflammatory genes [ 23 ], processes which are aggravated especially, post-ischemic insult as a result of reperfusion of white blood cells to the injured area [ 24 ]. As a result there is a rapid loss of reducing equivalents of the cell and hence an onset of oxidative stress. The oxidative stress further leads to the upregulation of expression of a wide variety of pro-inflammatory cytokines, including adhesion molecules, all of which contribute to tissue injury, apoptosis/necrosis [ 25 , 26 ]. Therefore maintenance of GSH pool and other antioxidant levels is critical to cell survival and adaptation to the ischemic injury [ 27 ]. In response to the battery of free radicals generated during ischemia, the cells initially neutralize the oxidative challenge via GSH mediated antioxidant mechanisms. However, a rapid decline in the levels of GSH soon follows which ultimately leads to tissue injury. Therefore it is imperative that any therapeutic intervention should be able to cater to this deficiency observed during ischemia/OGD. Our results show that RC, FC and TC were able to reverse the GSH levels, which was significantly depleted during OGD. This restorative/protective activity of RC, FC and TC could be partly attributed to their native antioxidant contents (GSH = 8.33 ± 0.5, 10.26 ± 0.55 and 6.94 ± 0.49, Vit C = 27.52 ± 0.93, 32.99 ± 1.03 and 41.86 ± 0.68 and Polyphenols = 18.33 ± 2.02, 11.88 ± 1.33 and 21.00 ± 1.26 mg/g extract respectively). However, it was not clear at this juncture as to how much of these antioxidants are actually bio-available, an area which is out of scope of the present study. On the other hand RC, FC and TC may exert such a restorative effect by increasing the synthesis of GSH in the cells. To test this hypothesis we studied the expression status of γ-GCS gene during OGD in both untreated and treated hippocampal slices. RC, FC and TC showed a positive inductive effect on the γ-GCS gene expression after 48 hours. Although significant, it is to be noted that the three drugs exhibited partial differences in their response towards the expression of the GCLC gene expression and in restoration of the GSH levels. This indicates that the drugs may act via other mechanisms that may have a sparing effect on the GSH levels. Scavenging of the free radicals is one such mechanism whereby depletion of GSH is prevented [ 28 , 29 ]. In order to test the later hypothesis we examined whether or not RC, FC and TC could directly interact with the free radicals such as O 2 - , OH . , NO and ONOO using pyrogallol, menadione, DETA-NO and Sin-1 as respective donors. Electron paramagnetic resonance study has revealed a significant scavenging effect of the three drugs on the free radicals chosen for the study. The results were more pronounced for OH . and NO radicals as depicted by the non-linear curve fit analysis. The effects on O 2 - , and ONOO radicals were also found to be quite promising. Therefore, direct scavenging of the free radicals is an important mechanism by which the drugs may exert their cytoprotective effect, not only by sparing GSH utilization by free radicals but also preventing the free radical mediated tissue damage. GSH depletion can also be brought about by its abnormal redirection towards neutralisation of the oxidants. Within the normal metabolic course of the cell, reduction of the prostanoid hydroperoxides to their respective hydroxides, is catalysed by the enzyme glutathione peroxidase (GPx), which requires GSH as one of the co-substrates for the reaction [ 30 , 31 ]. This is basically a protective mechanism of the cell against the harmful lipid peroxides generated during their synthesis and oxidative stress. In the present study we have recorded a significantly decreased GPx activity in OGD hippocampal slices as compared to the controls. The observed decrement may be attributed to the depleted GSH levels either due to increased utilisation and/or diminished activity of γ-GCS. Furthermore, diminished GPx activity, at least in part, indicates cellular accumulation of the lipid hydro peroxides, which can potentially turn on a chain reaction wherein more unsaturated lipids become targets for further peroxidative tissue injury. The ability of RC, FC and TC to enhance the GPx activity is therefore an important finding since one of the protective mechanisms of the herbs under study might be mediated via upregulation of the GPx activity. This effect may further be explained in view of the fact that the herbs themselves contain an appreciable amount of GSH and ascorbate. A large body of evidence suggests that another important intracellular enzyme Cu-Zn SOD is found to be neuroprotective in nature along with GPx and GSH [ 32 , 33 ]. We observed a significant drop in the cytosolic free levels of Cu-Zn SOD in the untreated OGD slices as compared to the stabilized and the three herbs RC, FC and TC significantly restored the levels of the antioxidant enzyme. However, it was not clear at this juncture as to the mechanism involved in such a restoration. Oxidative stress is known to induce the synthesis of Cu-Zn SOD as a defensive mechanism aimed at containing the oxidant levels generated during inflammatory/ ischemic conditions [ 34 ]. Cu-Zn SOD is one of the first lines of defense against free radicals such as O 2 - and NO and acts as a direct scavenger of these extremely potent hazardous species. We therefore investigated whether the antioxidant properties exhibited by the three herbs also involve their modulatory effect on the expression of the Cu-Zn SOD expression. The increased expression of the Cu-Zn SOD gene in RC, FC and TC treated OGD slices clearly confirm the positive modulatory effect the three drugs have on the cellular antioxidant system. Increased expression of Cu-Zn SOD in response to these herbs is a crucial finding especially since this enzyme is directly implicated in the scavenging of not only O 2 - but also the more potent toxicant ONOO [ 35 ]. Thus the three herbs show potent antioxidant properties via modulating the expression of the antioxidant genes such as GCLC and Cu-Zn SOD. The mechanism of antioxidant and neuroprotective effects reported above for RC, FC and TC are new findings. However, as to what components of the drugs bring about such cell-protective effects could not be fully ascertained. Preliminary analysis has revealed that all the three herbs have significant amounts of GSH and Vit C and another important antioxidant, polyphenol. In addition inductively coupled plasma spectroscopic analysis have also revealed the presence of important trace elements in the three herbs (personal observations (Zn- 13.81, 18.19 and 14.94, Cu- 3.16, 9.46 and 13.79, Vd- 30.00, 18.55 and 26.00, Se- 1.79, 3.33 and 0.28 and Mo- 0.39, 1.64 and 1.15 ppm in RC, FC and TC respectively). We therefore thought that the three herbs exert their ameliorative properties due to their antioxidant and trace element contents. In order to test this hypothesis we investigated the effect of GSH and Vit C on NO generation in OGD treated and untreated hippocampal slices. The amount of GSH and Vit C used were equivalent to that found in the three herbs. Our results indicate that GSH and the Vit C components of the herbs are at least partly responsible for the attenuation of NO generation in the treated OGD slices. The effects of RC, FC and TC were almost similar to those recorded for GSH and Vit C. NO is an important neurotransmitter in the brain and also can be rendered harmful due to unprecedented generation during hypoxic/ischemic/inflammatory conditions [ 20 , 36 ]. In addition, NO can combine with O 2 - to form a more toxic ONOO, which is extremely deleterious to the cells [ 37 ]. Hence our observation of the ability of the herbs to attenuate NO generation is not only new but also is indicative of the possible mechanism by which these herbs might exert their protective functions. It is interesting to note that RC, FC and TC also increase Cu-Zn SOD expression thus contributing to the decrease in ONOO formation due to an increased scavenging of O 2 - . Furthermore the three herbs were found to repress the expression of the iNOS gene, which is considered to be an important damaging factor during hypoxia/ischemia [ 37 ]. Thus the decrease in generation of NO by RC, FC and TC is not only due to direct scavenging by the herbs (fig 3 ) but also via the transcriptional modulation of the iNOS gene, which is induced during OGD. Conclusions In conclusion, RC, FC and TC exert cell/neuroprotective properties via preventing the depletion and increasing GSH levels by inducing GCLC expression, by reducing oxidant levels via direct scavenging, decreasing iNOS expression and by increasing the antioxidant gene Cu-Zn SOD. Further protective ability may be attributed to the enhanced activity of GPx brought about by the herbs. The antioxidant contents of the herbs appear to be important components for the observed effects. However, further investigations are required to ascertain the role of individual constituents in the efficacy of the above described properties of the herbs in order to ascribe potential pharmacological applications to these herbs. Competing interests Authors do not have any competing interest with anyone whatsoever. Authors' contributions AKR – This work is a part of AKR's PhD thesis and he has done all the experiments, data collection and processing and manuscript preparation. MGM – He is co-supervisor for AKR and was involved in the ideology, manuscript preparation and providing facilities for the work. SKB – He is the supervisor for AKR and was involved in the main ideology and study design of the work, manuscript preparation and performing EPR studies and training AKR for the respective techniques. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514713.xml
514698	Linear fuzzy gene network models obtained from microarray data by exhaustive search	Background Recent technological advances in high-throughput data collection allow for experimental study of increasingly complex systems on the scale of the whole cellular genome and proteome. Gene network models are needed to interpret the resulting large and complex data sets. Rationally designed perturbations (e.g., gene knock-outs) can be used to iteratively refine hypothetical models, suggesting an approach for high-throughput biological system analysis. We introduce an approach to gene network modeling based on a scalable linear variant of fuzzy logic: a framework with greater resolution than Boolean logic models, but which, while still semi-quantitative, does not require the precise parameter measurement needed for chemical kinetics-based modeling. Results We demonstrated our approach with exhaustive search for fuzzy gene interaction models that best fit transcription measurements by microarray of twelve selected genes regulating the yeast cell cycle. Applying an efficient, universally applicable data normalization and fuzzification scheme, the search converged to a small number of models that individually predict experimental data within an error tolerance. Because only gene transcription levels are used to develop the models, they include both direct and indirect regulation of genes. Conclusion Biological relationships in the best-fitting fuzzy gene network models successfully recover direct and indirect interactions predicted from previous knowledge to result in transcriptional correlation. Fuzzy models fit on one yeast cell cycle data set robustly predict another experimental data set for the same system. Linear fuzzy gene networks and exhaustive rule search are the first steps towards a framework for an integrated modeling and experiment approach to high-throughput "reverse engineering" of complex biological systems.	Background While similarity (homology) of DNA sequence between organisms can be used to propose potential gene functions, transcriptional regulation, and protein pathways (e.g., [ 1 ]), there are often major differences in the protein products, functions, and pathway involvement of genes with nearly identical sequences [ 2 ]. Consequently, sequence homology may be viewed as a means of generating an initial "draft" hypothesis for the gene network of a newly sequenced organism that can be built upon using high throughput experimental techniques such as DNA chips and microarrays for mRNA transcript profiling [ 3 ], protein abundance profiling with mass spectroscopy and 2-D gel electrophoresis [ 4 ], and protein-protein and protein-DNA binding assayed using SELDI mass spectrometry [ 5 ] and protein chips [ 6 ]. In addition, new genetic technologies, in particular small interfering RNA (siRNA) for selective gene suppression facilitate high-throughput massively parallel perturbation of the gene and protein networks of biological systems [ 7 ]. Given the potential scale and complexity of experiments and resulting data sets, biologists need a modeling and simulation framework to optimally design experiments and interpret results. The problem is not simply one of "reverse engineering" to find the optimal "best fit" gene, protein, and/or metabolite interaction model to explain a set of experimental results; rather, modeling should suggest the range of hypotheses that can potentially explain the results of one experiment and select the optimal next experiment to reduce the number of possible alternative hypotheses, with the goal of converging to a biological system model that can be used to predict the effect of molecular perturbations. A major challenge of modeling biological systems is that conventional methods based on physical and chemical principles require data that is difficult to accurately and consistently obtain using either conventional biochemical or high throughput technologies, which typically yield noisy, semi-quantitative data (often in terms of a ratio rather than a physical quantity) [ 3 ]. In particular, microarray gene expression ratios are ultimately obtained from pixel counts of relatively messy images. Boolean networks (e.g. [ 8 ]) are computationally simple and do not depend on precise experimental data, and thus they are suitable for handling both the complexity of biological networks and the challenge of generating and comparing multiple hypothetical networks as described in the above scheme. However, Boolean models have inadequate dynamic resolution to accurately describe the behavior of a biological network [ 9 ]. In contrast, differential equation models (e.g., [ 10 ]) can be computationally expensive and sensitive to imprecisely measured parameters. Even the lower throughput RT-PCR method for gene expression measurement (as described in [ 10 ]) cannot produce quantitatively precise data that can be accurately mapped to actual mRNA concentrations in the sample. Because of computational limitations, continuous modeling approaches (e.g., [ 10 , 11 ]) are limited to finding the single model that best fits experimental data given some set of constraints, such as a maximally sparse gene interaction network [ 11 ]. Fuzzy logic [ 12 ] provides a mathematical framework that is compatible with poorly quantitative yet qualitatively significant data. Fuzzy logic is a natural language for linguistic modeling, thus it is consistent with the qualitative linguistic-graphical methods conventionally used to describe biological systems. Fuzzy models are rule-based; accordingly, there is a potential scalability problem as the number of antecedents ("inputs" to the rule) and variable states ("resolution" of inputs and rule outputs) increase, causing combinatorial explosion. Non-scalable conventional fuzzy logic has previously been used to analyze microarray data [ 13 ]. However, because of the nonlinear scalability of the modeling method and resulting computational expense of generating rules for multiple inputs, this method allows for only one possible positive and one possible negative regulator for each gene, thus yielding few biologically meaningful insights and experimentally testable hypotheses. The problem of rule set combinatorial explosion is addressed by the union rule configuration (URC) developed by Combs and Andrew [ 14 ], which allows for linear growth in rule set complexity with both resolution (number of states) and number of inputs (rule antecedents) at the cost of having to represent nonlinear relationships between inputs as hidden layers [ 15 ]. In the linear (URC) fuzzy logic scheme, there are distinct fuzzy rules for each individual input to a given output. For example, given input variables A and B to an output C, there would be a set of rules relating A to C (e.g., "If A is LOW then C is LOW", "If A is HIGH then C is HIGH") and another set of rules relating B to C (e.g. "If B is LOW then C is HIGH", "If B is HIGH then C is LOW"). After each rule is applied individually, the intermediate evaluations of the fuzzy state of the output variable ("node") are aggregated by a fuzzy union (logical OR) operation (e.g. by summing or taking the resulting memberships in the fuzzy sets defining the state of the output). This contrasts with conventional fuzzy logic (or the "intersection rule configuration"), which has rules relating all combinations of inputs evaluated by a fuzzy intersection (logical AND). For the example with inputs A and B to output C, rules would read as, e.g., "If A is LOW and B is LOW then C is LOW", "If A is LOW and B is HIGH then C is HIGH", etc., leading to a combinatorial explosion avoided by the URC. The utility of linear (URC) fuzzy logic has been demonstrated in its ability to qualitatively model the lac operon of E. coli [ 16 , 17 ]. In our previous work, a URC fuzzy logic model was constructed from existing qualitative biological knowledge about the interaction of genes and limited quantitative data on protein and metabolite concentration and enzyme kinetics, showing the power of linear fuzzy logic to describe complex multi-component regulation. Here, the linear fuzzy logic method is extended to tackle the inverse problem of gene network reconstruction from real quantitative microarray data where there are many inputs. This involves both methods for mapping the experimental data to the fuzzy logic membership functions and a useful implementation of the URC fuzzy logic to represent the gene networks. In addition, a robust algorithm for performing searches through the exponentially large space of possible gene networks is presented. To address the problem of generating all plausible hypothetical network models that explain an experimental data set, we are initially proceeding with an exhaustive search of possible gene interactions to find those that fit the data within some error threshold. Thus, the problem we are tackling is of exponential complexity with O( m N ) growth in the number of possible rules for the behavior of a given "output" gene of a gene interaction node, where N is the number of (input) genes that can possibly control it and m is the number of possible rules describing the effect of each single input gene on the output gene. On the other hand, if a linear fuzzy logic scheme is not used, the problem would grow at an unacceptably high O( m N^N ) rate. The number of possible rules for each gene-gene interaction ( m ) is given by n n , where n is number of fuzzy sets that describe the state of a variable. Hence, we will constrain the size of the problem by (i) setting the minimum number of fuzzy sets to three, the minimum for meaningful resolution, (ii) limiting the number of possible input genes that are allowed to control the output of the output gene at each node of the fuzzy network model, and (iii) not allowing nonlinear gene interactions which would require hidden layers. The last condition is not particularly severe, as a typical nonlinear interaction (e.g., "xor") interaction between two regulatory proteins at a gene is mediated by an intermediate complex between the proteins that can be represented as an independent node in a network model. Therefore, "hidden layers" may generally be avoided by including more biological detail as explicit nodes in the model: for example, explicitly including the temporary interaction between proteins within a scaffolded cellular signal transduction complex, or by incorporating as model nodes the various topological states of a region of DNA influencing transcription factor binding, or in general, adding sufficient biological detail such that interactions between inputs can be linearly modeled. We apply partially scalable, linear fuzzy network modeling to a data set commonly used for demonstrating computational methods in systems biology, microarray experiments of yeast cell cycle gene expression [ 18 ]. These data were obtained in 1998, prior to subsequent technical and statistical advances to improve data quality. However, to keep our case study as general as possible and demonstrate the ability of the fuzzy logic approach to handle other similarly noisy data sets, we do not do any data processing other than the fuzzy modeling process (described in the Methods). Exhaustive search is used as a brute force "reverse engineering" method to find all possible gene network models that fit the data for a set of twelve genes known to participate in the yeast cell cycle. We show that the search converges to a small number of models describing the expression of each gene within a fit tolerance. Models found from the data for one particular yeast cell cycle time series are also capable of qualitatively predicting data from another time series experiment (i.e., one using a different cell synchronization method). In addition, given the constraints of the search algorithm (described in more detail in the Methods) and our limitation to pure transcriptional data, we find that the best fitting fuzzy network models collectively recover some direct and indirect functional relationships between genes predicted by interactions found by previous biochemical experiments as well as quantitative and statistical methods based on transcriptional correlations. Results Yeast cell cycle data set As a proof of concept, we have used exhaustive search to generate fuzzy gene networks based on yeast ( Saccharomyces cerevisiae ) cell cycle microarray time series data sets presented in [ 18 ] (which included data from [ 19 ]). Researchers frequently use these data sets to demonstrate and validate statistical and clustering analysis (e.g., [ 20 , 21 ]), mathematical modeling [ 22 , 23 ], and reverse engineering methods [ 21 , 24 ]. Biological details of the yeast cell cycle transcriptional network and some computational methods for its analysis are reviewed in [ 25 ]. S. cerevisiae cell cycle regulatory protein-DNA interactions were also the subject of a recent extensive experimental study [ 26 ] and there is a large amount of previously obtained biological knowledge on the interaction of yeast cell cycle proteins, i.e., information contained in the Yeast Proteome Database [ 27 ] and the KEGG pathway database [ 28 ]. Consequently, predicted transcriptional network models we derive for the Spellman et al . [ 18 ] data set can be tested against numerous independent data sets and compared with models obtained using other methods. We focus on the 12 key yeast cell cycle genes listed in Table 1 with descriptions taken from the Yeast Proteome Database. The protein products of these genes have been extensively studied using conventional biological techniques and are known to regulate each other and play key roles in controlling cell cycle. Consequently, observed correlations between the genes of Table 1 in cell cycle microarray data are most likely the result of real biological activity rather than noise. In addition, cell cycle gene subsets similar to this one have been the subject of other recent gene network modeling and reverse engineering publications (e.g., [ 21 , 24 ]). Figure 1 shows the current understanding of the interactions of the cell cycle protein subset. There are three sets of gene expression time series in [ 18 ] measured for cells synchronized by different methods, called the cdc15 , alpha , and cdc28 sets. We fit models on the basis of the cdc15 data set since it contains the least number of missing data. Time points in the cdc15 set for which there is missing data for one or more of the 12 genes are excluded from the rule search. We perform an exhaustive search with a maximum of 4 inputs per node, as detailed in the Methods. A Microsoft Excel workbook with the complete fitting data set is provided in Additional File 1 , including all the fuzzy rule models for each gene obtained from exhaustive search with an E MIN threshold of approximately 0.6. Results of fitting to data Figure 2 shows the number of rule models found in the exhaustive search that fit the expression time series of the CLN1 gene (using the cdc15 data set) at different error tolerance levels ( E MIN , as defined by Equation 4 in the Methods). It shows typical behavior for the exhaustive rule search. The number of fuzzy models that fit a gene expression time series decreases exponentially as the fit tolerance ( E MIN ) increases, up to a maximum tolerance above which no models fit the data. A successful search generally converges to a small number of distinct models at the maximum fit tolerance, representing "plausible" hypothetical transcriptional networks that can explain the available data. In some cases, though it did not occur for any of the genes analyzed here, the search fails and there are a large number of models with similar poor fit scores and no suitable subset of "plausible" models. The plausible model subset generally contains common rule patterns. For example, Table 2 lists the models for CLB5 expression with the highest fit scores found in the exhaustive search. The rules are in the format used for the example described in the Methods section. Table 3 shows three models for each gene in the network: the best fitting rule (highest score) and the two highest scoring rules with different combinations of input genes. The scores for each of the three models are provided in corresponding rows at the bottom of the table. Figure 3 shows the best fitting interaction network diagrams for each node gene from Table 3 . To test whether the linear fuzzy gene network models found for one set of experimental data (i.e., cdc15 synchronization time series) can accurately predict another set of results for the same system, we analyzed the microarray time series for alpha cell synchronization presented in [ 18 ]. There are some missing values for some genes at some time points in the alpha data set, which are set to zero and could potentially lead to discrepancies between the modeling and experimental data only at those points. Figure 4 shows the predicted time series for the expression ratio of four genes ( CLN1 , CDC28 , SWI6 , and CLB5 ) given the highest (except for CLN1 , second-highest) scoring models in Table 3 . (The second-best fitting model is used for CLN1 because it consists of four inputs, including all three inputs in the highest-scoring model along with another gene. Thus, it represents a more general "consensus" model for CLN1 .) These models fit the original cdc15 training data with very different calculated tolerances (as measured by the fit error E ) ranging from 0.510 ( CDC28 , Figure 4B ) to 0.930 ( CLN1 , Figure 4A ). Discussion Using exhaustive search, we found linear fuzzy networks that predict cdc15 cell cycle microarray data for the expression of most of the twelve yeast genes we analyzed. The rule search typically converged to a small set of "plausible" models at a given fit error ( E ) tolerance for each gene (with exponential convergence as shown in Figure 2 ). Even for genes for which no highly fitting model could be found, such as SWI4 , the best model (fitting at E = 0.620) predicts the qualitative behavior of independently measured alpha time series data (Figure 4C ). Moreover, models that are more predictive ( E > 0.8) of the cdc15 training data provide quantitatively accurate predictions of the alpha data (Figures 4A and 4D ). Notably, these consistently good fits for the alpha data set were achieved using exactly the same arctangent data normalization and fuzzification scheme applied to the cdc15 data set. This suggests that the fuzzy processing methods described here can be generally applied for data sets obtained from different microarray experiments, provided a roughly symmetric distribution of Log2 ratios about 0, such that sets 1 and 3 both remain meaningful – though the ratios could be re-centered if necessary. In general, our results demonstrate the ability of qualitative fuzzy rule models to interpret the results of quantitative data and make predictions that can be statistically analyzed. Consequently, these models can be used to pose experimentally testable hypotheses. Measurements of mRNA expression from microarray experiments complement information from additional gene knockout, DNA-protein and protein-protein experiments. A model based on pure transcriptional data will thus necessarily contain indirect relationships between proteins and miss other direct purely protein-protein interactions. However, gene network models can suggest functional roles and relationships for genes and proteins, and these models are necessary in complex system analysis to design and interpret further experiments that will specifically determine protein function and identify actual chemical interactions. To see what biological insights can be derived from fuzzy gene network models, we can examine areas of agreement and discrepancies between the best-fitting models found in our exhaustive search (shown in Table 3 and Figure 3 ) and the current understanding of the yeast cell cycle network (summarized in Figure 1 ). Focusing on CLN1 , we found positive regulation by CLN2 and negative regulation by CDC20 (Figure 3 ), which are correlations expected from biological knowledge (as shown in Figure 1 ) and found by Soinov, et al . using a supervised learning method [ 21 ]. In addition, the model for CLN1 includes a direct connection with CDC28 and an indirect connection with MBP1 (through regulation of the SBF complex) that are consistent with their relative positions in the cell cycle (Figure 1 ). The best-fit model for CLN1 depended solely on a positive interaction with CLN2 , revealing the strong co-transcriptional connection between CLN1 and CLN2 . The connection between CLB5 and CLB6 was also found in the model for CLB5 . Other successfully found interactions include the negative regulation of CDC6 by CLB6 and the positive regulation of CLB6 by MBP1 . Some biologically accurate relationships were found that were absent from the supervised learning analysis of [ 21 ]. Notably, the model successfully recovers the apparent inhibition of CLB5 by CDC20 , which is not shown in Figure 1 (based on the KEGG pathway) but arises from cdc20 protein presenting clb5 protein to proteases for degradation (as included in the model of [ 22 ], references within). There are several biological relationships that are not found in the best-fitting networks of Figure 3 , such as an interaction between SWI6 and SWI4 (which form a multiprotein complex). The best-fitting models for SWI4 include a repressing action by MBP1 , which is inconsistent with biological knowledge (Figure 1 ) suggesting that MBP1 and SWI4 activity should correlate (since they act at the same stage in the cell cycle). However, closer examination of the Spellman data set reveals that the amplitude of MBP1 transcription varied within a small range, and the measurement could have been very noisy, resulting in a potential error by the algorithm. (It should be noted that no correlation is identified between MBP1 and SWI4 by the supervised learning algorithm in [ 21 ].) In general, determining which relationships found in the fuzzy gene network represent biologically accurate interactions is a question that must be resolved by analyzing other data sets or from new experiments. The multiple plausible hypothetical input gene combinations can be used to optimally design experiments to add most information for least effort (time and cost) to revise fit errors and produce a new, more realistic set of hypothetical networks. Conclusions In this work, we describe partially scalable, linear fuzzy logic models for biological network modeling. We demonstrate our approach by developing network models that accurately predict transcriptional data from typically noisy and semi-quantitative microarray experiments. Looking at the transcription network alone provides us with a view of the system at the "gene interactions" level. As measurement technology rapidly advances, the methods we describe can be extended to comprehensive heterogeneous data sets. To address the problem of analyzing the complex results of an exhaustive fuzzy model search and designing optimal experiments, we are currently developing pattern recognition methods to better visualize and interpret potentially large sets of models. In addition, we are considering stochastic methods to accurately sample and characterize the "space" of all possible fuzzy models to (a) more efficiently identify the subset of plausible models and (b) identify common patterns among all the models to gain a better understanding of the system and its evolution. While it is tempting to develop methods to obtain a single "optimal solution" as in a classic inverse problem, this is not appropriate for complex biological systems. Scarcity of both data and biological understanding mean that at best experiments will merely limit the space of potential solutions. Biological system analysis is a dynamic reverse engineering problem, requiring continuous acquisition of new experimental data – data that should be acquired from experiments designed and informed by continuous modeling. Linear fuzzy rule network models are a promising methodology for an integrated modeling and experimental approach. Since fuzzy rule models are enumerable, methods developed for combinatorial optimization can be extended to them. Moreover, linear fuzzy network models can simultaneously contain both quantitative and qualitative information, providing a common framework for a broad range of biological data, including mass spectrometry analysis, RT-PCR, single cell imaging, metabolite profiling, and other technologies yet to be developed. Methods Converting between numerical data and fuzzy sets We use three fuzzy sets, Low (or 1 ), Medium ( 2 ), and High ( 3 ) to represent the magnitude of gene expression, as defined in Figure 5 . Fuzzification (conversion to fuzzy representation) of a numerical datum x is performed by finding the corresponding fuzzy set memberships y 1 , y 2 , and y 3 (with values ranging from 0 to 1.0) given the linear functions shown in Figure 5 , where Defuzzification (conversion back to numerical representation) is performed using the "simplified centroid method" [ 29 ], with point set definitions shown in Figure 5 . Following a fuzzy rule evaluation that returns fuzzy set memberships y = [ y 1 y 2 y 3 ] in sets 1 (Low), 2 (Med), and 3 (High) respectively, the estimated numerical result of the evaluation, , is given by the centroid for the points located at -1, 0, and +1 for each set respectively, or The fuzzy set definition and centroid defuzzification of Equations 1 and 2 were selected to maximize computational efficiency during exhaustive search: all 27 rules can be represented by easily implemented algebraic functions and it is possible to design the implementation to avoid as many costly if/then comparisons as possible. In addition, the scheme perfectly reproduces monotonic linear positive and negative interactions (i.e., the functions f ( x ) = x and f ( x ) = - x are quantitatively equal to monotonic fuzzy rules, which can be written using notation from the following section as [1 2 3] and [3 2 1] respectively) so it generally will not introduce systematic error in the model. To apply this scheme for defuzzification and fuzzification scheme, experimental data must be projected on to the interval -1.0 through +1.0. Thus, log base 2 expression ratios are normalized by taking the arctangent of each ratio and dividing by π/2, yielding a symmetric transformation covering the desired interval. Previous work normalized expression ratios by the maximum value found in the experiment [ 17 ] or used different fuzzy set definitions for each variable [ 16 ], but those approaches suffer from a lack of universality across data sets and makes it difficult to compare and integrate data from different experiments. On the other hand, the arctangent method is defined across infinity, so no data will be "out of range". It also takes into account the fact that gene expression ratios often "saturate", and the difference between different degrees of high and low ratios are not necessarily biologically significant (this is because of the optical methods for measuring microarrays and the exponential error introduced using RT-PCR). When used in conjunction with the overlapping fuzzy set mappings shown in Figure 5 , these "middle" values will tend to land in the Medium set ( 2 ). Comparing fuzzy predictions to numerical data The fuzzy rule relating the input of a single gene to an ouptut node gene can be expressed as a rule vector r . For example, the rule r = [3 2 1] corresponds to the linguistic rules: If Input is Low ( 1 ) then Output is High ( 3 ) If Input is Med ( 2 ) then Output is Med ( 2 ) If Input is High ( 3 ) then Output is Low ( 1 ) Given the fuzzified expression of an input gene y = [ y 1 y 2 y 3 ] obtained using Equation 1 and the general fuzzy rule r = [ r 1 r 2 r 3 ], the resulting fuzzified expression of the output gene z will be: In general, node behavior is the result of N input genes acting on the output gene simultaneously. In the linear fuzzy logic scheme, the rule for each input gene is evaluated separately, leading to intermediate outputs z i : These intermediate fuzzy values are summed algebraically to obtain the final resulting fuzzy value for node gene expression: This result is defuzzified using Equation 2 to evaluate the output of the node. For three fuzzy sets, there are 3 3 or 27 possible rules describing the effect of a single gene on another gene. Thus, if there are N input genes for a node, there are 27 N total possible rule combinations describing the behavior of the node gene. In general, no rule combination will be an exact fit to real experimental data. Given some tolerance to fitting error, there will be multiple possible rule combinations, representing plausible hypothetical gene network models. In our present work, we define the error of the fit for the M data of the output gene x = { x 1 , x 2 ,..., x M } as where is the set of defuzzified numerical predictions (typically log expression ratios) and is the mean of the experimental data set x . A perfect fit results in a maximum E of 1.0. This error score was chosen because while it is quantitative, it emphasizes the correlation in qualitative behavior between the fit and prediction instead of the absolute numerical fit, which can be difficult to model with the limited resolution of three fuzzy sets. We can use the fitting error to rank these models, and use rule patterns consistent throughout all plausible models as a basis for constructing the template of a final network model that can be tested experimentally. Example of fuzzy rule evaluation As an example to illustrate fuzzy gene networks using a simple rule combination, we consider three genes ( G1 , G2 , G3 ) with log base 2 expression ratios measured at three different times: G 1 = {-3.0 0 +3.0} G 2 = {0.3 0 -0.3} G 3 = {+1 0 -1.0} Using the arctangent normalization to project the ratios on [-1,1], we obtain G 1 = {-0.795 0 +0.795} G 2 = {+0.186 0 -0.186} G 3 = {+0.500 0 -0.500} which can be fuzzified using Equation 1 to yield: G 1 = {[0.795 0.205 0] [0 1.0 0] [0 0.205 0.795]} G 2 = {[0 0.814 0.186] [0 1.0 0] [0.186 0.814 0]} G 3 = {[0 0.5 0.5] [0 1.0 0] [0.5 0.5 0]} with vectors for each time point containing set membership in Low ( 1 ), Medium ( 2 ), and High ( 3 ). Consider the following rules for G1 and G2 as input genes to G3 : G 1: G 3 = [3 2 1] G 2: G 3 = [1 2 3] where the rules can be written in English as If G1 is Low ( 1 ) then G3 is High ( 3 ) If G1 is Med ( 2 ) then G3 is Med ( 2 ) If G1 is High ( 3 ) then G3 is Low ( 1 ) If G2 is Low ( 1 ) then G3 is Low ( 1 ) If G2 is Med ( 2 ) then G3 is Med ( 2 ) If G2 is High ( 3 ) then G3 is High ( 3 ) Now, the evaluations of the rules taken individually are G 1: G 3 = {[0 0.205 0.795] [0 1.0 0] [0.795 0.205 0]} G 2: G 3 = {[0 0.814 0.186] [0 1.0 0] [0.186 0.814 0]} The sum of two intermediate outputs (Equation 3) is the predicted fuzzy behavior of G3 for the three time points, which can be defuzzified using the point-centroid method (Equation 2) and transformed back to real numbers on [-1,1]: G 3 = {[0 1.019 0.981] [0 2.0 0] [0.981 1.019 0]} = {0.491 0 -0.491} These numbers can be transformed back to a Log2 expression ratio by inverting the normalization (multiplying by π/2 and taking the tangent): G 3 = {0.97 0 -0.97} Finally, we use Equation 4 and the experimental data for G3 to calculate the fit error for this rule combination: which compares to a maximum E = 1.0 for a perfect fit. Exhaustive network search In general, a possible model for a node can include any combination of the genes available to act as inputs. In the work described here, we consider potential interactions of 12 genes. Thus, a rule for any one gene can include as inputs any combination of any number of up to all 11 other genes. Since each input gene can influence the node by any one of the 27 possible fuzzy rules, there are approximately 10 16 possible rule combinations for each of the 12 genes, making the exhaustive search method practically impossible. Thus, the number of possible inputs to a node must have a maximum constraint to make exhaustive search tractable. Studies of network topology through the experimentally observed association of proteins suggest that in many cases only few regulatory proteins are observed to directly influence the expression of a gene [ 26 , 30 - 32 ]. For our transcriptional network searches, we use the constraint of up to 4 input genes to any node. Thus, for each node gene, each of the other 11 genes occurs as an input alone and also in combination with any of up to 3 of the other genes as multiple inputs. Our use of this input constraint does not necessarily restrict the full range of interactions that can be found for the genes in our network, since all possible combinations of 1 through 4 of the genes are searched sequentially. For example, in our fitting of rules to CLN3 , we considered the following potential input combinations: SIC1 alone, SIC1 and CLN1 together, SIC1 - CLN1-CLN2 , SIC1-CLN1-CLN2-CLN3 , CLN1 , CLN1-CLN2 , CLN1-CLN2-CLN3 , CLN1-CLN2-CLN3-SWI4 , CLN2 , CLN2-CLN3 , etc. If we include all combinations from 1 through 4 of the genes taken from the 11 total possible inputs, then the total search space for each of the 12 genes consists of approximately 10 8 rules (taking about 10 minutes on a PowerMac G4 using a single 450 MHz processor). Simulation files used to generate all the data presented here are available from the authors upon request. Authors' contributions BAS originated the concept of applying scalable (URC) fuzzy logic to modeling biological systems, implemented the scheme described within on the data set, and was the primary author. JPF conceived of the approach to use exhaustive search for biological network reconstruction. JNQ and AAQ developed and initially implemented the method of combinatorial input selection for the exhaustive network search, and AAQ contributed to the text. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Microsoft Excel spreadsheets of simulation results. See descriptive text in the workbook. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514698.xml
524029	β3-adrenoceptor agonist prevents alterations of muscle diacylglycerol and adipose tissue phospholipids induced by a cafeteria diet	Background Insulin resistance induced by a high fat diet has been associated with alterations in lipid content and composition in skeletal muscle and adipose tissue. Administration of β3-adrenoceptor (β3-AR) agonists was recently reported to prevent insulin resistance induced by a high fat diet, such as the cafeteria diet. The objective of the present study was to determine whether a selective β3-AR agonist (ZD7114) could prevent alterations of the lipid profile of skeletal muscle and adipose tissue lipids induced by a cafeteria diet. Methods Male Sprague-Dawley rats fed a cafeteria diet were treated orally with either the β3-AR agonist ZD7114 (1 mg/kg per day) or the vehicle for 60 days. Rats fed a chow diet were used as a reference group. In addition to the determination of body weight and insulin plasma level, lipid content and fatty acid composition in gastronemius and in epididymal adipose tissue were measured by gas-liquid chromatography, at the end of the study. Results In addition to higher body weights and plasma insulin concentrations, rats fed a cafeteria diet had greater triacylglycerol (TAG) and diacylglycerol (DAG) accumulation in skeletal muscle, contrary to animals fed a chow diet. As expected, ZD7114 treatment prevented the excessive weight gain and hyperinsulinemia induced by the cafeteria diet. Furthermore, in ZD7114 treated rats, intramyocellular DAG levels were lower and the proportion of polyunsaturated fatty acids, particularly arachidonic acid, in adipose tissue phospholipids was higher than in animals fed a cafeteria diet. Conclusions These results show that activation of the β3-AR was able to prevent lipid alterations in muscle and adipose tissue associated with insulin resistance induced by the cafeteria diet. These changes in intramyocellular DAG levels and adipose tissue PL composition may contribute to the improved insulin sensitivity associated with β3-AR activation.	Background Dietary fatty acids are known to influence the composition of stored triacylglycerol (TAG) and membrane phospholipids (PL) in adipose tissue [ 1 ]. More recently, it was demonstrated that the lipid profile in skeletal muscle reflected dietary lipids [ 2 - 4 ]. Furthermore, the modifications of fatty acid concentrations and composition in tissue lipids induced by a high fat diet has been associated with alterations in lipid metabolism and insulin sensitivity [ 5 , 6 ]. Indeed, enrichment of membrane PL with saturated fatty acids (SFA) was able to impair insulin action in skeletal muscle and adipose tissue, whereas a higher proportion of polyunsaturated fatty acids (PUFA) improved insulin sensitivity in these tissues [ 7 - 9 ]. TAG accumulation in skeletal muscle was also correlated with the development of insulin resistance, independent to the degree of obesity [ 10 - 13 ]. Intramyocellular TAG could represent only a marker of insulin resistance whereas intracellular accumulation of long chain acyl-coenzyme A, ceramide or diacylglycerol (DAG) were reported to directly alter the insulin action [ 14 ]. Chronic activation of the β3-adrenoceptor (β3-AR), which is predominantly expressed in white and brown adipose tissue, by selective agonists exerts both anti-obesity and anti-diabetic effects in rodent models of obesity [ 15 , 16 ]. Activation of this receptor has been reported to enhance energy expenditure via stimulation of thermogenesis in brown adipose tissue [ 16 ]. The improvement in glucose homeostasis induced by β3-AR agonists appears to be a consequence of increased insulin sensitivity in peripheral tissues rather than stimulation of insulin secretion by the pancreas [ 17 ]. Although, the expression of β3-AR in myocytes is still a matter of debate [ 18 - 20 ], obese rats treated with β3-AR agonists demonstrated an improvement of insulin sensitivity in brown and white adipose tissue as well as in skeletal muscle [ 17 , 21 , 22 ]. In adipose tissue this effect is believed to be mediated by the conversion of large adipocytes into small adipocytes, which are more sensitive to insulin [ 21 ]. In skeletal muscle, it seems more likely that the effects of β3-AR agonists on insulin sensitivity are mediated by alternate indirect mechanisms. The objective of the present study was to determine whether a selective β3-AR agonist could prevent alterations in the profile of skeletal muscle and adipose tissue lipids induced with the consumption of the cafeteria diet, previously reported to induce weight gain and hyperinsulinemia [ 22 , 23 ]. The selective β3-AR agonist ZD7114 was used in this study. When administered at 1 mg/kg/day, this compound has been shown to increase thermogenesis in rodents and dogs without increasing heart rate or β2-AR-mediated effects such as tremor [ 24 ]. ZD7114 pharmacological specificity was also demonstrated in brown adipocytes and smooth muscles [ 25 , 26 ]. As expected, we observed that a chronic treatment with the ZD7114 prevented the development of excessive fat mass and hyperinsulinemia induced by this diet. These preventive beneficial effects exerted by the β3-AR agonist were associated with a reduction of muscle DAG accumulation. In adipose tissue, ZD7114 treatment was able to limit the proportional reduction of PUFA into the PL pool, induced by the cafeteria diet. Our results indicate that a β3-AR agonist prevents some cafeteria diet-induced alterations of the fatty acid profile of lipids in skeletal muscle and adipose tissue. Furthermore, we propose that these changes may contribute to the improved of insulin sensitivity observed in rats treated with a β3-AR agonist during the development of obesity. Methods Animal study Male Sprague-Dawley rats were purchased from Charles River (L'arbesles, France) at 5–6 weeks of age. Animals were individually housed in temperature-controlled rooms (22°C) with a 12-h light-dark cycle. Ten days before the beginning of the study, 30 rats were provided a normal chow diet (Kliba-Nafalg, Switzerland) and free access to drinking water. At the end of this period, rats were weighed and pre-selected for their sensitivity to weight gain (i.e. rat presenting at least 35% weight gain after a 10-day selection period). The selected rats (n = 15) were equally randomized into 3 groups. A reference group, consisted of rats fed for 60 days with a standard pellet chow diet containing 28, 57 and 15% E from protein, carbohydrate and fat, respectively (REF group). The remaining rats were fed for 60 days with a cafeteria diet composed of 30 g of a mix containing salami, cookies, cheese, sausage, chips, chocolate and almonds in a proportion of 2:2:2:1:1:1:1 and 30 g of the reference group chow diet. This mixed diet contained 26, 27 and 47% energy as protein, carbohydrate and fat, respectively. At the beginning of the dietary intervention, both groups fed the cafeteria diet received daily, by gavage (0.5 ml/100 g body weight), either the selective β-3AR agonist ZD7114 at 1 mg/kg per day (CAF-ZD group) or water alone (CAF group) until day 60. Rats from the REF group (chow diet) also received a daily gavage of water (0.5 ml/100 g body weight). Body weight and food intake were recorded daily. Rats were fasted for 8 hours before sacrifice, performed under isoflurane anaesthesia. Tissues were immediately collected, weighed, frozen in liquid nitrogen and kept at -80°C until analysed. All procedures in the study were in compliance with the ethical committee of the "Service vétérinaire du canton de Vaud". Estimation of the proportions of the different lipid classes contained in the cafeteria diet (see Discussion) was performed using the USDA Nutrient Database. Lipid fatty acid composition Adipose tissue Fatty acid composition of lipids in adipose tissue was performed by Lipomics Technologies (West Sacramento, USA). Briefly, lipids were extracted from 30 mg of frozen epididymal adipose tissue in the presence of authentic internal standards by the method of Folch et al. [ 27 ] using chloroform:methanol (2:1, by vol.). After separation of individual lipid classes by preparative thin-layer chromatography, the TAG and PL were scraped and trans-esterified; the resulting fatty acid methyl esters were then separated and quantified by capillary gas chromatography as previously described [ 28 ]. Muscle The frozen gastrocnemius muscle was thawed and thoroughly dissected under stereomicroscope to remove extramyocellular adipose tissue. Lipids from lyophilized, finely powdered, dissected muscle (50 mg) were extracted according to the method of Folch [ 27 ]. PL (19:0), TAG (17:0) and DAG (15:0) internal standards (Varian; Zug, Switzerland) were added prior to lipid extraction. The lipids were loaded on a Chromabond NH2 cartridge (Varian; Zug, Switzerland), and neutral lipids were separated from free fatty acids and PL by sequential elution with chloroform/2-Propanol (2:1), 2% acetic acid in diethylether and methanol, respectively [ 29 ]. The neutral lipids were subjected to thin-layer chromatography using hexane:diethylether:acetic acid (70:30:1, by vol.) as solvent system to separate DAG from TAG [ 30 ]. The hydrolysis of TAG into DAG during sample analysis has been assessed and represented less than 0.5% of total DAG. The fatty acids from the phospholipids, TAG and DAG were converted to their methyl esters. Fatty acid methyl-ester separation was performed by automated gas-liquid chromatography (HP 6890 series) with FID detection (280°C); authentic standard mixtures of fatty acid methyl-esters (Nu-Chek-Perp; Lowell Mutter, USA) were injected to identify fatty acid methyl-ester peaks. Results are expressed in μmol fatty acids per gram lyophilised muscle. Plasma metabolites Plasma glucose and insulin were determined with commercially available kits purchased from Sigma (Buchs, Switzerland) and Crystal Chem Inc. (Downers Grove, USA), respectively. Plasma triglycerides and free fatty acids concentrations were analysed using kits from Roche Diagnostic (Basel, Switzerland) and Wako Chemicals (Richmond, USA), respectively. Statistical analysis Comparisons of the means of the dependent variables of each group were performed using a one-way ANOVA. Results Body weight and fat mass Male Sprague-Dawley rats were fed, during 60 days, either a chow diet used as a reference group (REF group), a cafeteria diet (CAF rats) or a cafeteria diet plus a daily gavage of the β3-AR agonist ZD7114 (CAF-ZD rats). At day 60, the mean body weight of CAF rats was significantly greater than that of REF rats (Table 1 ). On the other hand, CAF-ZD rats presented a significant reduction in mean body weight when compared with CAF rats (Table 1 ). Similar effects were observed on weight gain which was 46% greater in CAF rats than in REF rats (Figure 1 ; 213.2 ± 12.2 g vs. 312.3 ± 19.2 g in REF and CAF rats, respectively; p < 0.01), and reduced by 17% in CAF-ZD rats as compared to CAF rats (258.70 ± 12.70 g in CAF-ZD rats, p < 0.05). The enhancement of body weight in CAF rats was strongly associated with the weight increase of two main deep adipose depots, confirming the obesigenic properties of the cafeteria diet. Indeed, the epididymal and retroperitoneal fat pads in CAF rats were respectively 131% and 185% heavier than those of REF rats (Table 1 ). ZD7114 treatment decreased the weight of the two adipose tissue depots by about 45%, compared to CAF rats (Table 1 ). The weights of gastrocnemius skeletal muscle, liver and heart were not different between groups (Table 1 ). As expected, CAF rats had a higher energy intake compared with REF animals (106.03 ± 7.25 vs 78.49 ± 2.96 kcal/day). However, the anti-obesity effect of ZD7114 was not attributed to a reduction of energy intake (107.70 ± 7.30 kcal/day in CAF-ZD rats). Table 1 Body weight, tissues and fasting plasma metabolites REF CAF CAF-ZD Final body weight (g) 443.78 ± 21.42 540.20 ± 16.69 * 477.82 ± 12.09 + Retroperitoneal (g) 3.73 ± 0.44 10.65 ± 0.48 5.78 ± 0.23 + Epididymal (g) 4.65 ± 0.41 10.73 ± 0.43 5.96 ± 0.30 + Gastrocnemius (g) 2.69 ± 0.13 2.78 ± 0.01 2.79 ± 0.03 Heart (g) 1.25 ± 0.07 1.48 ± 0.01 1.46 ± 0.02 Liver (g) 13.53 ± 0.79 16.69 ± 0.15 15.47 ± 0.12 Glucose (mmol/l) 12.80 ± 1.51 12.10 ± 1.02 14.78 ± 1.11 Insulin (μU/ml) 19.75 ± 2.05 98.04 ± 16.01 43.27 ± 8.26 + Fatty acid (mmol/l) 0.47 ± 0.22 0.42 ± 0.68 0.30 ± 0.39 Triacylglycerol (mmol/l) 2.27 ± 0.30 4.59 ± 0.58 ** 4.11 ± 0.95 Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by * (p < 0.05) and ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by + (p < 0.05). Figure 1 Individual weight changes. Body weight was measured in rats (n = 5) fed a chow diet (REF) or a cafeteria diet alone (CAF) or treated with 1 mg/kg/day ZD7114 (CAF-ZD) at the beginning and at the end of the different interventions (day 60). Data are represented as individual values. Glucose and insulin plasma concentrations Measurement of insulin and glucose concentrations in plasma of fasted animals showed that CAF rats presented a marked hyperinsulinemia with a 4.6 fold increase in insulin concentration as compared to REF animals whereas the glucose level was not changed (Table 1 ). ZD7114 treatment limited the hyperinsulinemia induced by the cafeteria diet, as demonstrated by the 2.3 fold decrease in plasma insulin concentrations in CAF-ZD rats compared with the CAF group (Table 1 ). No significant changes in plasma glucose concentrations were observed between these two groups (Table 1 ). Adipose tissue lipids Lipid content PL and TAG contents per gram of tissue were measured in epididymal adipose tissue of rats fed with a chow diet or a cafeteria diet treated or not with ZD7114. No significant change was observed in the concentration of PL and TAG in adipose tissue of CAF rats when compared with REF animals (Table 2 ). Furthermore, ZD7114 treatment did not affect adipose tissue lipid content of CAF rats. Table 2 Adipose tissue lipid content μmole/g tissue REF CAF CAF-ZD Phospholipids 8.29 ± 1.28 10.59 ± 1.06 8.65 ± 0.87 Triacylglycerol 2476.34 ± 68.31 2462.63 ± 41.33 2271.57 ± 122.46 Data are the mean ± SEM. REF: rats fed a chow diet; CAF: rats fed a cafeteria die, CAF-ZD: rats fed a cafeteria diet and treated with ZD7114 Fatty acid composition Analysis of the fatty acid profile shows that cafeteria diet induced an increase in the proportion of monounsaturated fatty acids (MUFA), which was compensated for by a reduction in the percentage of PUFA in both adipose PL and TAG (Figure 2 ). Changes in the proportion of MUFA in PL and TAG were mainly due to the increase of oleic acid (1.7 and 2.0 fold increase in PL and TAG, respectively; Table 3 ). Reduction in the percentage of linoleic acid was mainly responsible for the decrease in proportion of PUFA in both PL and TAG (1.6 and 2.3 fold decrease for PL and TAG, respectively; Table 3 ). The proportions of arachidonic and α-linolenic acids were also slightly decreased in PL and TAG, respectively (Table 3 ). Although the global proportion of SFA was not modified in adipose tissue PL and TAG of CAF rats when compared with REF animals, the percentage of myristic and stearic acids were respectively enhanced in PL and TAG of CAF animals. Figure 2 Proportion of the different lipid classes in adipose tissue. Saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) were measured in epididymal adipose tissue phospholipid (PL) and triacylglycerol (TAG) of rats fed a chow diet (empty bars) or a cafeteria diet (black bars) alone or treated with ZD7114 (grey bars). Data are represented as mean ± SEM, and values significantly different to data measured in rats fed the chow or cafeteria diets are indicated by * (p < 0.05), ** (p < 0.01) or + (p < 0.05), respectively. Table 3 Composition of adipose tissue lipids % mole fatty acids REF CAF CAF-ZD Phospholipids Myristic acid (14:0) 3.04 ± 0.16 3.81 ± 0.19 * 2.96 ± 0.15 ++ Palmitic acid (16:0) 28.70 ± 3.10 30.72 ± 1.83 26.22 ± 1.70 Stearic acid (18:0) 15.99 ± 1.19 16.10 ± 0.75 16.93 ± 0.50 Palmitoleic acid (16:1n-7) 1.17 ± 0.28 1.35 ± 0.31 1.16 ± 0.29 Oleic acid (18:1n-9) 10.74 ± 0.94 18.17 ± 1.60 ** 16.26 ± 1.39 * Vaccenic acid (18:1n-7) 1.24 ± 0.12 0.89 ± 0.07 * 0.98 ± 0.10 Linoleic acid (18:2n-6) 21.21 ± 1.98 13.27 ± 1.20 ** 16.41 ± 1.55 * Eicosadienoic acid (20:2n-6) 2.16 ± 1.37 0.92 ± 0.92 0.37 ± 0.37 Arachidonic acid (20:4n-6) 7.69 ± 0.73 4.47 ± 1.23 * 8.90 ± 1.35 + Triacylglycerol Myristic acid (14:0) 1.23 ± 0.03 1.33 ± 0.08 1.39 ± 0.28 Palmitic acid (16:0) 21.80 ± 0.29 21.48 ± 0.63 21.02 ± 0.65 Stearic acid (18:0) 3.35 ± 0.15 5.27 ± 0.35 ** 4.84 ± 1.68 * Palmitoleic acid (16:1n-7) 2.50 ± 0.27 2.29 ± 0.34 2.44 ± 0.46 Oleic acid (18:1n-9) 24.37 ± 1.04 48.87 ± 1.35 47.09 ± 0.20 Vaccenic acid (18:1n-7) 1.98 ± 0.12 1.59 ± 0.12 1.73 ± 1.20 Linoleic acid (18:2n-6) 38.22 ± 1.36 16.59 ± 0.72 18.37 ± 0.16 α-linolenic acid (18:3n-3) 2.85 ± 0.16 0.67 ± 0.06 0.71 ± 0.20 Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by * (p < 0.05) and ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by + (p < 0.05) and ++ (p < 0.05). Only main fatty acids are presented. CAF-ZD: rats fed a cafeteria diet and treated with ZD7114. ZD7114 treatment did not change the proportions of any lipid classes measured in adipose TAG of CAF rats (Figure 2 ). However, in PL, the percentage of PUFA was significantly increased by 1.3 fold in CAF-ZD rats compared to CAF animals. This change was essentially due to a two-fold increase in the proportion of arachidonic acid measured in CAF-ZD rats (Table 3 ). A slight reduction in the percentage of myristic acid (1.2 fold decrease) was measured in CAF-ZD rats compared to CAF animals. Skeletal muscle lipids Lipid content PL, diacylglycerol (DAG) and TAG contents were measured in the gastrocnemius of REF animals and CAF rats with or without ZD7114 treatment (Table 4 ). Figure 3 Proportion of the different lipid classes in muscle. Saturated (SFA), monounsaturated (MUFA) and polyunsaturated fatty acids (PUFA) were measured in muscle phospholipid (PL), triacylglycerol (TAG) and diacylglycerol (DAG) of rats fed a chow diet (empty bars), a cafeteria diet (black bars) alone or treated with ZD7114 (grey bars). Data are represented as mean ± SEM, and values significantly different to data measured in rats fed the chow or cafeteria diet are indicated by * (p < 0.05), (p < 0.01) or + (p < 0.05), ++ (p < 0.01), respectively. The cafeteria diet clearly induced TAG and DAG accumulation in the gastrocnemius, as demonstrated by the respective 3.4 and 2.0 fold increases observed in CAF vs. REF rats (Table 4 ). Chronic treatment with ZD7114 did not significantly reduce the cafeteria diet induced-accumulation of TAG in muscle. In contrast, DAG accumulation was prevented as indicated by a 1.5 fold reduction of DAG levels in CAF-ZD when compared with CAF rats (Table 4 ). Values of intramyocellular TAG and DAG obtained in the present study (TAG: between 1 to 4 μmol/g fresh muscle; DAG: between 0.5 to 1 μmol/g fresh muscle) were similar to those previously described in rat skeletal muscles [ 31 ] (TAG: between 4 to 5 μmol/g fresh muscle; DAG: between 0.5 to 2.5 μmol/ g fresh muscle). Table 4 Muscle lipid content μmole/g tissue REF CAF CAF-ZD Phospholipids 60.31 ± 4.28 65.62 ± 1.26 70.80 ± 2.77 Triacylglycerol 5.40 ± 0.98 18.63 ± 3.24 15.65 ± 2.57 Diacylglycerol 2.07 ± 0.32 4.08 ± 0.24 2.66 ± 0.25 ++ Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by ++ (p < 0.05). Fatty acid composition Fatty acid profiles were determined in muscle PL, TAG and DAG. The gastrocnemius of CAF rats, compared with the REF group, presented an increase in the percentage of MUFA and a decrease in the proportion of PUFA in both TAG and DAG and to a lesser extent in PL (Figure 3 ). Variations in the percentage of oleic and linoleic acids were, respectively, responsible for the changes in the proportions of MUFA and PUFA in muscle TAG and DAG (Table 5 ). Modifications of the proportions of PUFA observed in muscle PL of CAF rats were due to slight decreases of both linoleic (1.4 fold increase) and docosahexaenoic (22:6 n-3) acids (1.3 fold increase), whereas the percentage of arachidonic acid (20:4 n-6) was increased by 1.2 fold. Table 5 Composition of muscle lipids % mole fatty acids REF CAF CAF-ZD Phospholipids Palmitic acid (16:0) 27.87 ± 0.50 26.17 ± 0.29 26.44 ± 0.57 Stearic acid (18:0) 20.21 ± 0.64 22.37 ± 0.54 * 21.64 ± 0.58 Oleic acid (18:1n-9) 6.84 ± 0.21 9.13 ± 0.48 9.12 ± 0.24 Linoleic acid (18:2n-6) 13.09 ± 0.65 8.98 ± 0.51 9.02 ± 0.28 Arachidonic acid (20:4n-6) 11.64 ± 0.46 15.07 ± 0.53 14.11 ± 0.22 Docosatetraenoic acid (22:4n-6) ND 0.61 ± 0.04 0.51 ± 0.02 Adrenic acid (22:5n-3) 2.04 ± 0.07 2.44 ± 0.18 2.10 ± 0.09 Docosahexaenoic acid (22:6n-3) 18.30 ± 0.95 15.23 ± 0.52 * 16.09 ± 0.35 Triacylglycerol Myristic acid (14:0) ND 1.66 ± 0.08 1.53 ± 0.06 Palmitic acid (16:0) 37.13 ± 2.06 25.26 ± 0.95 25.15 ± 0.81 Stearic acid (18:0) 7.94 ± 0.31 9.36 ± 0.45 * 9.38 ± 0.65 Palmitoleic acid (16:1n-7) ND 1.51 ± 0.14 1.31 ± 0.13 Oleic acid (18:1n-9) 29.00 ± 0.94 51.36 ± 1.83 50.14 ± 0.93 Linoleic acid (18:2n-6) 25.00 ± 1.98 10.86 ± 0.83 12.47 ± 0.66 Diacylglycerol Myristic acid (14:0) 1.92 ± 0.09 1.75 ± 0.07 1.85 ± 0.05 Palmitic acid (16:0) 25.55 ± 0.86 22.42 ± 1.22 24.88 ± 0.62 Stearic acid (18:0) 6.71 ± 0.45 8.74 ± 0.63 * 9.71 ± 0.08 Palmitoleic acid (16:1n-7) 2.58 ± 0.20 1.59 ± 0.14 1.26 ± 0.07 Oleic acid (18:1n-9) 28.90 ± 1.24 52.88 ± 1.25 46.45 ± 1.61 ++ Nervonic acid (24:1n-9) 3.92 ± 1.93 1.24 ± 0.42 2.71 ± 0.88 Linoleic acid (18:2n-6) 26.96 ± 2.22 10.14 ± 2.04 8.05 ± 0.69 α-linolenic acid (18:3n-3) 1.12 ± 0.18 ND ND Stearidonic acid (18:4n-3) ND ND 1.90 ± 0.41 Arachidonic acid (20:4n-6) 0.78 ± 0.06 0.46 ± 0.06 0.26 ± 0.04 ** + Data are the mean ± SEM. Values significantly different from those obtained in the group of rats fed a chow diet (REF) are indicated by * (p < 0.05) and ** (p < 0.01). Data measured in the group of rats treated with ZD7114 (CAF-ZD) and significantly different from those in the group of rats fed a cafeteria diet (CAF) are shown by + (p < 0.05) and ++ (p < 0.05). Fatty acids representing more than 1% in at least one group are presented. The influence of ZD7114 on the cafeteria-induced modification of fatty acid composition in the three lipid species was evaluated by comparing CAF rats with CAF-ZD rats. While ZD7114 did not affect the fatty acid profile of either TAG or PL in muscle of CAF rats, it induced changes in the fatty acid composition of muscle DAG. Indeed, the proportion of SFA was slightly (1.2 fold increase), but significantly, elevated in DAG of CAF-ZD rats with an increase in the proportion of palmitic and stearic acids (Table 5 ). Furthermore, the proportion of MUFA in DAG of CAF-ZD rat muscles was decreased compared with DAG of CAF rats. This change was essentially attributed to a decrease in the proportion of oleic acid (Table 5 ). Stearidonic acid (18.4 n-3) was only detected in DAG accumulated in muscles of ZD-CAF rats. No differences in the proportion of PUFA between DAG stored in muscles of CAF-ZD or CAF rats were observed (Figure 3 ). Discussion The present study demonstrates that feeding the cafeteria diet to rats not only promotes weight gain, hyperinsulinemia and hypertriglyceridemia, but also accumulation of lipids in skeletal muscle. Although TAG levels per gram of adipose tissue was not affected by the cafeteria diet, this dietary intervention strongly enhanced TAG accumulation in skeletal muscle, as previously described [ 4 , 5 ]. We show that this intramyocellular accumulation of TAG was associated with an increase in skeletal muscle DAG content. This finding is in agreement with the accumulation of DAG observed in human skeletal muscle cells incubated with saturated fatty acids in vitro [ 12 ]. DAG mass was also described to be increased in skeletal muscle biopsies obtained from normal volunteers in whom insulin resistance was produced by raising FFA levels during a lipid infusion [ 32 ]. However, to our knowledge, the present study demonstrates for the first time that a high fat diet, resembling the human Western diet, was able to increase DAG storage in skeletal muscle. Although both TAG and DAG accumulations in skeletal muscle were reported to be correlated with insulin resistance [ 4 , 5 , 33 ], DAG (a precursor of TAG synthesis) is proposed to directly impair insulin sensitivity by inactivating insulin receptor activity through activation of the protein kinase C [ 34 ]. The fatty acid composition of TAG stored in muscle was previously shown to be affected by dietary lipids [ 2 ]. Interestingly, we also find that the fatty acid composition of muscle DAG was modified by the cafeteria diet. Indeed, the increase in MUFA and the reduction of PUFA proportions, measured in both DAG and TAG, reflected the difference in lipid composition of the two diets (+ 19% MUFA and -28% PUFA in cafeteria diet vs. chow diet). It is noteworthy that qualitative changes in DAG were reported to affect the activity of DAG as a secondary messenger, since specificity of its fatty acyl moieties for the activation of protein kinase C has been described [ 35 , 36 ]. Although measurements of insulin sensitivity were not directly accessed in this study, our observation suggests that diet may regulate insulin response in muscle by modifying DAG composition. Fatty acid compositions of muscle and adipose tissue PL were also modified by the cafeteria diet. More specifically, the proportion of PUFA in PL was decreased and MUFA was increased in both tissues. These differences were more pronounced in PL from adipose tissue than from gastrocnemius, indicating that membrane phospholipids in adipose tissue are more susceptible to variations in dietary composition than skeletal muscle. Interestingly, reductions in the proportion of PUFA in PL from myocytes [ 7 ] and adipocytes [ 8 ] were reported to impair insulin action. As previously reported with Trecadrine, a selective β3-AR agonist [ 22 ], chronic administration of ZD7114 prevented the excess weight gain and fat accumulation induced by a cafeteria diet. This present report demonstrates that ZD7114 treatment reduced the hyperinsulinemia elicited by the cafeteria diet (by 2.3 fold), thereby suggesting an improvement of insulin sensitivity in the treated rats. This observation was associated with a significant reduction in the accumulation of skeletal muscle DAG, and a slight, non-significant decrease in TAG. It is tempting to suggest that the decrease in muscle DAG accumulation induced by the chronic administration of a β3-AR agonist could participate in the prevention of hyperinsulinemia. β3-AR agonist treatment significantly modified the fatty acid composition of DAG, causing a 1.2 fold reduction in the proportion of oleic acid. As mentioned before, these modifications in DAG composition could directly regulate insulin action in muscles. Since the presence of a β3-AR in skeletal muscle is still a subject of debate, it is difficult to elucidate the mechanism(s) by which the β3-AR agonist lowers DAG content in muscle and modifies its fatty acid composition. Although the content and the fatty acid composition of TAG stored in adipose tissue was not affected by β3-AR agonist administration, the fatty acid profile of adipose PL was modified by the treatment. The β3-AR agonist was able to restore the proportion of PUFA to a level similar to the REF rats by increasing the percentage of arachidonic acid by 2 fold. As mentioned above, a greater percentage of PUFA in PL has been reported to enhance insulin sensitivity in adipose tissue. The changes in PL composition induced by the β3-AR agonist may be associated with the previously reported improved insulin sensitivity in adipose tissue [ 8 ], and the decrease of hyperinsulinemia measured in the present study. Conclusions Whilst the effectiveness of β3-AR agonists is limited in humans, understanding the metabolic changes affected in rodents will provide insights into mechanisms, underlying insulin responsiveness in humans. The present study demonstrates that β3-AR agonist treatment not only limits the hyperinsulinemia induced by a cafeteria diet, but also partially prevents the associated alterations in adipose and muscle lipid composition. Particularly, activation of the β3-AR limits the intramyocellular DAG accumulation and the decrease in the proportion of PUFA in adipose tissue PL. This combined action may contribute to the beneficial effect of β3-AR agonists on insulin sensitivity. List of abbreviations CAF rats : Rats fed with a cafeteria diet; DAG : Diacylglycerol; MUFA : Monounsaturated fatty acid; PKC : Protein kinase C; PUFA : Polyunsaturated fatty acid; REF rats : Rats fed with a chow diet; SFA : saturated fatty acid. TAG : Triacylglycerol; ZD-CAF rats : Rats fed with a cafeteria diet and treated with the β3-AR agonist ZD7114.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524029.xml
340938	pRb Inactivation in Mammary Cells Reveals Common Mechanisms for Tumor Initiation and Progression in Divergent Epithelia	Retinoblastoma 1 (pRb) and the related pocket proteins, retinoblastoma-like 1 (p107) and retinoblastoma-like 2 (p130) (pRb f , collectively), play a pivotal role in regulating eukaryotic cell cycle progression, apoptosis, and terminal differentiation. While aberrations in the pRb-signaling pathway are common in human cancers, the consequence of pRb f loss in the mammary gland has not been directly assayed in vivo. We reported previously that inactivating these critical cell cycle regulators in divergent cell types, either brain epithelium or astrocytes, abrogates the cell cycle restriction point, leading to increased cell proliferation and apoptosis, and predisposing to cancer. Here we report that mouse mammary epithelium is similar in its requirements for pRb f function; Rb f inactivation by T 121 , a fragment of SV40 T antigen that binds to and inactivates pRb f proteins, increases proliferation and apoptosis. Mammary adenocarcinomas form within 16 mo. Most apoptosis is regulated by p53, which has no impact on proliferation, and heterozygosity for a p53 null allele significantly shortens tumor latency. Most tumors in p53 heterozygous mice undergo loss of the wild-type p53 allele. We show that the mechanism of p53 loss of heterozygosity is not simply the consequence of Chromosome 11 aneuploidy and further that chromosomal instability subsequent to p53 loss is minimal. The mechanisms for pRb and p53 tumor suppression in the epithelia of two distinct tissues, mammary gland and brain, are indistinguishable. Further, this study has produced a highly penetrant breast cancer model based on aberrations commonly observed in the human disease.	Introduction Aberrant retinoblastoma 1 (pRb) pathway activity, resulting from defects in pRb itself, cyclin-dependent kinase inhibitor 2A (p16 INK4a ), cyclin D1 (CCND1), or cyclin-dependent kinase 4 (CDK4), is observed in the majority of human sporadic cancers ( Marshall 1991 ; Weinberg 1995 ; Sherr 1996 ; Ortega et al. 2002 ). This pathway is commonly altered early in cancer development, indicating an ability to predispose cells to tumorigenesis. However, whether the mechanism(s) is similar among cell types is not known. Examination of pRb inactivation in specific cell types in vivo has been technically challenging due to the apparent functional compensation or redundancy among pRb, retinoblastoma-like 1 (p107), and retinoblastoma-like 2 (p130) in many cell types of the mouse ( Luo et al. 1998 ; Robanus-Maandag et al. 1998 ; Dannenberg et al. 2000 ; Sage et al. 2000 ). Thus, genetic inactivation of the Rb gene alone, either by conditional deletion ( Marino et al. 2000 ) or by the generation of chimeric mice harboring pRb-deficient cells ( Maandag et al. 1994 ; Williams et al. 1994 ) yields only medulloblastomas, pituitary, and thyroid tumors. We have begun to systematically examine the role of retinoblastoma protein family (pRb f ) inactivation in multiple cell types of the mouse by dominant expression of T 121 , a truncation mutant of simian virus 40 (SV40) T antigen that inactivates all three pRb-related proteins ( DeCaprio et al. 1989 ; Dyson et al. 1989 ; Ewen et al. 1989 ; Stubdal et al. 1997 ; Sullivan et al. 2000 ). In this report we determine the role of pRb inactivation in mammary adenocarcinoma predisposition, establish a role for p53 inactivation in subsequent mammary adenocarcinoma progression, and, together with our previous studies, provide a comprehensive comparison of these mechanisms in distinct epithelial lineages. pRb plays a critical role in eukaryotic cell cycle progression, when cells exit G0 or G1 and enter S phase, thereby acting as a crucial negative regulator of cellular proliferation and neoplasia ( Sherr and McCormick 2002 ). In quiescent or early G1-phase cells, pRb is hypophosphorylated and associates with specific members of the E2F transcription factor family, converting them to active transcriptional repressors ( Hamel et al. 1992 ; Weintraub et al. 1992 ). Gene repression is also mediated by pRb and p130 recruitment of histone deacetylase to promote formation of inhibitory nucleosomes ( Brehm et al. 1998 ; Luo et al. 1998 ; Magnaghi-Jaulin et al. 1998 ). The many proteins found in association with pRb suggest other regulatory mechanisms may also be involved ( Morris and Dyson 2001 ), although the biological potential for most of these interactions remains yet unproven. Cell cycle progression from G to S phase occurs when complexes of D-type cyclins/CDK4/CDK6 phosphorylate pRb, thereby derepressing E2Fs to direct transcription of DNA-replication machinery and nucleotide biosynthesis genes ( Dyson 1998 ). Like most human solid tumors, breast cancers harbor frequent alterations in the pRb pathway, including CCND1 overexpression in 45% ( Buckley et al. 1993 ), p16 INK4A loss in 49% ( Geradts and Wilson 1996 ), and pRb loss in 6% of breast tumors ( Geradts and Wilson 1996 ). In the Rb -deficient mouse mammary gland, p107 and/or p130 may play overlapping or compensatory roles, as they do during embryonic development, given that pRb is dispensable for normal mammary development and mammary tumor suppression. pRb-deficient embryonic stem cells participate in normal mammary gland formation in chimeric mice ( Maandag et al. 1994 ), and donor pRb −/− mammary precursor cells transplanted into wild-type mice can populate a normal mammary gland without evidence of neoplasia, even after multiple pregnancies ( Robinson et al. 2001 ). The interplay between pRb signaling and the tumor protein p53 pathway is also critical to the understanding of breast cancer biology. Since the pRb pathway is defective in a majority of human tumors and the p53 gene is mutated in about half of them, including approximately a fifth of sporadic breast cancers ( Nigro et al. 1989 ; Greenblatt et al. 1994 ), these aberrations often coexist. Whether loss of these tumor suppressor pathways collaborate in tumorigenesis is also cell type-specific. In a brain epithelial tumor model, we previously demonstrated that, in the absence of pRb f function, inactivation of p53 significantly decreases apoptosis and accelerates tumor growth in vivo ( Symonds et al. 1994 ). However, in astrocytic brain tumors induced by pRb f inactivation, tumor progression is not accelerated by reduced p53 activity; rather, the phosphatase and tensin homolog ( PTEN ) regulates the apoptosis, and reduction in its function accelerates tumor growth ( Xiao et al. 2002 ). In this report, we extend our analysis of pRb function in vivo and examine the consequence of pRb f loss specifically in mammary epithelium. These studies serve not only to provide insight into the cell specificity of tumor suppression mechanisms, but also to model the stepwise evolution of breast adenocarcinomas that harbor defects in this pathway. Results Generation of Mice with Inducible pRb f Deficiency in Mammary Cells Seven founder mice were generated in which the T 121 gene was regulated by the whey acidic protein (WAP) transcriptional signals ( Figure 1 ; see Materials and Methods ). Of these, two founder animals died spontaneously of unknown causes, while the transgenic progeny of the third line died prematurely, also of unknown cause ( Figure 2 A). The extent to which the transgene contributed to these deaths was not investigated further; however, ectopic transgene expression was detected in several tissues (data not shown). Characterization of female mice of the four remaining lines is the focus of this report. Figure 1 Diagram of the WAP-T 121 Transgene and Protein The fragment consists of the 2.4 kb WAP promoter (hatched) and the mutant SV40 T-antigen coding region (white box) containing two deletions, the 196-bp amino-terminal deletion, which abolishes small t antigen production, and the dl 1137 deletion, which truncates T antigen. Both the J domain and the LXCXE domain are required for pRb family inactivation (see Materials and Methods ). Figure 2 Expression of T 121 Protein in WAP-T 121 Mice and a Summary of Gross Phenotypes As expected, each line showed mammary-specific expression following lactation induction, while line 4 showed more widespread expression, with protein detected in brain and kidney. Mice from the higher-expressing lines 3 and 4 failed to nurse because of lactation defects. Mammary glands of adult female mice from all four lines showed elevated proliferation and apoptosis. Glands from line 1 and 2 mice were hyperplastic, while glands from lines 3 and 4 were atrophic. Lines 3 and 4 later developed carcinomas and other neoplasms. T 121 protein was detected by Western blot analysis in lactating mammary glands of animals from all four lines (B), although the lower-expressing lines 1 and 2 required immunoprecipitation with anti-T-antigen antibody prior to Western blot analysis (right panel in [B]). Brain tumor extract (see Materials and Methods ) was used for a positive control, and nontransgenic mammary tissue extract was used for a negative control. A timecourse analysis of T 121 expression (C) shows lactation-induced expression peaking at 5 d postpartum. Abbreviations: Adeno-Ca, adenocarcinoma; AP, elevated apoptosis in mammary gland; At, atrophy; dpc, postcoital; FTN, failure to nurse; Hyp, hyperplastic acini; MG, mammary gland; MIN, mammary epithelia neoplasia; ND, not determined; nt, nontransgenic; pp, postpartum; Pr, elevated proliferation in mammary gland; pw, post-weaning. Footnotes: a Mosaic founder animal. b At earlier stages, development defects attributed to atrophy, while MIN and adenocarcinoma were observed at terminal stages. c Approximately half of progeny died of unknown cause. T 121 Is Expressed in Lactating Mammary Western immunoblotting analyses of mammary gland extracts demonstrated that this tissue expresses T 121 protein at the expected size in all four lines ( Figure 2 B). T 121 expression in lines 1 and 2 was only revealed following immunoprecipitation using an anti-T-antigen antibody prior to Western blot analysis, indicating lower levels of T 121 (right panel in Figure 2 B). A survey of select tissues showed that detectable expression was restricted to the mammary gland in lines 1–3, while expression was more widespread in the higher expressing line 4 (data not shown) and included brain and kidney expression. As expected, T 121 expression was induced by lactation with highest levels observed 5 d postpartum ( Figure 2 C). Southern blot analyses indicate that mice in line 3, which was used as a representative line for extensive characterization, harbor approximately ten copies of the transgene at a single insertion site (data not shown). Impact of Rb f Inactivation in Mammary Epithelium Representative histological analysis of lactating mammary glands (day 1) from single-pregnancy females of the line 2 founder (F 0 ) and a line 3 F 1 mouse shows that the impact of Rb perturbation is severalfold. Compared to an age- and parity-matched control tissue, the normal architecture of the lactating mammary tissue is disturbed. In contrast to normal tissue where acini consist of a single layer of secretory epithelia with milk-filled lumen ( Figure 3 A), transgenic animals have a lower density of acini ( Figure 3 K), consistent with atrophy, and are often atypical ( Figure 3 I). T 121 -positive mammary epithelial cells were associated with abnormalities ( Figure 3 B, 3 F, and 3 J). The line 2 F 0 animal was mosaic for T 121 protein expression with distinct regions of expressing and nonexpressing cells ( Figure 3 F), whereas T 121 expression in the line 3 animal was in secretory epithelium distributed throughout the gland ( Figure 3 J). Increased proliferation, indicated by proliferating cell nuclear antigen (PCNA) staining, was also observed in transgenic mammary glands ( Figure 3 C, 3 G, and 3 K), concomitant with increased levels of apoptosis assayed by TUNEL staining ( Figure 3 D, 3 H, and 3 L). Quantification of T 121 expression and apoptosis revealed higher protein expression levels (see Figure 2 B) correlate with higher percentages of apoptotic cells ( Figure 4 A). Consistent with a model for cell-autonomous functioning of T 121 , the pattern of abnormalities of morphology, proliferation, and apoptosis in the mosaic animal mimicked the regionalized T 121 expression pattern, and conversely, where T 121 protein was absent, the tissue appeared normal. Figure 3 Mammary-Specific Inactivation of the pRb Pathway Induces Extensive Abnormalities Histologic comparisons of nontransgenic (A–D), mosaic (F 0 line 2 [E–H]), and transgenic (F 1 , line 3 [I–L]) lactating mammary glands reveals that T 121 expression results in increased proliferation and apoptosis. Hemotoxylin and eosin staining shows acini of the normal lactating gland are composed of a single layer of secretory epithelial cells (A) with milk-filled lumen. Consistent with atrophy, transgenic animals have a lower density of acini demonstrated by the presence of lipid-filled adipocytes (asterisk in [K]). Acini composed of T 121 -expressing cells are atypical. Many are collapsed and composed of tall columnar epithelia of large hyperchromatic cells with papillary tufting (arrows in [I]). Transgene-expressing cells have large pleomorphic nuclei (open arrows in [G]) as compared to nuclei of nonexpressing cells (arrows in [G]). Staining for T 121 expression (blue in [B]–[J]) indicates the line 2 F 0 animal is mosaic, showing localized expression (F), whereas the transgene expresses throughout the gland of an F 1 line 3 animal (J). Increased proliferation assayed by PCNA staining (red) is also localized in the mosaic founder (G), but found throughout the F 1 transgenic gland (K). Similarly, TUNEL staining (brown) demonstrates increased apoptosis in transgenic animals (H and L); moreover, the regionalized apoptosis in the mosaic gland (H) strongly suggests that transgene expression and not precocious involution is the cause. All samples are from primiparous females on lactation day 1. Figure 4 Reduced p53 Activity Decreases Apoptosis but Does Not Increase Proliferation Representative apoptosis levels of each mouse line correlate with T 121 expression as indicated by the percentage of TUNEL positive cells (A). Decreasing levels of p53 activity correlate with lower levels of apoptosis in transgenic mammary glands (B). The mean percentage of apoptotic cells in p53 wild-type transgenic glands was 21%; in p53 heterozygous animals, 9%; and in p53 null animals, 5% (B), indicating that 75% of the apoptosis is p53-dependent. Apoptosis levels are further reduced to 2% in terminal stage tumors (B, Tumors). The percentage of PCNA staining cells remains unchanged in p53 heterozygous or nullizygous animals (C), indicating that reduction of p53 activity levels had no significant impact on cell proliferation. Samples were derived from primiparous animals on lactation day 1, except as indicated as tumor samples (B). Transgenic animals in (B) and (C) were from line 3. Role of p53 in Apoptosis To investigate the impact of germline loss of p53 on apoptosis levels in Rb f -deficient mammary glands, we mated line 3 animals to p53 null mice to generate transgenic and nontransgenic females of distinct p53 genotypes (+/+, +/−, −/−). Transgene expression was induced by a single pregnancy, and mammary glands were examined on lactation day 1. As expected, nontransgenic mammary glands showed no appreciable apoptosis regardless of p53 status ( Figure 4 B). However, in transgenic animals, decreased levels of p53 activity were correlated with lower levels of apoptosis. The mean percentage of apoptotic cells in p53 wild-type transgenic glands was 21%; in p53 heterozygous animals, 9%;and in p53 null animals, 5% ( Figure 4 B), indicating that 75% of the apoptosis is p53-dependent. That we could detect haploinsufficiency of p53 for apoptosis is remarkable, since in the previously characterized T 121 -expressing choroid plexus epithelium, apoptosis levels were the same in p53 heterozygous and wild-type backgrounds ( Lu et al. 2001 ). This observation indicates that there is a threshold for p53 levels in eliciting apoptosis and that either the threshold is different between cell types or that the absolute functional p53 level is distinct. Such differences could have significant impact on the requirements for tumorigenesis. Role of p53 in Proliferation In two other transgenic mouse models of breast cancer, where tumors were initiated by activated Harvey rat sarcoma viral oncogene homolog (v -Ha-ras ) ( Hundley et al. 1997 ) or wingless -related murine mammary tumor virus (MMTV) integration site 1 ( Wnt-1 ) ( Donehower et al. 1995 ), inactivation of p53 did not result in a reduction of apoptosis; rather, loss of p53 was associated with increased proliferation of the mammary epithelium. To determine whether p53 inactivation also impacted mammary cell proliferation induced by Rb f inactivation, glands from primiparous lactating (day 1) mice were assessed for the expression of nuclear PCNA. Unlike the tumors initiated by activated Ras or Wnt-1 , p53 heterozygosity or nullizygosity had no significant impact on the level of cell proliferation ( Figure 4 C). This experiment indicates that p53 can have distinct mechanisms of action depending on the nature of the initiating lesion. pRb Inactivation Predisposes to Tumorigenesis All females from higher-expressing lines (lines 3 and 4) failed to nurse pups because of lactation defects and developed mammary tumors after multiple pregnancies. Because line 4 mice expressed T 121 in nonmammary tissues, further characterization focused on line 3. For this line, the median time following initial transgene induction until a palpable tumor appeared was 10 mo, and within 16 mo, all mice developed palpable tumors ( Figure 5 A). Interestingly, latency in this line on a BALB/cJ background (see Materials and Methods ) was reduced to a median time of 8.5 mo ( p = 0.0077; Figure 5 A) indicating the presence of modifier alleles. The condensed timeframe for tumor development in this strain will also be valuable for future preclinical studies using this model. However, all further studies in the current report were carried out on the original B6D2F1 background. Figure 5 Mammary Tumor Onset and Growth Are Accelerated by p53 Reduction Among line 3 animals, the median time following initial transgene induction until a palpable tumor appeared was 10 mo, and within 16 mo, all mice developed palpable tumors (red line in [A]). In p53 +/− transgenic animals (blue line in [A]), mammary tumors were detected significantly earlier ( p < 0.0003) with a median onset of 6 mo. Among mice with BALB/cJ background (black line in [A]), median mammary tumor latency (8.5 mo) was significantly shorter ( p = 0.0077) compared to mice of the hybrid BDF1 background strain and indistinguishable ( p = 0.2466) from WAP-T 121 ;p53 +/− mice. Once palpable, WAP-T 121 ;p53 +/− tumors grew faster than the p53 wild-type counterparts (B). The average growth rates for p53 +/+ (black solid) and p53 +/− (dashed) are indicated. The median onset for mammary tumors in line 4 was 14 mo ( n = 3; data not shown), which indicates that the transgene and not its insertion caused tumorigenesis. With two exceptions, line 3 WAP-T 121 mice, regardless of p53 status, developed a single palpable tumor (87% of p53 +/+ , n = 15; 78% of p53 +/− , n = 9). A single mouse with either two or three palpable tumors was also observed in both p53 +/+ and +/− backgrounds. At least one additional nonpalpable tumor was visible during necropsy in approximately one-third of all tumor-bearing mice. While the two lower-expressing lines, lines 1 and 2, were able to nurse pups and appeared grossly normal, both had hyperplastic lobular alveoli associated with increased levels of proliferation and apoptosis. However, females from low-expressing lines did not develop adenocarcinomas after at least four pregnancies and 20 mo of age (line 1, n = 2; line 2, n = 6) (data not shown). Most terminal stage tumors in either wild-type or p53 +/− backgrounds were adenocarcinomas ( Figure 6 A, 6 B, and 6 E); however, we also observed four pilar tumors ( Figure 6 C and 6 E) and one spindle cell carcinoma ( Figure 6 D and 6 E). Terminal-stage mammary adenocarcinomas resembled poorly to moderately differentiated invasive ductal adenocarcinoma in humans. Morphologically, we designate these tumors as mixed solid and glandular carcinomas with necrosis and fibrosis. Poorly differentiated solid tumors ( Figure 6 A) are composed of nests of epithelial cells with large pleomorphic nuclei and delicate chromatin patterns with inverted nuclear:cytoplasmic ratios, while glandular tumors ( Figure 6 B) are composed of irregular glands with varying degrees of differentiation. While most animals had a single tumor mass, the adenocarcinomas were multifocal, with solid tumors consisting of subclones of distinct expansile masses, and with only two exceptions, glandular tumors were coincident with solid tumors. The adenocarcinomas were malignant, infiltrating dense, fibrous connective tissue, and were accompanied by strong peripheral immune response ( Figure 6 A). Figure 6 Tumor Morphologies Hemotoxylin and eosin staining of WAP-T 121 (C and D) and WAP-T 121 p53 +/− (A and B) (also representative of WAP-T 121 ) tumor sections shows that terminal stage adenocarcinomas have varied morphologies. Poorly differentiated solid tumors were comprised of nests (A) or cords of epithelial cells (Tu) that infiltrate a fibrous stroma and were accompanied by necrosis (arrow) and strong immune response (arrowheads). Moderately differentiated glandular tumors (B) consisted of irregular, disorganized glands. In animals of wild-type p53 background, four pilar tumors (C), distinguished by swirls of laminar acellular keratin (arrow), and a single spindle cell carcinoma (D) were also observed. For comparison, a lactating gland from a wild-type animal is shown in Figure 3 A. The percentage of animals displaying each of the phenotypes is summarized in (G). Since many tumors shared multiple morphologies, the sum exceeds 100%. Mammary Tumor Onset and Growth Are Accelerated by p53 Reduction Since 75% of the apoptosis induced by Rb f inactivation was mediated by p53 and was indeed reduced even in p53 +/− mice, we investigated the impact of p53 loss on tumor onset and growth kinetics. Animals harboring either one or two p53 null alleles were monitored for mammary tumors. As expected, a subset of p53 +/− and p53 −/− mice developed nonmammary tumors (either thymic lymphomas or sarcomas), consistent with published reports ( Jacks et al. 1994 ; Sandgren et al. 1995 ; Dannenberg et al. 2000 ). All p53 −/− mice ( n = 4) succumbed to these tumors by 4 mo of age, prior to developing palpable mammary tumors, so acceleration of this phenotype could not be assessed. In p53 +/− animals, mammary tumors were detected significantly earlier (see Figure 5 A; p = 0.0003) compared with p53 +/+ mice. Furthermore, once palpable, WAP-T 121 ;p53 +/− tumors grew significantly faster than the p53 wild-type counterparts (see Figure 5 B). The observation of four pilar tumors in p53 +/+ animals and none in p53 +/− animals is a statistically significant difference (Fisher–Freeman–Halton's exact test, p = 0.0177) and suggests that the reduction of p53 activity drives tumors to the adenocarcinoma phenotype. Taken together, these studies indicate that p53 heterozygosity leads to increased tumor growth rates and/or progression and may alter the spectrum of tumor morphologies. Selective Pressure for p53 Inactivation during Adenocarcinoma Development Since apoptosis was significantly reduced in WAP-T 121 ;p53 +/− mammary tissue compared with that of WAP-T 121 ;p53 +/+ mice, it was possible that p53 heterozygosity was sufficient for tumor acceleration. To assess whether this was the case or whether there was selective pressure for p53 inactivation during tumor progression, real-time PCR analysis was employed to determine the status of the wild-type p53 allele in WAP-T 121 ;p53 +/− tumors. Of ten tumors, eight showed loss of the wild-type p53 allele ( Table 1 ), indicating that the apoptosis reduction observed in WAP-T 121 ;p53 +/− mammary epithelium was not sufficient for tumor progression. Significant selective pressure favored cells that had completely inactivated p53, indicating that tumor progression requires further reduction of apoptotic activity and/or that p53 loss contributes to tumor progression through additional mechanisms that confer selective advantage. Assessment of apoptosis levels in terminal tumors showed apoptosis levels were indeed reduced in comparison to preneoplastic tissue (see Figure 4 B). Table 1 p53 LOH among the Majority of p53 +/− Tumors Real-time PCR was performed in duplicate to determine the status of the wild-type p53 alleles in the mammary tumors or tissues as indicated. Analysis of standard samples indicates that copy numbers of 2, 1, and 0 are indicated by 2 -ΔΔCt values of greater than or equal to 0.7, 0.2–0.7, and less than 0.2, respectively ( Lu et al. 2001 ). Of ten WAP-T 121 ;p53 +/− tumors, eight show LOH of p53 gene, while all three WAP-T 121 ;p53 +/+ tumors retained both p53 alleles. Abbreviation: Tg, transgenic a Tumor samples were derived from line 3 animals, except tumor 1, which was derived from a line 4 animal b ΔΔC t = [sample C t ( p53 ) − sample C t ( β-actin ) ] − [ p53 +/+ control Ct ( p53 ) − p53 +/+ control C t ( β-actin ) ]. C t = the number of cycles required to reach a threshold value, which is set within the exponential phase of the logarithmic scale amplification plot Comparative Genomic Hybridization Reveals Recurrent Chromosomal Imbalances in Tumors, but Limited Chromosomal Instability Among the multiple mechanisms of tumor suppression attributed to p53, a common hypothesis is that p53 prevents genetic instability. Indeed, studies using other mouse models indicate loss of p53 function in tumors often correlates with chromosomal instability. These include other breast cancer models such as Wnt-1p53 +/− ( Donehower et al. 1995 ) and MMTV-ras p53 +/− ( Hundley et al. 1997 ) and p53 +/− thymic lymphomas and sarcomas ( Venkatachalam et al. 1998 ). In marked contrast, our study of p53 deficiency in an evolving brain epithelial tumor showed that tumorigenesis progresses without chromosomal instability, indicating p53 loss contributes via alternative mechanisms ( Lu et al. 2001 ). To determine whether this difference was due to cell-type specificity, differences in initiating mechanisms, or differences in experimental approaches, we analyzed the genome of mammary WAP-T 121 ;p53 +/− tumors. We employed two methods of comparative genomic hybridization (CGH): chromosome-based CGH (cCGH) (Panel I in Figure 7 ) ( Kallioniemi et al. 1992 ) and microarray CGH (aCGH) (Panel II in Figure 7 ) ( Solinas-Toldo et al. 1997 ; Pinkel et al. 1998 ). Figure 7 CGH Analysis Shows Limited Genomic Instability Twelve tumors were analyzed by CGH: ten by cCGH (Panel I, A–J), eight by aCGH (Panel II, B, C, E, and H–L), and six by both procedures (Panels I and II, B, C, E, and H–J). In Panel I, green and red lines adjacent to the ideograms indicate relative gain or loss, respectively. Tumor sample identities are indicated by letters above gain and loss lines. Only a single sample (Panel I, D) shows loss of Chromosome 11. Telomeric sequences of many chromosomes are increased, most frequently Chromosomes 5 and 15. Recurrent losses are seen on Chromosomes 10 and X. For aCGH (Panel II), the genomic map is depicted with chromosomes horizontally aligned centromere to telomere. The relative fluorescence intensities (tumor:normal) are indicated along the vertical axis. Individual BACs are plotted according to their physical map position versus relative fluorescence, with sample identities indicated by a unique symbol for each tumor. To simplify visualization, only BACs with relative intensities greater than 1.25 (gains) or less than 0.75 (losses) are shown. X Chromosome values were halved to account for sex-mismatched samples. Changes spanning the entire length of the chromosome are readily detected on Chromosomes 6, 8, 10, 15, 18, and X. None of the clones showing loss on Chromosome 11 spans the p53 locus. The original p53 background of the animal and the p53 LOH status of each tumor are also indicated in the legend. Twelve mammary tumors were assayed by CGH: ten by cCGH, eight by aCGH, and six by both procedures. Both assays revealed limited genomic imbalances ( Figure 7 ), yet only a single tumor showed loss of Chromosome 11 (which harbors p53 ). Among samples tested by both methods, there is strong concordance among large chromosomal changes, encompassing multiple cytological bands to whole chromosome lengths. For example, there is apparent whole chromosomal duplication of Chromosomes 6 and 15 in tumor C and of Chromosomes 8 and 18 in tumor H, monosomy of Chromosome 10 in tumor J, and loss of X Chromosome in tumors E and H (all or partial, respectively). Making comparisons among imbalances spanning shorter chromosome lengths was more difficult, mainly due to the challenge of reconciling cytological and physical maps. Furthermore, technical limitations may account for real differences between the two assays: small imbalances detected by one to several bacterial artificial chromosome (BAC) clones are irresolvable by cCGH; on the other hand, the relatively low density of BAC clones may not adequately sample smaller regions detected by cCGH. Nevertheless, on average, about five imbalances per tumor were detected by cCGH. This number is comparable to the number of changes observed in myelocytomatosis oncogene (c -myc )-induced mouse mammary tumors ( Weaver et al. 1999 ) and human tumors ( Ried et al. 1995 ), yet less than the number of changes seen in breast cancer 1 ( Brca1 )-deficient mouse tumors (8.0) and more than v- erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER2/ neu )-induced tumors (2.8) ( Montagna et al. 2002 ; Weaver et al. 2002 ). Discussion Common Mechanisms for Tumor Progression in Epithelial Cells of Distinct Origin Here we report that loss of pRb family function in mammary epithelium predisposes to malignant adenocarcinoma. Using a single transgenic allele, we have thus far inactivated the pRb pathway in several cell types in the mouse: brain choroid plexus epithelium, astrocytes, and mammary epithelium. In each case, despite the marked differences among these divergent cell types, pRb inactivation causes a similar response, initially evoking increased proliferation and apoptosis and, ultimately, predisposing to tumorigenesis ( Chen et al. 1992 ; Saenz-Robles et al. 1994 ; Symonds et al. 1994 ; Xiao et al. 2002 ). Not surprisingly, the long latency of mammary adenocarcinomas indicates that additional events are required for tumor progression. We show that mammary epithelium is similar to brain epithelium ( Symonds et al. 1994 ; Lu et al. 2001 ) in its requirement for p53 activity in the apoptotic response to aberrant proliferation caused by pRb f inactivation. Previous models using wild-type large T antigen ( Li et al. 1996b ; Husler et al. 1998 ; Green et al. 2000 ; Schulze-Garg et al. 2000 ) are unable to address the relative contribution of pRb and p53, since T antigen also binds and inactivates p53. As in brain epithelium, we show here that when the mammary tumor phenotype is initiated by pRb f inactivation, most of the apoptosis is mediated through p53. Furthermore, as in brain epithelium, heterozygosity for a null p53 allele significantly shortens tumor latency (discussed further below). Importantly, the Rb f deficiency-induced apoptotic response and inhibition of tumor progression are not universally dependent on p53. In astrocytes, we recently showed that PTEN, and not p53, modulates these same responses to Rb f inactivation. In contrast to the p53-dependent apoptosis of mammary epithelial cells in response to pRb f deficiency, apoptosis associated with normal mammary involution subsequent to lactation does not require p53 ( Li et al. 1996a ). Thus, the “wiring” of the apoptotic response within this cell type is not global, but rather depends on the signal. Although loss of p53-dependent apoptosis accounts for the acceleration of mammary tumorigenesis in WAP-T 121 ;p53 +/− mice, in models expressing either activated v-Ha-ras ( Hundley et al. 1997 ) or Wnt-1 ( Jones et al. 1997 ), earlier tumor formation in p53 heterozygous and homozygous null mice is accounted for by increased proliferation rather than attenuated apoptosis. An important caveat to this comparison is that the latter studies compared apoptosis in terminal tumors in which loss of apoptosis might have been selected regardless of initial p53 status, leaving open the possibility that tumor growth rates in these models reflect the combined effects of increased proliferation as well as reduced apoptosis. Nevertheless, there is a clear difference in WAP-T 121 mammary gland in that, unlike the Ras and Wnt-1 models, proliferation levels do not depend on p53 status. Taken together, these observations indicate that the specific cellular response to an oncogenic stimulus depends on the nature of the initial insult. Given that the pRb pathway is directly disrupted in T 121 -expressing cells, this could be explained if these other initiating events evoke p53-dependent growth arrest which, in part, functions upstream of pRb. High Selective Pressure for p53 Inactivation in the Transition to Aggressive Mammary Adenocarcinoma Most of the apoptosis induced by pRb f deficiency in both mammary (75%) and brain (85%) epithelia is p53 -dependent as determined by comparing p53 +/+ and p53 −/− tissue. However, while p53 heterozygosity had no impact on the level of apoptosis in the brain epithelium, in the mammary gland the level was reduced by half in p53 +/− tissue. Given that apoptosis is the basis for selective inactivation of p53 in the brain tumor model ( Lu et al. 2001 ; X. Lu and T. Van Dyke, unpublished data), it was possible that the pressure was relieved or reduced in WAP-T 121 ;p53 +/− mice. However, aggressive adenocarcinoma growth was accelerated with 100% penetrance, and 80% of these tumors underwent selective loss of the wild-type p53 allele, just as in the brain tumor model ( Lu et al. 2001 ). This result indicates that tumor progression requires more than a simple reduction in the level of apoptosis; it follows that p53 may contribute to tumor suppression by multiple mechanisms. While both mammary and brain carcinomas show high rates of p53 loss of heterozygosity (LOH), the mechanism of loss may be distinct. Chromosome loss clearly explains p53 LOH in the brain carcinoma model ( Lu et al. 2001 ) where nearly all tumors (greater than 90%) are monosomic for Chromosome 11, whereas only a single mammary tumor analyzed by CGH showed Chromosome 11 loss. Alternative mechanisms that may explain p53 LOH in the mammary tumors include somatic recombination or chromosomal reduplication following mitotic nondisjunction. Whether these alternative routes of LOH represent bona fide tissue-specific phenomena or are due to relatively small sample sizes will require further analyses. Interestingly, most mammary tumors derived from Brca1 -deficient mice lost p53 ; however, regions distal to p53 were amplified ( Weaver et al. 2002 ). Thus, it is possible that mammary tumor promoting factor(s) is located on distal Chromosome 11, selecting against loss. Limited Chromosomal Instability in the Absence of p53 Genomic instability is a hallmark of most human solid tumors, and a widely held view is that p53 represses instability to suppress tumorigenesis, although evidence for this activity has been mostly correlative. Contrary to this model, we demonstrated previously that in the absence of p53 activity in brain epithelia, tumors progress without chromosomal instability; except for Chromosome 11 loss, in a p53 +/− background these carcinomas are diploid ( Lu et al. 2001 ). Here we show that mammary tumors similarly harbor limited genome-wide alterations. While the number of aberrations within the mammary tumors is small, it is intriguing that some changes are recurrent, suggesting that their accrual is causal in tumorigenesis. T 121 -induced mammary carcinomas harbor more genomic imbalances than brain tumors (approximately five versus approximately one). One explanation for this observation is that, because the brain is a vital organ, animals succumb to their illness when the brain tumor is at a relatively earlier stage at which fewer changes have accumulated. However, chromosome content of choroid plexus tumors passaged further in xenografts remained stable (X. Lu and T. Van Dyke, unpublished data). The converse experiment, analyses of early mammary tumors subsequent to p53 loss, will be required to determine the kinetics of chromosomal changes in this tissue. Pocket Protein Redundancy Chimera and tissue-grafting experiments with pRb-deficient cells indicate the absence of pRb alone is not sufficient for abnormal mammary development or tumor formation ( Maandag et al. 1994 ; Robinson et al. 2001 ). Yet mammary-directed overexpression of CCND1, an upstream regulator of pRb f , leads to mammary adenocarcinoma ( Wang et al. 1994 ). Given other recent studies indicating the possibility for compensation of pRb function by p107 and/or p130 ( Dannenberg et al. 2000 ; Sage et al. 2000 ) and the clear redundancy of function in some murine cell types ( Robanus-Maandag et al. 1998 ; Xiao et al. 2002 ), it is likely that the discrepancy among our results can be explained by overlapping functions of other family members, p107 and/or p130. In our studies, T 121 abrogates the activities of all Rb family members by a dominant interfering mechanism. A subtly distinct alternative explanation is that the acute loss of pRb signaling, rather than a chronic loss as of pRb during mammary development, as in the chimera and grafting models, accounts for the difference. Cell culture experiments that support this hypothesis were recently reported ( Sage et al. 2003 ). In this model, p107 and p130 may be more responsive to pRb regulatory signals during development than in the terminally differentiated tissue; therefore, the developing tissue more easily accommodates for the absence of pRb in the pool of available pocket proteins. In the WAP-T 121 model, the gland undergoes normal development and then is subsequently subjected to acute pRb pathway loss. We presume that this scenario more closely mimics the situation of spontaneous somatic loss in adult human breast. The test of this alternative hypothesis awaits analyses of tissue-specific inactivation of pRb and the paralogous pocket proteins using conditional alleles. A Model for Mammary Tumorigenesis Initiated by Targeting the pRb Pathway The WAP-T 121 model is a significant addition to the current repertoire of preclinical mammary tumor models exploring the role of pRb pathway in tumorigenesis. Despite the prevalence of pRb pathway defects in human sporadic cancers, mice harboring germline mutations of p16 INK4a do not develop mammary cancer ( Krimpenfort et al. 2001 ; Sharpless et al. 2001 ). In addition, mammary-directed expression of CCND1 is only mildly oncogenic ( Wang et al. 1994 ), and as mentioned above, inactivation of pRb alone is not sufficient for tumorigenesis. Although the WAP promoter was a convenient means of directing mammary-specific expression for an initial assessment this model, it also presents the major shortcoming to this model in that expression of T 121 is linked to lactogenic hormone activity, as in most existing murine mammary tumor models. Future improvements aim to direct expression of T 121 through hormone-independent methods. Finally, the advantage over wild-type T antigen models is that WAP-T 121 uncouples the simultaneous inactivation of pRb and p53 and permits an assessment of the relative contributions of the individual oncogenic pathways. Testing the combinatorial effects of Rb loss and other breast cancer mutations (e.g., BRCA1 and BRCA2 ), along with the further characterization of WAP-T 121 tumors, should help provide additional insights into human breast cancer biology. Materials and Methods Derivation and characterization of transgenic mice. The 2.4 kb WAP promoter region was isolated from a WAP-TGFα construct (a gift from David Lee, University of North Carolina at Chapel Hill, United States [ Sandgren et al. 1995 ]) and was cloned upstream of a 2.4 kb KpnI–SalI fragment of the dl 1137′t plasmid ( Chen et al. 1992 ). We targeted T 121 expression to mammary gland using the WAP promoter, which is induced late in pregnancy and expressed during lactation ( Pittius et al. 1988 ) (see Figure 1 ). T 121 contains the first 121 amino acids of the SV40 T antigen (see Figure 1 ) that encodes a J domain and a pRb-binding domain, which together are sufficient to cause transformation by inactivating the pRb f proteins ( DeCaprio et al. 1989 ; Dyson et al. 1989 ; Ewen et al. 1989 ). Importantly, in contrast to other wild-type T antigen constructs encoding the entire SV40 early region ( Husler et al. 1998 ; Green et al. 2000 ; Schulze-Garg et al. 2000 ), small t antigen expression is absent due to a deletion that removes the splice acceptor site. The importance of this is demonstrated by the recent observation that small t antigen alone is sufficient for tumorigenesis in the mammary gland ( Goetz et al. 2001 ). Furthermore, p53 and EP300 (E1A-binding protein p300), which map to the carboxyl half of T antigen, are also abolished, thus permitting assessment of pRb f inactivation without the confounding effects of altering additional suppressor pathways. An EcoRI fragment containing the full transgene (see Figure 1 ) at a concentration of 4 ng/μl was injected in to fertilized eggs harvested from B6D2F1 (Jackson Laboratory, Bar Harbor, Maine, United States) mice as described previously ( Yan et al. 1990 ). Transgenic mice were identified by PCR amplification of a 160 bp fragment using primers 5′-GAATCTTTGCAGCTAATGGACC-3′ and 5′-GCATCCCAGAAGCTCCAAAG-3′ with toe-derived genomic DNA as template. Cycling profile was as follows: 94°C, 2 min; 35 cycles of 94°C, 20 s; 62°C, 45 s; 72°C, 45 s; and final incubation of 72°C, 2 min. TgWAP-T 121 mouse lines were maintained by crossing to nontransgenic B6D2F1 mice (Jackson Laboratory) and therefore are designated as B6;D2-Tg(WAP-T 121 ) Tvd. To study the effect of background differences, WAP-T 121 males were backcrossed to BALB/cJ (Jackson Laboratory) female mice. To increase sample size, tumor onset analysis for BALB/c background mice combined data for N6 ( n = 6), N7 ( n = 1), and N9 ( n = 4) generation mice. For tumor induction, female mice, unless noted as virgin, were housed with male mice to maximize the number of pregnancies, because WAP promoter activity is lactation-dependent ( Pittius et al. 1988 ). To study the effect of p53 mutation on mammary tumorigenesis in WAP-T 121 mice, male WAP-T 121 + mice were mated to p53 +/− females ( p53 tm1Tyj ; Jackson Laboratory). p53 genotypes were determined by PCR using two reactions ( Lowe et al. 1993 ), one that amplifies the neomycin insertion site (neomycin primer: 5′- TCCTCGTGCTTTACGGTATC-3′, p53 primer: 5′-TATACTCAGAGCCGGCCT-3′; 525 bp product) and a second that amplifies the endogenous p53 allele (substituting 5′-ACAGCGTGGTGGTACCTTAT-3′ for the neo primer, 475 bp product). Cycling parameters were the same as the above WAP-T 121 reaction. We performed the cross WAP-T 121 − ;p53 +/− × WAP-T 121 + ;p53 +/− , and transgenic female mice that were p53 +/+ , p53 +/− , or p53 −/− were used for analyses while nontransgenic littermates served as controls. Western immunoblotting analysis. Protein expression levels were assayed as previously described ( Symonds et al. 1993 ). Fresh or flash-frozen tissue samples were homogenized in lysis buffer (50 mM Tris [pH 8.0], 5 mM EDTA, 150 mM NaCl , and 1% NP-40) using a Polytron ® homogenizer (Kinematica, Littau-Lucerne, Switzerland). Total protein (10 μg) was electrophoresed through a 15% polyacrylamide denaturing gel and then transferred to nitrocellulose membrane (15 V, 30 min). Alternatively, for low-expressing lines, immunoprecipitation was performed prior to electrophoresis as previously described ( Symonds et al. 1991 ). The filter was preincubated in 3% bovine serum albumin, followed by incubation with primary antibody against SV40 T antigen (PAb419 at a dilution of 1:5,000; Harlow et al. 1981 ). The filter was then washed, followed by incubation at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG (Amersham Biosciences, Little Chalfont, United Kingdom). The enhanced chemiluminescence method (Amersham Biosciences) was used for autoradiography. Histopathology and immunohistochemistry. Mammary tissue and tumor samples were dissected from WAP-T 121 transgenic or age- and parity-matched B6D2F1 animals. A portion of each tumor was flash-frozen in liquid nitrogen and the remaining tissue was fixed in 10% phosphate buffered formalin, embedded in paraffin, cut to a 5-μm thickness, and stained with hemotoxylin and eosin or immunostained using the Vector ABC system (Vector Laboratories, Burlingame, California, United States) for histopathological examination. Apoptosis levels were evaluated by TUNEL assay ( Gavrieli et al. 1992 ) essentially as described in Symonds et al. (1994 ). Real-time PCR. Quantitative real-time PCR analysis was performed using a TaqMan approach on DNA derived from terminal tumors to determine the status of the wild-type p53 allele as previously described ( Lu et al. 2001 ). The primers for the p53 allele were 5′-ATGGCCATCTACAAGAAGTCACAG-3′ and 5′-ATCGGAGCAGCGCTCATG-3′. The sequence of the p53 probe was 5′-ACATGACGGAGGTCGTGAGACGCTG-3′. The primers for the internal control β-actin gene were 5′-AAGAGCTATGAGCTGCCTGA-3′ and 5′-ACGGATGTCAACGTCACACT-3′. The sequence of the β-actin probe was 5′-CACTATTGGCAACGAGCGGTTCCG-3′. Each 25-μl reaction mixture contained 50 ng of DNA template, 18 nM p53 primers, 80 nM β-actin primers, 8 nM probe, and 12.5 μl of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, California, United States) containing AmpliTaq Gold polymerase, deoxynucleoside triphosphates, and PCR buffer. The cycling conditions were 50°C for 2 min and 95°C for 10 min for 1 cycle, and 95°C for 15 s and 60°C for 1 min for 40 cycles. The reactions were performed using an ABI 7700 Sequence Detection system (Applied Biosystems), and the data analyzed using Sequence Detector 1.7 (Applied Biosystems) and standard protocols ( http://www.appliedbiosystems.com ). The copy number of each sample was determined by calculating ΔΔCt based on the formula ΔΔC t = [sample C t(p53) − sample C t( β-actin ) ] − [p53 +/+ control Ct (p53) − p53 +/+ control Ct ( β-actin ) ], where C t is the number of cycles required to reach a threshold based on linear amplification. Analyses of standard samples (L. Chin, Harvard University, Cambridge, Massachusetts, United States, personal communication) indicate copy numbers of 2, 1, and 0 are indicated by 2 -ΔCtn values of greater than 0.6, 0.15–0.6, and less than 0.15, respectively. Standard samples analyzed along with experimental samples confirmed the accuracy of these assignments. Statistical analyses. Kaplan–Meier survival analysis was used to determine median tumor latencies (StatsDirect, Camcode, Sale, United Kingdom), and the Log-Rank (Peto, StatsDirect) test was performed to evaluate significance. The equivalence of tumor morphology distributions was tested using the Fisher–Freeman–Halton's exact test. CGH. Genomic DNA was extracted from end-stage tumors (1 cm in diameter) or tails using a DNeasy genomic tip (Qiagen, Valencia, California, United States) and further purified by proteinase K digestion followed by phenol/chloroform extraction, ethanol precipitation, and resuspension in sterile H 2 O. cCGH was performed as described in Kallioniemi et al. (1992 ), Donehower et al. (1995 ), and Lu et al. (2001 ). aCGH was performed as described in Snijders et al. (2001 ). For both methods, genomic DNA from tumor and normal tissue was labeled with different fluorochromes and then cohybridized together with Cot-1 DNA to either normal metaphase chromosomes from cultured cells (cCGH) or microarrayed BAC clones containing mouse genomic DNA (aCGH). Nonequivalent fluorescence intensities indicate relative imbalances of genomic DNA. Aneuploidy and partial chromosome gains and losses are detectable by cCGH with approximately 10 Mb resolution. Graphical output of cCGH data was generated using the National Cancer Institute and National Center for Biotechnology Information Spectral Karyotyping SKY and Comparative Genomic Hybridization CGH Database ( http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi ). For aCGH, approximately 1,500 BAC clones span the entire mouse genome with 2–20 Mb spacing. Tumor DNA and normal DNA were sex-mismatched; thus, the X Chromosome served as an internal control, while normal tail DNA was used as a negative control. Gains or losses were scored based on tumor:normal fluorescence ratios that were greater than 1.25 or less than 0.75, respectively. Supporting Information Accession Numbers The accession numbers for the genes and gene products discussed in this paper are Brca1 (LocusLink ID 12189), CDK2 (LocusLink ID 1017), CDK4 (LocusLink ID 1019), CDK6 (LocusLink ID 1021), c -myc (LocusLink ID 17869), cyclin D1 (LocusLink ID 595), cyclin E (LocusLink ID 898), E2F (InterPro ID IPR003316), HER2/ neu (LocusLink ID 13866), histone deacetylase (LocusLink ID 3065), p16 INK4a (LocusLink ID 1029), p53 (LocusLink ID 7157), p107 (LocusLink ID 5933), p130 (LocusLink ID 5934), p300 (LocusLink ID 2033), PCNA (LocusLink ID 18538), pRb (LocusLink ID 5925), PTEN (LocusLink ID 5728), v -Ha-ras (LocusLink ID 3265), WAP (LocusLink ID 22373), and Wnt-1 (LocusLink ID 22408). These databases may be found at www.ncbi.nlm.nih.gov/LocusLink/ (LocusLink), and www.ebi.ac.uk/InterPro/ (InterPro).	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC340938.xml
212688	Functional Analysis of RSS Spacers	null	Based on sheer numbers, microbes should rule the world. Most don't cause disease, but those that do have the advantage of multiplying and mutating at a much faster rate than any multicellular organism can. So how does a slowly reproducing, trillion-celled organism like a human protect itself? By having the right weapon for the job—and that requires an incredibly diverse arsenal. A new study by a team of researchers from Yale University School of Medicine, Duke University Medical Center, and Mount Sinai School of Medicine demonstrates how the creation of that arsenal depends on a complex series of interactions between key genetic elements and proteins during the formation of the white blood cells called lymphocytes. Two heavy hitters of the immune system—B and T cells—each produce unique protein receptors that specifically recognize and mediate the killing of the variety of potential foreign invaders, or antigens, such as bacteria, viruses, and parasites. (B cells make immunoglobulin, or antibodies, and T cells make T-cell receptors.) But these lymphocytes are unlike other cells: instead of making proteins from genes they inherited, they custom-make their genes by recombining fragments of their genes into new configurations. This genetic reshuffling process, called V(D)J recombination, yields the diversity of molecules necessary to combat the billions of different antigens they might encounter. The V, D, and J refer to different clusters of DNA sequences that follow specific rules of recombination. While the products of recombination vary, the method does not. The fragments are spliced and then reassembled in a highly regulated process directed and controlled by a stretch of DNA (called a recombination signal sequence, or RSS) next to the gene fragment. The recombination process, the researchers show, relies on complex interactions among different parts of the signal sequences and the proteins that regulate them at key steps along the recombination pathway. Each RSS is made up of three components: the nonamer, which controls the ability of proteins to bind to the gene fragments and initiate recombination; the heptamer, which directs the splicing of the gene fragment; and the spacer, which regulates how the gene fragments are recombined. Mutations in the DNA sequence of each of the three RSS components show that all play a critical role in the ability of the gene fragments to recombine appropriately. While it has been established that spacers, as their name suggests, ensure that the space between the nonamer and heptamer is correct, the researchers show that spacers also regulate recombination activity by providing protein-binding sites along the DNA sequences that affect recombination. While the nonamer is the most important determinant of recombination, changes in the spacer, these researchers demonstrate, produced dramatic changes in the ability of the gene fragments to recombine. Past studies have shown that recombination depends on the presence and sequence of specific nucleotides, but the quality of that recombination, the researchers say, can't be understood simply by analyzing those nucleotides in isolation. Generally speaking, highly conserved sequences have functional importance. But it would be a mistake, they suggest, to think that just because a nucleotide sequence isn't highly conserved, it's not biologically important. Using a computer model to predict how different protein-gene interactions affect recombination, the researchers demonstrate that a fuller understanding of the process depends on observing how all these elements—including those that aren't highly conserved—interact throughout the recombination process.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC212688.xml
545212	Volume Status in Severe Malaria: No Evidence Provided for the Degree of Filling of the Intravascular Compartment	null	The study by Planche et al. [1] provides important new information addressing intracellular volume depletion in children with severe childhood malaria, but does not address the question of whether intravascular volume depletion (hypovolemic shock) is present. Using sophisticated methodology to determine total body water and extracellular water, they demonstrate a 6.7% deficit in total body water and an 11.7% deficit of intracellular water, providing an important indication of the volumes of fluid that may be required to optimize hydration. The data, however, do not address the degree of filling of the intravascular compartment, nor should they be used to answer the question about the state of tissue and organ perfusion. Indeed, we believe that these new data present no conflict with our previously reported findings. Using methods to study critical illness physiology that are widely employed within pediatric intensive care units for interpretation of circulatory status, we have demonstrated evidence for hypovolemia in 53 Kenyan children with severe malaria complicated by metabolic acidosis [2] . Our children were younger, had longer capillary refilling times (>3 s), lower central venous pressures (mean 2.9 cm H 2 O) and higher creatinines (>80 µmol/l): all features of compensated hypovolemic shock. Furthermore, hypotension (systolic BP < 80 mm Hg) was present in 44% of children with severe acidosis (base deficit >15). These findings also indicate important baseline differences in two cohorts of children studied. We agree that reconsideration of guidelines for acute fluid management is warranted, particularly when current recommendations await an adequate evidence base. Nevertheless, conflicting opinions on the question of volume status in children with severe malaria can be satisfactorily resolved only through prospective randomized trials that include both fluid resuscitation and control groups. While the design and conduct of such trials will involve considerable challenges, optimal fluid management will never be resolved on the basis of theoretical consideration alone.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545212.xml
523829	New Drugs for Neglected Diseases: From Pipeline to Patients	The Drugs for Neglected Diseases Initiative is a new, public- sector organization dedicated to drug discovery	In wealthy countries, state-funded research has yielded breakthroughs in molecular biology, chemistry, and engineering. These advances have been taken up by the pharmaceutical industry and applied to drug development for a growing range of illnesses and conditions. As a result, patients have access to new drugs that are better tolerated, more specific, and more effective than old ones. In poor countries, however, millions of people have yet to experience the benefits wrought by science. The deadly infectious diseases that plague them, such as sleeping sickness, Chagas disease, and visceral leishmaniasis, fail to arouse the interest of drug developers. The Drugs for Neglected Diseases Initiative (DNDi) is a new, not-for-profit organisation set up to correct this fatal imbalance by developing new drugs for these forgotten patients. Dropped off the Radar Screen Most of the drugs still used to treat ‘neglected diseases’ were developed in colonial times. These are often expensive, difficult to administer, and hard to tolerate; several of them are also becoming ineffective because of increasing parasite resistance. Very few new alternatives have been developed in the past decades: between 1975 and 1999, 1,393 new drugs were made available to the public, but only 16 of these were meant for neglected diseases [ 1 ]. What makes the lack of drugs more difficult to accept is that scientists know an enormous amount about kinetoplastids, the organisms responsible for sleeping sickness, Chagas disease, and leishmaniasis [ 2 ]. The wealth of knowledge generated in this field could easily be used for drug development if the treatment of neglected diseases were perceived as financially attractive. But populations affected by neglected diseases have no purchasing power, so there is no financial incentive for drug companies to develop the drugs. The basic mechanics of the market-driven system are failing to help these populations. So most scientific research stops at the publication stage or falls through the gaps at different stages of the drug development pipeline ( Figure 1 ) [ 3 ]. Figure 1 The Drug Development Pipeline Because of the gaps in the development pipeline, potential new drugs for neglected diseases often stay stuck at an early stage of development. (Photos: World Health Organization/P.Virot and World Health Organization/Eric Miller) Whose Role Is It, Anyway? It is dangerous to oversimplify the causes of this situation. What share of responsibility for the world's health is borne by the pharmaceutical industry, which has the know-how and the resources for innovation? Aren't international organisations also partly responsible? After all, they are the ones who allocate major funding for health programmes and encourage research programmes. And what about public research institutions in rich countries that generate the knowledge used by industry? Governments have the power to influence their research priorities and drug development decisions, either through funding or direct involvement. Unfortunately drugs for neglected diseases are low priority for governments [ 4 ]. They tend to prioritise research with potential commercial applications instead. Responses to the Crisis All is not doom and gloom. In the past few years, there has been some movement on research and development (R&D) for neglected diseases. Despite its broad mandate and limited resources, the Special Programme for Research and Training in Tropical Diseases (TDR)—established and funded by the World Health Organization, the World Bank, and the United Nations Development Programme—can be credited with several important successes in the fight against malaria and leishmaniasis. The Medicines for Malaria Venture and the Global Alliance for TB Drug Development were set up as public–private partnerships to tackle malaria and tuberculosis. These partnerships were made possible by the fact that malaria and tuberculosis are global diseases, affecting patients in the North and South, so there was enough of a market to persuade industry to develop new drugs for these diseases. A different solution, however, was needed for diseases that are limited to tropical countries, are of no military or strategic interest to wealthy countries, and are not supported by markets or patients' organisations capable of attracting the attention of politicians. This is the kind of solution put forward by the DNDi. A Collaborative Not-for-Profit DNDi is a not-for-profit organisation designed to mobilise resources for R&D of new drugs for neglected diseases. Many people and organisations around the world share an ambition to redress the lack of new treatments for neglected diseases, and bring the benefits of science to forgotten patients. Several of them came together to create DNDi: one humanitarian organisation—Médecins Sans Frontières; five research institutions—the Oswaldo Cruz Foundation from Brazil, the Indian Council for Medical Research, the Kenya Medical Research Institute, the Ministry of Health Malaysia, and the Pasteur Institute from France; and the TDR ( Box 1 ). Box 1. From Pipeline to Patients—Some Key Organizations DNDi: http://www.dndi.org TDR: http://www.who.int/tdr Medicines for Malaria Venture: http://www.mmv.org Global Alliance for TB Drug Development: http://www.tballiance.org Oswaldo Cruz Foundation: http://www.fiocruz.br Indian Council for Medical Research: http://icmr.nic.in/home.htm Kenya Medical Research Institute: http://www.kemri.org Ministry of Health Malaysia: http://dph.gov.my/ Pasteur Institute: http://www.pasteur.fr/externe Médecins Sans Frontières: http://www.msf.org The initiative is a virtual organisation with a growing network of academic and R&D expertise at its disposal. The different players involved in DNDi are bringing their knowhow in parasitology and clinical trials, their experience treating neglected patients, and their drug manufacturing capacity. They are pooling these resources to move drugs stuck in the pipeline all the way to the patients themselves. Pharmaceutical companies have a particularly important role to play: they possess vast repositories of molecules, the means to move from development to industrial production, and highly specialised teams of researchers. Their contribution will be crucial to the success of DNDi. Matching Needs and Opportunities DNDi is a needs-driven initiative—in other words, the needs of patients suffering from neglected diseases are paramount in its search for new drugs to treat them. The organisations that make up DNDi have firsthand knowledge of these needs because they work with patients in disease-endemic countries throughout the developing world. The initiative is taking this knowledge of patient needs, matching it with opportunities in R&D, and pushing the most relevant projects through the pipeline. Ultimately, neglected patients will have access to drugs targeting their specific diseases, drugs that were designed with them specifically in mind—such as short-course, low-toxicity treatments that don't require hospitalisation, or tablets to swallow rather than injections. To identify opportunities in R&D that are both relevant to patient needs and that meet required criteria of scientific merit, DNDi is sending out calls for letters of interest to the scientific community via advertisements in journals and posted on the DNDi website ( http://www.dndi.org ). These have already pinpointed several promising projects. DNDi is also proactively contacting scientists working on infectious diseases, and surveying published literature for research of interest. In the Pipeline DNDi's project portfolio currently holds nine projects at different stages of development to address identified needs for the treatment of visceral leishmaniasis, sleeping sickness ( Box 2 ), Chagas disease, and malaria ( Figure 2 ). At discovery stage, DNDi is working on validating the kinetoplastid enzyme dihydrofolate reductase as a potential target for leishmaniasis, trypanosomiasis, and Chagas disease, and on identifying inhibitors of the kinetoplastid enzymes trypanothione reductase and protein farnesyltransferase. It is also conducting high throughput screening on whole cell trypanosomes to discover novel lead compounds. Box 2. New Drugs for Sleeping Sickness Only a few drugs exist to treat sleeping sickness, and they are toxic or difficult to administer. Melarsoprol kills one in 20 patients. Eflornithine requires four daily infusions over 14 days. Given these limited options, DNDi is focusing on identifying new compounds that can cross the blood–brain barrier to treat second stage sleeping sickness. DNDi is using high throughput screening on whole cell trypanosomes to discover novel lead compounds, and is working to identify and optimise inhibitors of the enzyme protein farnesyltransferase. The initiative is working on validating the kinetoplastid enzyme dihydrofolate reductase as a drug target. Identifying trypanothione inhibitors is also relevant to other trypanosome parasites. These are long term projects. Nifurtimox, a drug for Chagas disease, has been used to treat sleeping sickness since the 1970s in some isolated places. It has never been extended to more people because no one has studied its safety or effectiveness. DNDi will assess its short-term usefulness by conducting clinical trials on a treatment combination of eflornithine and nifurtimox. DNDi will continue to explore other short- and medium-term projects. Figure 2 DNDi Projects DNDi's project portfolio contains nine projects spread out across the drug development pipeline for the treatment of leishmaniasis, sleeping sickness, Chagas disease, and malaria. HAT, human African trypanosomiasis (sleeping sickness); VL, visceral leishmaniasis. The R&D of new drugs is time-consuming and expensive if the process starts at the early discovery stage, because of the associated risk of project attrition along the way. DNDi is therefore investing resources in several pre-development and development projects as well. These include developing fixed dose combinations of artesunate/amodiaquine and artesunate/mefloquine for use against chloroquine-resistant malaria in Africa and Asia, respectively; pushing for registration of paromomycin for use against visceral leishmaniasis in Africa; assessing combinations of existing drugs for visceral leishmaniasis; and evaluating the usefulness of nifurtimox in combination with eflornithine in the treatment of sleeping sickness. Advocacy for Change Governments can—some might say should—influence drug development choices. DNDi strongly believes that governments in both developed and developing countries should take an active interest in the R&D of new drugs for neglected diseases. In parallel to its own drug development activities, DNDi is working to raise awareness of the neglected disease crisis among key policy- and decision-makers, for instance the European Commission and the National Institutes for Health in the United States. Conclusion In the poorer countries in the world, over 350 million people are at risk from neglected diseases. Currently available treatments are inadequate or nonexistent, and new solutions are urgently needed. DNDi is working to ensure that the advances of science that have brought health and comfort to wealthy nations also benefit these neglected populations.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC523829.xml
555954	Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID): Frequently asked questions	The exponential development of Patient-Reported Outcomes (PRO) measures in clinical research has led to the creation of the Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID) to facilitate the selection process of PRO measures in clinical research. The project was initiated by Mapi Research Trust in Lyon, France. Initially called QOLID (Quality of Life Instruments Database), the project's purpose was to provide all those involved in health care evaluation with a comprehensive and unique source of information on PRO and HRQOL measures available through the Internet. PROQOLID currently describes more than 470 PRO instruments in a structured format. It is available in two levels, non-subscribers and subscribers, at . The first level is free of charge and contains 14 categories of basic useful information on the instruments (e.g. author, objective, original language, list of existing translations, etc.). The second level provides significantly more information about the instruments. It includes review copies of over 350 original instruments, 120 user manuals and 350 translations. Most are available in PDF format. This level is only accessible to annual subscribers. PROQOLID is updated in close collaboration with the instruments' authors on a regular basis. Fifty or more new instruments are added to the database annually. Today, all of the major pharmaceutical companies, prestigious institutions (such as the FDA, the NIH's National Cancer Institute, the U.S. Veterans Administration), dozens of universities, public institutions and researchers subscribe to PROQOLID on a yearly basis. More than 800 users per day routinely visit the database.	Review In clinical research it has become increasingly common to assess the patients' perspective of their symptoms and their impact on their daily life as a tool for determining treatment and a means of evaluating the outcome of the treatment chosen [ 1 , 2 ]. The added value of measuring Patient-Reported Outcomes (PRO) starts to be recognized by key players in the field of clinical research [ 3 ]. How patients perceive their health, and the impact of their treatment on their life can provide insight to clinicians previously unavailable [ 4 - 6 ]. However the successful application of PRO studies is dependant on the selection of the appropriate questionnaires for a given application [ 7 , 8 ]. They must be selected according to the domains they measure and the populations and pathologies for which they are designed. Practical issues, such as the availability of different translations, copyrights, and access to instruments are also major criteria in the choice of instruments. The search for the most appropriate instruments is hindered by the substantial increase of PRO questionnaires developed in the past ten years. A recent search on PubMed, matching "quality of life" and "questionnaires" shows a striking growth of 450% between the last two decades (Figure 1 ). Figure 1 PubMed Search: Number of references with Quality of Life-AND-questionnaire from 1966 to 2004 In order to facilitate the selection process the project of a Patient-Reported Outcome and Quality of Life Instruments Database (PROQOLID) was initiated by Mapi Research Trust in Lyon, France. Initially called QOLID (Quality of Life Instruments Database), the project's purpose was to provide all those involved in health care evaluation with a comprehensive and unique source of information on PRO and Health-Related Quality of Life (HRQOL) measures available through the Internet. In collaboration with Dr. Marcello Tamburini (Director, Unit of Psychology, National Cancer Institute, Milan, Italy), the developer of the QLMed.org web site, PROQOLID was launched at the beginning of 2002. PROQOLID was created by the systematic collection of over 470 validated HRQOL and PRO instruments and their subsequent ordering into categories (e.g. pathologies, conditions, population). Through the structured presentation of synthesized, reliable and constantly updated data on PRO instruments, the PROQOLID database aims to present an overview of existing PRO instruments including all relevant and updated information on each. By providing this information the PROQOLID database facilitates access to the instruments and their developers and eases the process of selecting the instrument appropriate for a given application. Instruments can be chosen through a powerful interactive search function that will allow for the rapid selection of an appropriate instrument. What follows is an overview of the proper usage of the PROQOLID web site through a series of FAQ's collected directly from users of the web site. The PROQOLID database can be accessed on the Internet at . Access What are the differences in access to PROQOLID's database between members and guests? The access to PROQOLID is organized in two levels. The guest or free level is available to all visitors at no charge. This level provides brief information for each instrument, including • Full name of the instrument and acronym • Author(s) • Objective • Pathology • Disease • Type of instrument: Coping, Disability/physical functioning, Health status, Psychosocial/psychological, Quality of life, Satisfaction, Social functioning, Symptom/functioning, Utility, Work • Population: Adolescent, Adult, All, Caregivers, Female, Geriatrics, Male, Pediatrics, Terminal patients • Mode of administration: Caregiver-administered, Interviewer-administered, Nurse-rated, Physician-rated, Proxy-administered, Self-administered, Telephone-administered • Number of items • Original language • List of existing translations • Existence of a database: Yes / No • Time recall Since 1995, Mapi Research Trust has been a non-profit organization promoting the use and development of Patient Reported Outcomes. In an effort to accomplish this, a significant part of PROQOLID has been made accessible to all users free of charge. In order to further develop and improve the database and provide additional information on the available instruments, Mapi Research Trust requests a financial participation in order to access PROQOLID'S advanced (or members') level. By subscribing to PROQOLID, you are supporting the continuous collection and update of this unique PRO resource. Membership options are available for pharmaceutical or commercial companies, non-profit organizations, universities, individual academic researchers and students. Benefits to members include a greater degree of practical information on the instruments and, when available, includes the review copy of the instrument, its translations and the user manual, most of them in PDF format. Detailed information of the advanced level available include for each instrument • Name of the instrument (full and abbreviated) • Name and contact information of the Authors • Contact person for information on, or permission to use, the instrument in its original language • Copyright information • Detailed conditions of use (e.g. fee, written permission, user agreement etc.) • Review copy of the original instrument (when possible). Depending on the author's wish, original instruments may be used under specific conditions such as an access fee or signed agreement • Bibliographic references of the original instrument • Contact person for information on, or permission to use the translations • Review copy of the available translations (when possible) • Bibliographic references of the available translations (when possible) • Dimensions covered by the instrument • Time for completion • Age range • Scoring: response options, available scores, weighting, score direction and Minimal Important Difference (MID) or Minimal Clinically Important Difference (MCID) • Existence of a user manual and copy of the user manual (when possible) • Link to the PRO database identification form, when available • Methodology of development • Internal consistency reliability • Related websites • Other bibliographic references Users Who uses PROQOLID? The web site is available to anyone having an interest in the development, availability and use of Patient-Reported Outcomes (PRO). Through the power of the Internet the PROQOLID project intends to provide this information to the world. Every major pharmaceutical company, non-profit organizations such as the US Food and Drug Administration, the NIH's National Cancer Institute, the Veterans Administration as well as dozens of Universities, researchers and students worldwide subscribe to the advanced level of PROQOLID on a yearly basis. The PROQOLID database is routinely visited by over 800 users per day, thereby educating clinicians, researchers, students, and the world about the availability and proper usage of PRO instruments. Content How are the instruments organized in the PROQOLID database? The PROQOLID database was created in an effort to provide a means to facilitate the search process for and provide more efficient searches of any given PRO instrument. By organizing instruments in the PROQOLID database by several easy to understand categories, both time and energy are saved by the user. The different categories can be located on the Search page of the web site or by accessing directly from the tool bar at the top of the page. The different categories are as follows: Alphabetical The purpose of the Alphabetical list is to provide an overview of all existing PRO instruments. Over 1000 instruments are listed in alphabetical order according to their abbreviated name (or acronym). Some of the instruments are only listed, and these are displayed in standard font. For the remaining instruments access is available by simply clicking on the green link containing the abbreviated name of the instrument. Instruments can be accessed through an interactive letterbox at the top of the page. For example if the instrument begins with "D", simply click on the "D" at the top of the page and all instruments beginning with that letter will be displayed alphabetically. Generic Instruments The generic instruments are listed by alphabetic order on a separate web page. Pathology/disease A specific web page is dedicated to each pathology, and the instruments are listed either as generic instruments of the pathology or as disease-specific. The classification is structured based on Medline's Medical Subject Headings (MeSH) to ensure that the concepts are widely accepted. Please note that some diseases may be part of several pathologies. For example the disease "dementia" is part of both the pathologies "Neurology" and "Psychiatry/Psychology". Population The web page lists the instruments as they apply to specific populations including Adolescent, Adult, All, Caregivers, Female, Geriatrics, Male, Pediatrics, and Terminal patients. Author's name Instruments are grouped alphabetically according to the author's name, and as in the alphabetical list a letterbox is provided at the top of the page. Search engine You may search for instruments according to 10 criteria, including the name of the instrument, the pathology, the population or the available languages. The various criteria may be crossed referenced using the following Boolean Operators: AND, OR, NOT. How many instruments are contained in PROQOLID? The PROQOLID database was developed and is updated in close collaboration with the instruments' developers. It currently describes over 470 PRO instruments according to a structured format. The list is currently growing at the rate of fifty instruments per year. The database also includes review copies of 350 original instruments and 350 translations, most of them in PDF format. In order to determine the available translations simply access the instrument on the web site and all existing translations are conveniently listed. Also available through PROQOLID are over 125 associated User Manuals, and the description of 80 separate PRO databases. A fifth update of the whole database is underway and will include new information for each questionnaire on the reproducibility (or test-retest reliability) and clinical validity. In addition the PROQOLID website contains links to 150 external Internet resources relevant to the field of PRO usage and development. A general question and answer section on PRO is also included as well as on line full text articles on the development, validation, and linguistic adaptation of many of the PRO instruments published in medical journals. What are the instruments criteria to be eligible for inclusion in PROQOLID? To be eligible for inclusion in the database an instrument must be the subject of a publication that describes its development and/or validation. There is no charge to authors who wish to insert their instruments in PROQOLID nor are authors paid for their participation in this program. The Mapi Research Trust in Lyon, France determines ultimate decision for inclusion. Search How does the Search Engine work? Besides the search by categories of instruments listed above (i.e. alphabetical list, generic, pathology/disease, population and author's name), an interactive search engine is included in the PROQOLID web site. All of the instruments contained on the web site can be accessed from this location. Searches can be made by: • Abbreviated Name • Full Name • Author • Pathology • Disease • Type of Instrument • Population • Mode of Administration • Inclusion of a PRO Database • Language In an effort to increase the effectiveness of each search the ability exists to include up to nine (9) sub-parameters per search with the stipulation of "AND" or "OR". For example one could enter Author "=" Anderson J "OR" Author "=" Anderson R "AND" Type of Instrument "=" Quality of Life, "AND" Language "=" French, or any other combination that would suit the users needs. Through this function a user is able to drastically narrow the number of instruments displayed in the results window, thereby saving time and effort. If questions exist on the functioning of the search engine, or any questions about the PROQOLID database a contact page is provided with names and e-mail addresses for Mapi Research Trust and individual site managers in both Europe and North America. Additionally a video including a demo of PROQOLID can be seen on the home page. Update Who maintains the PROQOLID database? Mapi Research Trust has maintained the PROQOLID database for three years. The information contained in the website for each instrument is updated at least once a year in collaboration with the instruments' developers and over fifty new instruments are added to the website each year. Mapi Research Trust has listened to the needs of the Pharmaceutical Industry, Industry Regulators, health care professionals, and patients. With the passing of time the organisation has developed into an intricate team of professionals whose single goal is to define and unite the various requirements of each of these groups in order to provide better communication and understanding of each groups needs. PROQOLID achieves to translate these objectives into a concrete application which ultimate goal is the improvement of the patients' quality of life and health outcomes. Conclusion The Patient-Reported Outcome and Quality of Life Instruments (PROQOLID) database aims to present an overview of existing PRO instruments. PROQOLID currently describes more than 470 PRO instruments in a structured format. It includes review copies of over 350 original instruments, 120 user manuals and 350 translations. Most are available in PDF format. The database is updated in close collaboration with the instruments' authors on a regular basis. Fifty or more new instruments are added annually. By providing this information the PROQOLID database facilitates access to the instruments and their developers and eases the process of selecting the most appropriate instrument for a given application. Instruments can be chosen through a powerful interactive search function. The PROQOLID database can be accessed on the Internet at . Authors' contributions MPE conceived the database and participated in its implementation and helped to draft the manuscript. CA helped in the conception of the database and drafted the manuscript. LLP has contributed in the design, coordination and follow up of the database. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555954.xml
535939	Can mutations in ELA2, neutrophil elastase expression or differential cell toxicity explain sulphasalazine-induced agranulocytosis?	Background Drug-induced agranulocytosis, a severe side effect marked by a deficit or absolute lack of granulocytic white blood cells, is a rare side-effect of the anti-inflammatory drug sulphasalazine. Mutations in the human neutrophil elastase gene ( ELA2 ), causing increased intracellular concentration of this serine protease, inhibits neutrophil differentiation in severe congenital neutropenia (SCN). Since the clinical symptoms of agranulocytosis and SCN are similar, we hypothesized that it may origin from a common genetic variation in ELA2 or that sulphasalazine may affect human neutrophil elastase activity and protein expression. Methods We screened for genetic differences in ELA2 in DNA from 36 patients who had suffered from sulphasalazine-induced agranulocytosis, and compared them with 72 patients treated with sulphasalazine without blood reactions. We also performed in vitro studies of the blood cell lines HL60 and U937 after sulphasalazine exposure with respect to cell survival index, neutrophil elastase protein expression and activity. Results None of the mutations in ELA2 , which previously have been reported to be associated with SCN, was found in this material. Protein expression of human neutrophil elastase in lymphoma U937 cells was not affected by treatment with concentrations equivalent to therapeutic doses. Cell survival of lymphoma U937 and promyelocytic leukemia HL-60 cells was not affected in this concentration range, but exhibited a decreased proliferative capacity with higher sulphasalazine concentrations. Interestingly the promyelocytic cells were more sensitive to sulphasalazine than the lymphoma cell line. Conclusion Neutrophil elastase expression and ELA2 mutations do, however, not seem to be involved in the etilogy of sulphasalazine-induced agranulocytosis. Why sulphasalazine is more toxic to promyelocytes than to lymphocytes remains to be explained.	Background Sulphasalazine (SA) has anti-inflammatory, immunosuppressive and antibiotic actions, and is a component in the therapy of Crohn's disease, ulcerative colitis and rheumatoid arthritis. Bacterial enzymes in the colon split sulphasalazine into sulphapyridine and 5-aminosalicylic acid before it is absorbed. Sulphapyridine acts as a sulphonamide antibiotic, whereas 5-aminosalicylic acid is believed to be the anti-inflammatory metabolite. Common side/toxic effects are vomiting, skin rash and headache. The incidence of the hematological adverse effects associated with sulphasalazine is generally low, but the reactions can be severe and sometimes fatal. The risk of sulphasalazine-induced agranulocytosis, i.e. profoundly depressed circulating neutrophils is highest within the first three months of sulphasalazine-treatment, with a fatality rate of 6.5 % [ 1 ]. Clinical symptoms of agranulocytosis include fever, malaise and susceptibility to infections. Patients with arthritic disorders have a greater risk of developing sulphasalazine-induced agranulocytosis than patients with inflammatory bowel diseases. Severe congenital neutropenia (SCN) and cyclic neutropenia (CN) occur both as inherited and as sporadic diseases. SCN has a constant low neutrophil number if left untreated, whereas CN manifests with cyclic oscillations of neutrophil number with a 21-day cycle. Recently, diverse heterozygous mutations in ELA2 , encoding human neutrophil elastase, have been identified in a majority of the cases with CN and two-thirds of the cases with SCN [ 2 ]. In this study, we hypothesized that sulphasalazine-induced agranulocytosis, with clinical symptoms similar to congenital neutropenia, may arise from genetic variation in the human neutrophil elastase gene. We genotyped 108 sulphasalazine-treated patients for ELA2 , one third which of had experienced sulphasalazine-induced agranulocytosis. We, furthermore, tested for cytotoxic doses of sulphasalazine, and studied protein expression of human neutrophil elastase in sulphasalazine-treated blood cell lines. Methods Subjects Patients were treated with sulphasalazine (Salazopyrin, Pharmacia, Sweden) for inflammatory joint diseases and inflammatory bowel disease. The cases with sulphasalazine-induced agranulocytosis were originally collected through the Swedish Medical Products Agency's register of adverse side effects [ 3 ]. The control group had been treated with sulphasalazine without adverse effects for at least 3 months. From the original patient material consisting of 39 cases and 75 controls, DNA was available for 36 cases and 72 controls. The patient journals were studied for information concerning neutrophil differentiation in bone marrow aspirates. The study was approved by the Ethics Committee of the Medical Faculty at Uppsala University, registration number 95–200. Mutation analysis Genomic DNA was extracted from whole blood using standard techniques. Fragments covering exons 2–5 of ELA2 were amplified by PCR using primer pairs listed in Table 1 . The selection of exons 2–5 and some of the flanking intron sequences was based on previously reported mutations in cases with SCN and CN [ 2 , 4 ], as outlined in Figure 1 . Products for exon 2–5 were amplified with 1.5 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), activated by 15 min at 95°C followed by 4 cycles 94°C 30 sec, 65°C 30 sec, 72°C 1.5 min and 35 cycles of 94°C 30 sec, 67°C 30 sec, 72°C 1.5 min with a final extension of 10 min at 72°C. The exception was amplification of exon 5, where a 2°C lower annealing temperature was used. All primers contained a consensus M13 sequence to enable sequencing with the same primer, included in BigDye Primer sequencing kit from Applied Biosystems, Stockholm, Sweden. Applied Biosystems 310 analyzer and Sequence Analysis software was used for all sequencing. Thus, the 36 cases and 72 controls were analyzed for genetic mutations in ELA2 . Table 1 Primer sequences for PCR amplification of ELA2 exon 2–5 ELA2 target sequence Primers Exon 2 F 5'-tgtaaaacgacggccagtgggaggggacaggctccttgg-3' Exon 2 R 5'-caggaaacagctatgaccaccgggacgcggggtccgagc-3' Exon 3 F 5'-tgtaaaacgacggccagtcaggcccgtcgccggatggg-3' Exon 3 R 5'-caggaaacagctatgacctccgtcgcagcctccaccct-3' Exon 4 F 5'-tgtaaaacgacggccagtgtgacgcgctgacgatctgt-3' Exon 4 R 5'-caggaaacagctatgaccgcagtaccgggctgggagcg-3' Exon 5 F 5'-tgtaaaacgacggccagtcagtccagcttccccacctt-3', Exon 5 R 5'-caggaaacagctatgaccgacctactgaccattttcaac-3' PCR primers sequences for ELA2 exon 2–5, for following sequencing reactions with BigDye primer, Applied Biosystems. Figure 1 Outline of reported mutations in ELA2 exon-sequences in patients with severe congenital neutropenia and cyclic neutropenia Outline of mutations previously reported [2, 4] in ELA2 exons 1–5. The SNP S173 [6] is indicated as an extended arrow and represents base number 4890 in accession number Y00477 and is a base C→A substitution. Cell culture The lymphoma cell line U937 and the promyelocytic cell line HL-60 (American tissue culture collection) were cultured in Dulbeccos modified Eagles Medium, DMEM (Sigma) supplemented with 10 % fetal bovine serum (SVA, Uppsala, Sweden), L-glutamine and penicillin-streptomycin (Sigma). Western blot Equal numbers (8 × 10 6 ) of U937 cells were grown in 75 cm 2 dishes in complete medium containing 0, 125 and 250 μM sulphasalazine for 24 h. For protein isolation, cells were washed in PBS and lysed in buffer containing 1% Triton X-100, 50 mM Tris-HCl pH 8.0 and protease inhibitor cocktail (Sigma) and were kept on ice for 30 min. Lysates were centrifuged for 10 min at 10 000 × g, and protein concentration was determined using BioRad protein assay. Criterion precast gels (BioRad, Sweden) were used to perform SDS-page with 20 μg protein loaded per well. After gel transfer to a nitrocellulose membrane, the membranes were blocked over night in 5 % dry milk in TBS-Tween. Primary antibody against human neutrophil elastase (Calbiochem, Sweden) was diluted 1:1000 in 5 % dry milk in TBS-T. After 2 h incubation, and four sets of washing, a secondary antibody was added (1:5000) and blots were developed using ECL (ECL Western blotting system, Amersham, Sweden). Western blot analysis of human neutrophil elastase expression was performed twice. Elastase activity assay Cells (HL-60 and U937) treated with 0, 125, 250 and 500 μM of for 24 h were lysed with 100 μl of buffer containing 100 mM Tris-HCl pH 7.4, 1 mM MgCl 2 , 0.1 % Triton X-100. After homogenization, 300 μl of 1.4 M NaCl in 0.1 % Triton X-100 was added and samples were centrifuged at 15 000 × g, for 15 min at 4°C. The supernatants were transferred to new tubes and assayed for elastase activity using Suc-Ala-Ala-Ala-pNA (Sigma) as a substrate. For each assay we took 25 μl sample, mixed with 100 μl buffer containing 100 mM Tris-HCl pH 8.5, 1 M NaCl, 500 mM MgCl 2 and 0.1 % Triton X-100. To this, 50 μl of substrate was added, to a final concentration 1 μM. After 30 min of incubation in room temperature, absorbance was read at 405 nm and the concentration was calculated from a standard curve of elastase (Sigma). Cell survival index For the cell viability assay, we used a fluorometric microculture cytotoxicity assay (FMCA) previously described by Larsson et al [ 5 ]. Briefly, 20 000 cells/well were plated in 96-well plates (NUNC, DK) in complete medium with addition of increased concentrations of sulphasalazine (0, 125, 250, 500, 750 and 1000 μM) and incubated for 72 h in a humidified atmosphere used in regular cell culturing. All samples were plated in triplicates and three wells with cell culture medium served as blanks. As controls we had cells without additions and cells only with solvent, in this case 0.5 M NaOH, with equal molarities as in the wells with the highest sulphasalazine-concentration. At the end of the 72 h incubation period, plates were centrifuged (200 × g, 5 min) and medium was aspirated in a microtitre plate washer, washed with PBS and 100 μl of 10 μg/ml of fluorescein diacetate (Sigma, Sweden), was added. This dye exclusively binds intact cell membranes of viable cells. After 1 h incubation at 37°C, the fluorescence was read in the Fluoroscan 2 (Labsystems OY, Finland) at 480 nm excitation and 530 nm as emission. The results are presented as survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Thus, a low numerical value indicates high sensitivity to the cytotoxic effect of sulphasalazine. Effective concentration is defined as the concentration when 50 % of the cells are viable (EC 50 ). Statistics Two-tailed Student's t-test was used to compare subject characteristics and results from cell culture between cases and controls. Frequencies of subject characteristics male versus females was tested with Chi 2 -test with one degree of freedom, using Minitab 14. A p -value less than 0.05 was denoted with (), p < 0.01 with (*) and was considered as statistically significant. Results Subjects The characteristics of the subjects are presented in Table 2 . The agranulocytosis cases were significantly older than the control patients ( p = 0.023). The white blood cell count (WBC) before sulphasalazine-treatment did not differ between cases and controls, nor did the dose of sulphasalazine. Bone marrow aspirates had been taken from 10 patients (cases). In all samples, the myelopoesis was seriously reduced and a maturation arrest at the promyelocyte-myelocyte stage of neutrophilic differentiation was seen. Table 2 Characteristics of subjects Cases (n = 36) Controls (n = 72) p -value Age range (median) 11–77 (55) 13–90 (47) 0.023 WBC before a 9.3 ± 4.7 8.5 ± 2.5 0.26 Dose of sulphasalazine (gram/day) 2.2 ± 0.6 2.0 ± 0.4 0.13 Male : Female 17 : 19 33 : 39 0.891 a WBC data only available for 28 cases and 67 controls. Cases are defined as the patients treated with sulphasalazine, who were diagnosed with agranulocytosis and controls were defined as patients treated with sulphasalazine without hematological side effects within the first three months of sulphasalazine treatment. The statistics of differences between cases and controls in age, white blood cell count (WBC) before sulphasalazine treatment and dose of sulphasalazine was calculated with 2-tailed Student's t-test. The frequency of males versus females was calculated with Chi 2 test, one degree of freedom. Significance level was set at 0.05. Mutation analysis None of the previously reported mutations in ELA2 was found in this material, although we found a silent single nucleotide polymorphism, called S173 [ 6 ] that corresponds to a C4890A substitution in Genbank accession number Y00477 (marked with extended arrow in Figure 1 ). The incidence of the S173 polymorphism did not differ between controls and cases, 0.31 for both, and S173 has previously been detected in healthy subjects [ 6 ]. No correlation between the S173 polymorphism and white blood count before sulphasalazine-treatment was found (Table 3 ). Table 3 White blood count (WBC), before sulphasalazine treatment, in subjects with or without the S173 polymorphism WBC Subjects with S173 (n = 34) 8.74 ± 2.51 Subjects without S173 (n = 74) 8.66 ± 3.51 White blood count (WBC) was estimated by the local physicians before starting with the sulphasalazine treatment. The average WBC in subjects with or without S173 is presented as the mean value ± SD. It was no statistical difference between the groups. Elastase expression, elastase activity and cell survival after sulphasalazine exposure to HL-60 and U937 By western blot analysis, we analyzed neutrophil elastase protein expression in U937 cells. No difference in human neutrophil elastase expression was detected after treatment with 125 and 250 μM sulphasalazine (Figure 2 ), compared to controls. The elastase activity in HL-60 and U937 cells was not affected by increasing sulphasalazine concentration, ranging from 0 to 500 μM, and expressed as elastase activity/μg protein (data not shown). For the cell survival index, each FMCA experiment was performed three times separately with similar results (inter-assay variation less than 10 %). Concentrations below 250 μM sulphasalazine did not affect the survival index of U937 and HL-60 cells (Figure 3A,3B ), but at 500 μM of sulphasalazine, the survival index of HL-60 cells decreased to a third (Figure 3A ). The U937 was only marginally affected at 500 μM sulphasalazine-concentration, but cellular survival decreased with approximately 40 % at 750 μM of sulphasalazine (Figure 3B ). The effective concentration (EC 50 ) of sulphasalazine was approximately 370 μM for HL-60 cells and 820 μM for U937 cells. Figure 2 Western blot analysis of human neutrophil elastase (hNE) expression after sulphasalazine exposure U937 cells were incubated for 24 h with 0, 125 and 250 μM sulphasalazine, followed by cell lysis and protein isolation. 20 μg of protein was applied in each lane, transferred to nitrocellulose membrane and incubated with human neutrophil elastase antibody. Figure 3 Survival index of HL-60 and U937 cells, incubated with increasing concentrations of sulphasalazine Survival index of HL-60 (A) and U937 cells (B) treated with increasing concentrations of sulphasalazine (0–1000 μM) for 72 h and measured with FMCA. Survival index, defined as fluorescence in test wells/ fluorescence in control wells (blank values subtracted) × 100. Discussion Idiosyncratic drug-induced agranulocytosis can be due to several different mechanisms of action, including immunological, toxic and genetic [ 7 , 8 ]. Toxic drug-induced neutropenia is often dose-dependent, whereas immunological and genetic causes are less related to dose. In our study, bone marrow aspirates from patients with sulphasalazine-induced agranulocytosis revealed maturation arrest of neutrophils at the promyelocyte-myelocyte stage. These findings resemble promyelocytic maturation arrest seen in severe congenital neutopenia (SCN) and cyclic neutropenia (CN) [ 9 ]. In the majority of cases with SCN and CN, germline mutations in the human neutrophil elastase gene ( ELA2 ) are implicated as the primary abnormality [ 2 , 4 ]. The focus of this study is therefore on the human neutrophil elastase gene as a possible cause of sulphasalazine-induced agranulocytosis. We found a coding synonymous polymorphism in ELA2 , which, however, was equally represented among cases and controls. Heterozygous mutations in ELA2 act in a dominant manner, interfering with sub-cellular trafficking of neutrophil elastase, and leading to an accumulation of neutrophil elastase in the cytosol [ 10 ]. For normal neutrophil cell maturation, the proliferative action of the granulocyte colony stimulating factor (G-CSF) is necessary [ 11 ]. When G-CSF is exposed to active elastase enzyme in vitro, G-CSF is rapidly cleaved and rendered inactive [ 11 ]. In theory, SCN and CN are caused by an accumulation of neutrophil elastase, leading to an inactivation of G-CSF and a negative feedback on granulopoiesis, which causes neutropenia. Other proteins, connected to expression and transportation of human neutrophil elastase, have also been linked to SCN disease. In canine cyclic hematopoieses, lack of the intracellular transport protein AP3β causes accumulation of canine neutrophil elastase in the cytosolic compartments [ 12 ], and mutations in ELA2 may disrupt the AP3β-recognition site [ 13 ]. Furthermore, mutations in the proto-oncogene GFI1 , a transcriptional repressor of ELA2 , causes over-expression of neutrophil elastase in mice, thus, making them neutropenic [ 14 ]. During maintenance therapy with sulphasalazine, trough serum sulpha concentration is on average approximately 100 μM at the Department of Clinical chemistry and pharmacology, Uppsala University hospital. To avoid toxic effects, trough serum concentration of sulpha should stay below 600 μM [ 15 ]. Our in vitro data suggest a decreased cell survival of sulphasalazine at concentrations around 500 μM. Interestingly promyelocytic leukemia HL-60 cells were more sensitive to sulphasalazine than lymphoma U937 cells, with EC 50 values of 370 μM and 820 μM, respectively. Human neutrophil elastase expression in lymphoma U937 cells did not differ after sulphasalazine at 125 and 250 μM, indicating that human neutrophil elastase production is not affected by sulphasalazine at subtoxic levels. Conclusions In conclusion, neutrophil elastase does not appear to be involved in the etiology of sulphasalazine-induced agranulocytosis. No causative ELA2 mutations were found, and therapeutic concentrations of sulphasalazine did not increase the expression of human neutrophil elastase. High concentrations of sulphasalazine were toxic to white blood cells in vitro; however, there is no evidence that this toxicity is mediated through human neutrophil elastase. Promyelocytic cells were more sensitive to sulphasalazine than lymphoma cells, and the reason for this difference may also explain sulphasalazine-induced agranulocytosis. Competing interests The author(s) declare that they have no competing interests. Authors' contributions AJ carried out the molecular genetic studies, participated in the sequence alignment, drafted the manuscript and carried out the in vitro experiments. MW participated in the design of the study and performed the statistical analysis. HM conceived the study, and AJ, MW and HM participated in its design and coordination. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535939.xml
524175	Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment	Background Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment. Methods Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β 1 -adrenergic receptor blocker atenolol for twelve weeks. Results The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele. Conclusions Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control.	Background Hypertension is a complex trait caused by multiple environmental and genetic factors interacting through the cardiac, vascular and endothelial systems. Several drug classes with different mechanisms of action, including inhibitors of the renin-angiotensin-aldosterone system (RAAS), calcium channel blockers, adrenergic receptor blockers and diuretics, are available for treatment of hypertension. However, the response to antihypertensive treatment is highly variable between individuals, which makes it difficult to predict the efficacy of a specific drug in the individual patient [ 1 - 3 ]. Currently, there are no clinically useful biochemical or metabolic markers for predicting the individual responses to antihypertensive treatment [ 4 - 6 ]. Twin studies have estimated that as much as half of the variability in blood pressure levels between individuals is due to genetic factors [ 7 , 8 ]. Based on the abundance of single nucleotide polymorphisms (SNPs) in the human genome [ 9 ], it can be expected that one or more SNPs occur in each of the genes encoding components of the blood pressure regulating systems, and that they are the genetic factors influencing individual blood pressure levels. Coding SNPs affecting the function of enzymes and receptors in pathways of blood pressure regulation, or regulatory SNPs, affecting the expression levels of genes, are likely to explain part of the variability of the response to antihypertensive treatment. Hence, these functional SNPs, or other SNPs inherited in linkage disequilibrium with them, could be potential pharmacogenetic markers for predicting the response to a certain drug, and thus guide the selection of the optimal drug for each individual patient [ 10 - 12 ]. The RAAS and the sympathetic nervous system play key roles in blood pressure regulation. We have earlier shown that polymorphisms in the angiotensin converting enzyme gene [ 13 ] and a SNP in the aldosterone synthase gene [ 14 ] are related to changes in blood pressure during treatment with the angiotensin II receptor blocker irbesartan, whereas two SNPs in the angiotensinogen gene were associated to the reduction in blood pressure by the β 1 -adrenergic receptor blocker atenolol [ 15 ]. Dyslipidemia with high levels of serum triglycerides and free fatty acids, and elevated serum cholesterol levels and low levels of high-density lipoprotein cholesterol are common in hypertensive patients. Association has been found between disturbance in lipid metabolism and hypertension, but so far no attempts have been made to relate variables reflecting lipids, or the genes involved in lipid metabolism, to the individual response to antihypertensive treatment. We have recently developed a microarray based minisequencing system for parallel genotyping of multiple SNPs in blood pressure regulating candidate genes [ 16 ]. Here we analysed the relationships between the genotypes of SNPs in the apolipoprotein A-IV, apolipoprotein A-V, apolipoprotein B-100, low density lipoprotein receptor, hepatic lipase and lipoprotein genes and reductions in blood pressure in hypertensive patients randomised to monotherapy with either irbesartan or atenolol. We found that SNP alleles in the apolipoprotein B gene and the low density lipoprotein receptor gene were associated to the antihypertensive response after twelve weeks of treatment. Methods Study population DNA extracted from blood samples from 97 hypertensive patients from the double blind parallel group "Swedish Irbesartan Left Ventricular Hypertrophy Investigation versus Atenolol" (SILVHIA) trial [ 17 ] were analysed. Men and women above the age of 18, having primary mild to moderate hypertension and left ventricular hypertrophy were enrolled in the trial and randomised to receive either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β 1 -adrenergic receptor blocker atenolol once daily as monotherapy. The dose was doubled after six weeks if the diastolic blood pressure was ≥ 90 mmHg. Blood pressure was measured by trained nurses using a mercury sphygmomanometer, after the patients had rested for at least 10 min in the seated position. Left ventricular hypertrophy was defined as left ventricular mass index of > 131 g/m 2 for men and > 100 g/m 2 for women, assessed by echocardiography. The data presented relates to the change in blood pressure after 12 weeks of treatment. For details on the SILVHIA trial, see Malmqvist etal [ 17 ]. Baseline characteristics for the patients are presented in Table 1 . The study was approved by the ethics committees of all participating centres of the SILVHIA trial and that of the Medical Faculty of Uppsala University. Table 1 Characteristics of the hypertensive patients in the two treatment groups. Irbesartan group 2 Atenolol group 2 Number of patients 48 49 Age (years) 54 ± 8 54 ± 8 Gender (proportion females) 37% 31% Height (m) 1.74 ± 0.09 1.73 ± 0.09 Weight (kg) 83 ± 15 82 ± 14 Smokers trial start (%) 29 18 Baseline fs-cholesterol (mM) 6.1 ± 1.0 5.8 ± 1.1 Baseline fb-glucose (mM) 5.7 ± 3.1 5.2 ± 2.5 Pre-treatment SBP 1 (mmHg) 164 ± 18 160 ± 20 Pre-treatment DBP 1 (mmHg) 104 ± 7 103 ± 8 Change in SBP at 12 weeks (mmHg) -16 ± 20 -11 ± 16 Change in DBP at 12 weeks (mmHg) -9.0 ± 11 -12 ± 7.7 1 Systolic blood pressure (SBP) and diastolic blood pressure (DBP) 2 Data are mean ± SD SNP markers and genotyping procedure In our previous study [ 16 ], 98 SNPs were selected from the NCBI (dbSNP, ) and the SNP Consortium (TSC, ) databases and validated in a pooled DNA sample representing the Swedish population. A subset of these SNPs located in genes involved in lipid metabolism and that were polymorphic in the Swedish population were included in the study presented here, together with one additional SNP in the apolipoprotein A-V gene. See Table 2 for information on the SNPs. Table 2 Investigated polymorphisms given as gene names, acronym and GenBank accession number. Gene name and acronym 1 dbSNP ID 2 Amino acid alteration SNP name 3 Apolipoprotein A-IV rs5092 Thr/Thr A1449G APOA-IV; J02758 Apolipoprotein A-V rs662799 Promoter C31455T APOA-V; AC074203 Apolipoprotein B-100 rs1801701 Arg/Gln G10108A † APOB; M19828 † ; M19810 § rs1367117 Thr/Ile C711T § Low density lipoprotein receptor rs688 Asn/Asn C16730T LDLR; AF217403 rs5925 Val/Val C2000IT Lipase, hepatic rs6083 Asn/Ser A110G LIPC; M35429 Lipoprotein rs328 Ser/Term C9040G LPL; AF050163 rs312 Intron G7315C rs314 Intron A7360G 1 Gene name and acronym, GenBank accession number for the sequence used in the design of primers for the PCR and minisequencing reactions. 2 SNP identification number in the NCBI SNP database dbSNP, . 3 Corresponding to the nucleotide position in the gene sequence referenced in the first column and the nucleotide variation in the coding strand. Fragments comprising the SNPs were amplified in multiplex PCR described previously [ 16 ]. A microarray based minisequencing single nucleotide primer extension assay, in which one or two of four ddNTPs labelled with the fluorophore Tamra (Perkin Elmer Life Sciences, Boston, MA, USA) were incorporated by the Thermo Sequenase™ DNA-polymerase at each SNP site. The incorporated ddNTPs were detected using a fluorescence scanner, and the fluorescence signals were extracted. A signal intensity fraction, obtained by dividing the fluorescence signal intensity for allele 1 with the sum of the fluorescence signal intensities for allele 1 and allele2, was used to assign the individual genotypes. The SNP APOA-V C31455T was genotyped using a microtiter plate minisequencing assay with tritium detection [ 18 ]. Statistical analyses Analysis of covariance (ANCOVA) with each SNP as factor, baseline blood pressure as covariate and the change in blood pressure as response, was performed. The analyses were performed by treatment group and blood pressure measurement (systolic and diastolic blood pressures). Correction for multiple testing was performed by calculation of critical p-values corresponding to a nominal type I error of 5% using a permutation test [ 19 ]. Two tailed significance levels were used. Results and discussion We explored possible associations between individual genotypes of ten SNPs and reduction in systolic and diastolic blood pressure as response to treatment with atenolol or irbesartan (Figure 1 ) in samples from the SILVHIA trial [ 17 ]. In the irbesartan group, a change in systolic blood pressure appeared to be related to genotype for the SNPs ApoA-IV A1449G, ApoA-V C31455T and ApoB C711T. In the atenolol treatment group, presence of the C-allele of the SNP LDLR C16730T was associated to the reduction in systolic blood pressure. Figure 1 Effect of SNP genotype on the change in blood pressure after 12 weeks of treatment for the ten SNPs. For each of the SNPs, the pattern of change in blood pressure related to genotype is illustrated for the systolic blood pressure (SBP) and diastolic blood pressure (DBP) in separate panels. In each panel, the mean change in blood pressure is shown for the SNP genotypes in the atenolol treatment group on the left, and the corresponding results in the irbesartan treatment group are given on the right. The error bars corresponds to the standard error of the mean. The p-values indicating significance for the APOA-IV A1449G, APOA-V C31455T, APOB C711T and LDLR C16730T SNPs are given in the corresponding panels. The number of individuals of each genotype is shown above the bars in each panel. Correction for multiple testing using a permutation test [ 19 ] yielded critical p-values of 0.004 and 0.007 for systolic blood pressure after atenolol and irbesartan treatment, respectively, and 0.006 and 0.007 for the diastolic blood pressure, corresponding to the significance level of p = 0.05. After the permutation test, carriership of the C-allele of the C711T SNP in the apolipoprotein B gene remained significantly associated to the reduction in systolic blood pressure (p = 0.004) in the irbesartan treatment group (Figure 1 ) while the individuals homozygous for the T-allele showed no reduction in systolic blood pressure. The same pattern of response related to genotype was seen for diastolic blood pressure, although it did not reach statistical significance. In the atenolol treatment group, the SNP C16730T in the low density lipoprotein receptor gene showed a trend of association to the reduction in systolic blood pressure. Presence of the C-allele was related to blood pressure reduction (p = 0.006), while the individuals homozygous for the T-allele (n = 9) actually showed an increase in systolic blood pressure (Figure 1 ). A similar response pattern was not seen for the diastolic blood pressure during atenolol treatment (p = 0.44) (Figure 1 ). There were 39 carriers of the favourable C-allele of the APOB C711T SNP in the irbesartan treatment group. The average reduction in systolic blood pressure for these individuals was 19 mmHg, compared to 0 mmHg for the individuals lacking this allele. In the atenolol treatment group, the individuals carrying the favourable C-allele of the SNP LDLR C16730T showed an average reduction of 14 mmHg in systolic blood pressure compared to an increase of 7.5 mmHg for the individuals homozygous for the T-allele. The SNP C711T in the apolipoprotein B gene is located in the coding region of the gene, and alters a threonine residue to an isoleucine residue in the protein. This SNP is located in the amino-terminal part of the enzyme and has been suggested to affect the dimerisation of apolipoprotein B and low density lipoprotein during cholesterol transport [ 20 ]. The C16730T SNP in the LDLR gene results in a synonymous amino acid change, however this SNP could be in linkage disequilibrium with another functional SNP potentially influencing the response to drug treatment. Irrespectively if the tested SNPs are actually functional, our findings imply a potential connection between lipid metabolism and response to antihypertensive treatment. We have recently found circulating apolipoprotein B to be the most powerful predictor of endothelium-dependent vasodilation of the commonly used markers of cholesterol metabolism [ 21 ]. It is not evident, however, why apolipoproteins predict the response to irbesartan, and not to atenolol treatment, as these drugs appear to improve endothelium-dependent vasodilation to a similar extent [ 22 ]. Lipid abnormalities, commonly seen in hypertension, have been considered to be connected to the blood pressure level by the common denominators obesity and insulin resistance. Other studies have suggested a more direct effect of lipids in blood pressure control, as infusion of the fat emulsion Intralipid together with heparin increases blood pressure in healthy subjects [ 23 - 25 ]. This effect is more pronounced in the normotensive subjects with a family history of hypertension [ 26 ]. It has also been shown that an acute elevation of free fatty acids alters heart rate variability, an index of cardiac autonomic nervous system balance [ 27 ], suggesting that lipid metabolism may be involved in the regulation of cardiovascular autonomic tone. Thus our results that indicate involvement of components of lipid metabolism in the response to antihypertensive treatment are supported by cross-sectional epidemiological studies. In our earlier exploratory study, 74 SNPs with a minor allele frequency over 5%, including nine of the SNPs analysed here were tested as predictors of blood pressure regulation in the SILVHIA study samples using a multiple regression model [ 16 ]. The main aim of this study was to establish the microarray-based genotyping system. Analysis of twenty-eight SNPs from this panel that are located in genes from the renin-angiotensin aldosterone system identified a SNP in the aldosterone synthase gene (CYP11B2 T267C) and two SNPs in the angiotensinogen gene (AGT G1218A and T1198C) that appeared to be associated to blood pressure reduction [ 14 , 15 ]. A limitation in these previous studies was that correction for multiple testing was not applied, whereas in the current study we used a permutation test. A remaining weakness in our study is the small number of samples available for analysis, which does not allow detection of small to medium size gene effects, and results in uncertain estimation of the the magnitude of the effects detected. Moreover, in a small study there is the risk of a non-representative group of patients with respect to gender, age, and genotype distribution. Despite these limitations, we detected a significant effect of the SNP C711T in the apolipoprotein B gene and the SNP C16730T in the low density lipoprotein receptor after correction for multiple testing. The pharmacogenetically interesting results from our study need to be replicated in other studies. As the C711T SNP in the apolipoprotein B gene predicted response to treatment with irbesartan, and the C16730T SNP in the low density lipoprotein receptor gene appeared to predict response to atenolol treatment, our results point at possible use of SNPs in genes encoding components of lipid metabolism in pharmacogenetic panels for selecting the optimal drug for each patient. To our knowledge our study is the first one to investigate the relationship between polymorphisms in genes involved in lipid metabolism and the response to antihypertensive treatment. Competing interests The authors declare that they have no competing interests. Authors' contributions UL performed the development of genotyping technology, genotyping lab work, interpretation of data and had a substantial role in writing the manuscript. LL provided clinical expertise, participated in selection of candidate genes and contributed to writing. LK provided clinical expertise, established a database of the SILVHIA phenotypes, and in writing. LB performed the statistical analysis. TK provided the SILVHIA samples and contributed to writing the manuscript. A-CS contributed by planning and supervision of the project, and to writing the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524175.xml
529323	In Times of Stress, Mutate Early and Often	null	For a human, the normal response to stress is to reduce it through some purposeful action, be it indulging in chocolate or calling in sick, at a rate which we can vary to fit the circumstances. For a strain of bacteria faced with stress, the choice is often more stark: it must mutate or die. Among evolutionary theorists, an important question has been whether the rate of mutation is fixed, or instead can adaptively increase in response to stress, thereby increasing the likelihood of a favorable mutation. Something like this latter possibility was envisioned by Darwin, but fell out of favor among some neo-Darwinists, for whom a steady rate of mutation was more in keeping with their overall model of evolutionary gradualism. This debate is taken up in a new study in this issue by P. J. Hastings and colleagues, who examined the mechanism by which Escherichia coli lacking the ability to digest lactose, called lac − mutants, regain that ability when presented with lactose as their only food source. It has been known for some time that the reversion of lac − mutants to a lac + state can be achieved by either of two genetic events: amplification, which creates numerous copies of the nonfunctional lac gene, and point mutations, which give rise to functional versions of the gene (many non-useful mutations also occur; thus, there is no directed mutation, in keeping with standard Darwinian evolution). According to the gradualist view, amplification precedes mutation, and the rapid appearance of lac + cells is explained by a normal mutation rate acting on multiple copies of the gene. In contrast, according to the hypermutation view, amplification and mutation are independent events, and lac + cells arise quickly because the mutation rate has increased. While some results from previous studies have supported the gradualist interpretation, the experiments of Hastings et al. show that hypermutation is the most plausible explanation. A variety of procedural improvements allowed them to analyze more individual cells at an earlier stage of colony development. For instance, they analyzed colonies composed of as few as one hundred cells, rather than the ten thousand cells in prior experiments, and even nascent colonies at the two-cell stage. Sectored colonies of lac + and lac − E. coli The study produced clear evidence that point mutations arise very early in the development of lac + colonies, before amplification can account for the number of lac + revertants observed. Amplification is not only independent from mutation, but occurs relatively late under starvation. The researchers found that amplification, but not point mutation, requires the presence of a particular DNA polymerase, further strengthening the case that amplification need not precede mutation. They also showed that amplification by itself does not induce a so-called SOS response. The SOS system includes a group of genes that cause an increase in mutation in response to stress, and one hypothesis arising from the gradualist model was that amplification turned on the SOS response. Based on their data, the authors reject the strict gradualist model for the adaptive mutation mechanism in the Lac system. They propose that amplification and hypermutation are independent responses to stress, each of which increases the likelihood of adaptive change. They also suggest that a stress-induced increase in the rate of point mutations may have implications for a variety of mutation-related phenomena, from tumor formation to development of resistance to antibiotics and chemotherapeutic drugs.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529323.xml
545206	Are Anticapsular Antibodies the Primary Mechanism of Protection against Invasive Pneumococcal Disease?	Background Antibody to capsular polysaccharide has been the basis of several vaccines that offer protection against invasive disease from Streptococcus pneumoniae . The success of such vaccines has led to the inference that natural protection against invasive pneumococcal disease is largely conferred by anticapsular antibody. If this is so, one would expect that the decline in disease from different serotypes would vary significantly, and that the appearance of substantial concentrations of anticapsular antibodies would coincide temporally with the decline in age-specific incidence. Methods and Findings Using incidence data from the United States, we show that, on the contrary, the decline in incidence with age is quite similar for the seven most important serogroups, despite large differences in exposure in the population. Moreover, only modest increases in antibody concentration occur over the second and third years of life, a period in which serotype-specific incidence declines to less than 25% of its peak. We also present detailed data on the distribution of antibody concentrations in Israeli toddlers, which are consistent with the United States findings. The same conclusion is supported by new data on age-specific incidence in Finland, which is compared with published data on antibody acquisition in Finnish toddlers. Conclusion We suggest some additional studies of the mechanisms of protection that could distinguish among potential alternative mechanisms, including acquired immunity to noncapsular antigens, maturation of nonspecific immune responses, or changes in anatomy or exposure.	Introduction The protective effects of antibody to pneumococcal capsular polysaccharides have been appreciated since the development of serum therapy, in which passively transferred, serotype-specific antipneumococcal serum reduced mortality from pneumococcal pneumonia by half [ 1 ]. The development of pneumococcal polysaccharide vaccines for adults [ 2 ] and the efficacy of pneumococcal polysaccharide–protein conjugate vaccines in infants and children [ 3 , 4 ] have confirmed that active immunity to the polysaccharide can provide excellent protection against invasive disease from pneumococci of the same serotype, and in some cases protection against cross-reacting serotypes within the same serogroup. While the ability of passive or vaccine-induced anticapsular antibodies to protect against pneumococcal disease is clear, less is known about the natural development of immunity to pneumococcal disease in unimmunized persons. In unimmunized populations, the incidence of invasive disease follows a well-known age distribution, peaking in the first 2 y of life, declining by more than an order of magnitude by the second and third decades of life, and then rising at an accelerating pace, with incidence in persons over 70 y approaching that in infants [ 5 ]. The reason for the decline in incidence has not been conclusively determined, yet it is often suggested that the acquisition of anticapsular antibodies plays a critical role in this decline [ 6 , 7 ]. Indeed, it has been proposed that the human immune system sees each serotype of Streptococcus pneumoniae as a distinct, independent pathogen [ 8 ]. The hypothesis that protection from invasive pneumococcal disease is caused by the acquisition of anticapsular antibodies directed against each of the pneumococcal serotypes yields two simple predictions about the age-specific epidemiology of pneumococcal disease. First, it predicts that the age-specific timing of the decline in invasive disease should be different for different serotypes: those that are rare, poorly immunogenic, or both should decline later in life than those that are common and immunogenic. Second, it predicts that protection against invasive disease from a given serotype should coincide temporally with the acquisition of anticapsular antibody to that serotype, both at an individual level and at a population level. We tested these predictions using data from the United States, Finland, and Israel. Methods United States Dataset Incidence of invasive pneumococcal disease was measured in eight sites around the United States participating in the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance between 1994 and 1999. The data used here are restricted to those periods during which serotyping was routinely performed: 1994–1999 for the Georgia site, 1995–1999 for the Minnesota site, and 1998–1999 for all other sites [ 5 ]. Data were not available on the timing of anticapsular antibody acquisition in these same populations, but we compared the timing of the decline in pneumococcal disease against previously published data on age-specific prevalence of anticapsular antibody levels greater than 0.2 mcg/ml [ 9 ]. Israel Dataset Antibody concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (with absorption by cell wall polysaccharide but not by 22F polysaccharide) in blood samples that were obtained from 130 toddlers at enrollment and at approximately 12 and 24 mo after enrollment in a double-blind, controlled trial of a nine-valent pneumococcal conjugate vaccine. The toddlers analyzed for this study were those in the control group, which received meningococcal C conjugate vaccine; the details of the trial [ 10 ] were previously described. Preliminary analyses of these data confirmed previous findings [ 11 ] that ELISA measurements were highly correlated (and therefore likely revealed cross-reactions) for all pairs of serotypes, except for type 14, for which correlations were minimal, consistent with previous findings of little cross-reaction. For this reason, we chose to analyze age trends only in serotype 14 antibodies. Finland Dataset Mandatory reporting from all microbiological laboratories in Finland to the National Register of Infectious Disease ( http://www3.ktl.fi/stat/ ) identified all blood and cerebrospinal fluid isolates of S. pneumoniae obtained in the years 1995–2001. Incidence within 6-mo age groups was calculated using population denominators obtained from Statistics Finland (Helsinki, Finland). Since the primary purpose of examining incidence in Finland was to compare age-specific rates against published distributions of antibody concentrations for the same age groups [ 12 ], we restricted our attention to serotype 14 and serogroup 6, for which subsequent investigations suggested antibody measurements in Finland were relatively unaffected by cross-reactions [ 13 ] (ELISA measurements for published data from Finland used a special type 6B polysaccharide that was found to minimize cross-reactions [ 13 ]). Results United States Findings Figure 1 shows the age-specific incidence of invasive pneumococcal disease, by capsular serogroup, obtained from population-based active surveillance in the United States prior to the introduction of the conjugate vaccine. Figure 2 shows age-specific incidence by type of infection, for the same age range. Figure 1 Age-Specific Incidence of Invasive Pneumococcal Disease in the United States by Serogroup, Based on Data from Active Bacterial Core Surveillance Serogroups 4 and 23 are shown only up to 48 mo, after which incidence is less than 1/100,000 person-years. All serogroups besides those in the heptavalent vaccine are shown combined as non-vaccine serogroups (NVG). Figure 2 Age-Specific Incidence of Invasive Pneumococcal Disease in the United States by Disease Type, Based on Data from Active Bacterial Core Surveillance Meningitis incidence is plotted only up to 30 mo, after which it remains at or below 1/100,000 person-years. “Pneumonia” indicates bacteremic pneumonia, while “bacteremia” indicates nonfocal bacteremia. “Total” includes other invasive diagnoses. Incidence peaks between the ages of 9 and 15 mo, and falls in an approximately parallel fashion thereafter, for each of the seven most important serogroups (which are those included in the seven-valent conjugate vaccine) and for the remaining serogroups put together. The same pattern is observed for both pneumonia and bacteremia. For each serogroup, incidence by age 24 mo is approximately half that in the peak age group, and by 36 mo, incidence for each serogroup has fallen to 10%–25% of its peak. The consistent timing of the pattern across multiple serogroups argues for a common mechanism, rather than for independent acquisition of immunity to each serogroup as a separate event. Since most individuals do not suffer from invasive pneumococcal disease in this age range, carriage or mucosal disease (otitis media) from pneumococci may be the immunizing event for anticapsular antibodies in the general population [ 12 ] (although in principle immunity to some serogroups could be generated in response to cross-reacting antigens from other bacterial species or other sources [ 14 ]). Different serogroups have vastly different frequencies among pneumococci isolated from carriage [ 12 , 15 , 16 , 17 ] and otitis media [ 12 , 18 ]; for example, serogroups 4 and 18 and the non-vaccine serogroups are isolated far less commonly than several of the other pneumococcal types identified in Figure 1 . One could postulate that these differences in frequency of carriage are offset by differences in immunogenicity; however, there is little evidence that serotypes 4 or 18C are more immunogenic than other, far more common serotypes [ 4 , 15 ]. One could also postulate that the frequency of isolation of serotypes from carriage depends on duration as well as incidence, so that the serotypes for which carriage appears rare are simply carried for a shorter duration. While the data to address this speculation are limited, the duration of carriage of types 4 and 18C seems to be comparable to that of other, more frequently carried serotypes [ 15 , 16 ]. Thus, the most parsimonious interpretation of the data on the timing of the decline in age-specific susceptibility is that one or more common mechanisms are responsible for the decline in disease from all serotypes. Testing the second prediction against data is hampered by the fact that, to our knowledge, no study has characterized the age-specific distribution of antibody concentration in a large population using the currently accepted methodology, which includes absorption with both cell wall polysaccharide and serotype 22F polysaccharide [ 13 , 19 ]. Analyses by Soininen and colleagues have found that antibodies measured by standard ELISA in unimmunized children are highly cross-reactive between different serotypes, and that cross-reactive antibodies lack opsonophagocytic function and often appear in the absence of any documented exposure to a given capsular serotype. As a result, age-specific antibody concentration data for any given serotype are “contaminated,” to a greater or lesser degree, by cross-reactive antibodies with other specificties. The most important exception to this problem occurs for antibodies to serotype 14, for which cross-reaction is minimal [ 11 ]. A recent publication describes the age-specific proportion of children in the United States with anti-type-14 polysaccharide antibody concentration exceeding the putative protective concentration of 0.2 μg/ml ( Figure 3 of [ 9 ]). At 12 mo, 90%–95% of the population falls below this level, and at 24 mo, 80%–85% remains below it—despite a 40%–50% drop in disease incidence from 12 mo to 24 mo. At 36 mo, 75% of children remain below the putative protective level, although by this age incidence has fallen more than 80% from its 12-mo peak. In summary, if the 0.2-μg/ml concentration were truly the threshold for “protection,” the 20%–30% reduction in the unprotected population between ages 12 and 36 mo would be inadequate to explain the 90% decline in disease incidence. Clearly, 0.2 μg/ml is not a precise dividing line between being “protected” and “unprotected,” a threshold that (if it exists) may vary by serotype, but given the available data, there is reason to doubt that anti-type-14 antibody alone is responsible for the decline in disease in this age range. Figure 3 Box-Whisker Plot of Anti-Type-14 Polysaccharide Antibodies in Israeli Toddlers, by 6-Mo Age Groups Central boxes indicate median and 25th and 75th percentiles; whiskers indicate upper and lower adjacent values. Israel Findings To assess whether the limitations of the United States antibody described above—i.e., the availability of only one cutoff point for antibody concentrations—might be providing an incomplete picture of the distribution of antibody levels by age, we examined an additional dataset from Israeli toddlers. For the reasons described above, we examined only antibodies to serotype 14, for which the distribution of concentrations by age is shown in Figure 3 . These data indicate that between the ages of 12–17 and 36–41 mo, the median antibody concentration increases by about 2-fold. These data are broadly consistent with those published for the United States; antibody levels rise very gradually, though detectably, during the second and third years of life. It is difficult to believe—albeit not impossible—that the dramatic declines in disease incidence over these years are explained simply by this small rise in antibody concentrations. Finland Findings Incidence of serogroup 6 and serotype 14 invasive pneumococcal disease by 6-mo age groups in Finland, shown in Figure 4 , is broadly similar to that found in the United States, albeit with lower absolute incidence for both serogroups. Peak incidence occurs in the 12–17-mo age group, and incidence declines to 25%–30% of its peak rate by 24–29 mo of age. This decline in incidence may be compared against the cumulative distributions of antibody concentrations in Finnish toddlers shown in Figure 2 of [ 12 ]. Between ages 12 and 24 mo, there is a discernible increase in the concentration of antibodies in the population, but the median concentration increases by only about 2-fold in this period. Moreover, the proportion of the population with antibody concentration below any particular threshold that may indicate protection changes little in this period. For example, the proportion of the population with anti-type-14 antibody concentrations less than 0.2 μg/ml declines from approximately 55% to approximately 40%, and the proportion with less than 0.5 μg/ml is reduced from about 95% to about 80%. Similar patterns are seen in the Finnish antibody data for type 6B [ 12 ]. Thus, as in the Israeli data, only a very small shift in the distribution of type-specific anti-polysaccharide antibody concentration is observed during the second year of life, yet incidence of invasive disease from the serotypes in question declines substantially. Figure 4 Age-Specific Incidence of Invasive Pneumococcal Disease Caused by Serogroups 6 and 14 in Finland, Based on Active Laboratory-Based Surveillance Discussion We have assessed two lines of epidemiological evidence, analyzed ecologically, that bear on the role of anticapsular polysaccharide antibody as the determinant of protection against invasive disease that develops during the second and third year of life. The simultaneous and approximately parallel nature of the decline in disease incidence for the seven most important serogroups in the United States suggests that one mechanism, rather than seven independent mechanisms, account for the declines in invasive disease from these serogroups. Moreover, only a slight increase in anticapsular antibody concentration is measurable in Finnish, United States, and Israeli toddlers during the same age range. As we discuss below, each of these lines of evidence is subject to caveats, but we believe that, taken together, these observations make a strong case for the importance of one or more factors other than acquisition of anticapsular antibodies in the development of protection against pneumococcal disease. There are several possible candidates for mechanisms that could explain this age-related decline in pneumococcal disease. These include the following: acquisition of antibodies or cellular immune responses to noncapsular pneumococcal “species” antigens; age-related changes in host biology that are not related to acquired immunity, such as maturation of the innate immune system or changes in anatomy or receptors for pneumococcal attachment; changes in other risk factors, such as exposure; or changes related to other microorganisms, including changes in the resident flora or changes in the incidence of viral infections. Systemic antibodies to several pneumococcal protein antigens, which are conserved across pneumococcal strains and serotypes, develop following pneumococcal carriage and otitis media and are present by the beginning of the second year of life [ 20 , 21 ]. In both Finland [ 20 ] and Kenya [ 21 ], there is an increase in the concentration of antibodies to the pneumococcal proteins pneumolysin and pneumococcal surface protein A over the first two or more years of life. In Kenya, antibodies to another conserved protein, pneumococcal surface adhesin A, showed similar distributions in the first, second, and subsequent years of life, while in Finland, levels of these antibodies were already high (equivalent to adult levels) in the first year of life, and increased above these levels in the second year. In mice, either passively transferred human serum IgG against pneumococcal surface protein A or vaccine-induced antibodies to pneumococcal surface protein A and/or pneumolysin are protective against invasive disease. Such data are consistent with the hypothesis that antibodies to these, or perhaps other, conserved pneumococcal proteins are in part responsible for the decline in invasive disease in the second and subsequent years of life. A number of investigators have tested the hypothesis that antibodies to the pneumococcal teichoic acid, known as cell wall polysaccharide (CWPS), are capable of protecting individuals against pneumococcal invasive disease. While studies in animals [ 22 ] and humans [ 23 ] have failed to find a protective effect of antibodies to CWPS or its components, a recent study showed that passive transfer of human IgG against phosphorylcholine, a component of CWPS, could protect mice against invasive pneumococcal infections [ 24 ]. Notably, such antibodies might be elicited by a number of bacteria in addition to pneumococci, such as Haemophilus influenzae, which also produce phosphorylcholine. We are unaware of studies on the timing of acquisition of anti-CWPS antibodies. We have recently shown that mice that are exposed thrice at weekly intervals to intranasal colonization with encapsulated pneumococci are protected against subsequent carriage, that this protection is effective for heterologous as well as homologous capsular types, and that it is effective even in MuMT mice, which lack the ability to produce antibodies (Malley R, Trzcinski K, Srivastava A, Thompson CM, Anderson PW, et al., unpublished data). We have also shown that intranasal immunization with unencapsulated, killed pneumococci protects against nasopharyngeal colonization, in a fashion that is independent of antibody but requires CD4+ T cells at the time of challenge. The relevance of cellular immune mechanisms in protecting humans against pneumococcal colonization or disease is not known. Another candidate for a factor that may be changing with age is susceptibility to viral infections, especially influenza, which may predispose to pneumococcal colonization [ 25 ] or disease [ 26 , 27 ]. Recent evidence from clinical trials of pneumococcal conjugate vaccines shows that the vaccines can reduce the incidence of infections such as bronchiolitis that are usually associated with viruses [ 28 ] and of documented, virus-associated pneumonia [ 27 ]. These findings raise the possibility that the decline in pneumococcal disease with age reflects, in part, a decline in the incidence or severity of viral infections, so that fewer such infections lead to secondary pneumococcal disease. Exposure to pneumococci probably changes in some fashion over the first 5 y of life. However, for changes in exposure to account for the sharp drop in disease incidence following the first birthday, it would be necessary for exposure also to drop severalfold per year over this age range. Studies of pneumococcal carriage do show gradual changes in the prevalence and serotype composition of the nasopharyngeal flora in these years, but the prevalence of carriage changes much more gradually than the incidence of invasive disease [ 29 ]. We are not aware of data that bear strongly on the plausibility of other possible mechanisms for the age-related decline in pneumococcal disease, such as changes in anatomy, physiology, receptor expression, or resident bacterial flora. However, factors other than antibody—such as innate or acquired cellular immune responses, age-related anatomical changes, or changes in exposure to pneumococci—cannot be ruled out, and more than one factor may be involved. Indeed, the peak of pneumococcal meningitis incidence in the 3–6-mo age group (see Figure 2 ) suggests that the mechanism of protection against meningitis may differ from those against pneumonia and bacteremia. Although we suggest that anticapsular antibody is not primarily responsible for the age-specific decline in invasive pneumococcal disease, there is no question that the capsule is an important virulence factor that interacts with the innate and acquired immune system in a number of ways. It is clear that the pneumococcal capsule interferes with various host clearance mechanisms [ 30 ]. It would be unsurprising if different capsular types were differentially effective in permitting pneumococci to evade phagocytosis and other host defenses [ 31 ] (M. Melin, H. Jarva, S. Meri, and H. Käyhty, unpublished data). If this were the case, then one could envision that certain capsular types might in fact follow a different age-specific incidence. In particular, recent analyses suggest that serotypes 1 and 5 have relatively stable incidence over a range of age groups (W. P. Hausdorff, D. R. Feikin, and K. P. Klugman, unpublished data). The evidence adduced here is subject to several limitations. With respect to the relative timing of acquisition of protection against different serotypes, one could postulate that because some of the most common pneumococcal serotypes, such as 6B, 19F, and 23F, are also among the least immunogenic [ 12 ], the effective exposure of the immune system is more consistent across serogroups than it appears from serogroup frequency alone. However, this pattern is not general; for example, serotype 14 is both very common and highly immunogenic [ 12 ]. With respect to the absolute timing of protection relative to the acquisition of antibody, one could argue that low levels of anticapsular antibody, perhaps of low affinity, may be present and even active at levels below those that can be reliably detected by current assays, or that B cell memory may be present and protective at an earlier age than that at which high levels of antibody are measurable. Inferences about protective antibody concentrations from animal studies and from concentrations achieved by vaccines suffer from several uncertainties. Making allowances for all of these limitations, we nonetheless believe the data suggest that mechanisms other than anticapsular antibody are primarily responsible for the age-specific decline in pneumococcal invasive disease that starts at the age of 1 y. The likelihood that mechanisms other than anticapsular antibody confer immunity to pneumococcal disease has important implications with respect to vaccine design. As experience with conjugate pneumococcal vaccines in children unfolds, it is becoming increasingly clear that such a strategy suffers from several limitations, including the possibility of serotype replacement (already confirmed in several clinical trials), a modest effect on nasopharyngeal colonization, limited serotype coverage, cost, and difficulties in production that have led to shortages since licensure. A better understanding of the mechanisms that underlie natural immunity to pneumococcus could pave the way for the development of more effective, species-specific pneumococcal vaccines. Patient Summary Background Streptococcus pneumoniae is a common bacterium that lives in the upper respiratory tract of many children, and some adults. The bacterium generally causes no harm in healthy individuals, but in some circumstances it can cause mild infections, such as ear infections, or more severe ones, such as lung infection (pneumonia), bloodstream infection (bacteremia), or infection of the lining of the brain (meningitis). These more severe forms, called invasive pneumococcal disease, occur especially in children, elderly people, and others with weakened immune systems. The bacterium exists in different versions, or serotypes. The different versions of the bacterium each have a different outer shell (the so-called bacterial capsule). Scientists have developed vaccines against Streptococcus pneumoniae that protect against the most common serotypes. These vaccines consist of a cocktail made up of material from the capsules of the most common serotypes. This material causes the body's immune system to produce antibodies that can fight Streptococcus pnemoniae and protect vaccinated individuals against disease caused by the common serotypes. In many developed countries vaccination is recommended for all children and elderly people. Why Was This Study Done? Most people get exposed to many different versions of the bacterium over the course of their lives. These encounters cause little or no disease in most people, and the risk of disease declines sharply and remains low through middle age, before climbing again in the elderly. Based on experience with vaccines, scientists have thought that this “natural” protection that develops with age was also based on antibodies against the bacterial capsule. The authors of this study wanted to test whether this was actually true. What Did the Researchers Do? If in the healthy population protection against invasive disease is in fact due to anticapsular antibodies, one can make certain predictions about the frequency of invasive disease among certain age groups. The researchers tested those predictions against actual disease records from the United States, Israel, and Finland. What Did They Find? The actual records did not match the predictions very well, suggesting that natural protection against invasive pneumococcal disease is not based on anticapsular antibodies alone. What Does This Mean? These results suggest that there are elements of natural protection against invasive pneumococcal disease that we do not understand yet. Moreover, these elements seem to involve more general protection against various forms of the bacterium rather than individual protection against particular serotypes. What Next? Given the importance of the disease, we should try to understand all elements of natural protection. Such understanding might help researchers develop better vaccines to prevent invasive pneumococcal disease, and maybe even improve treatment of patients who have become ill. More Information Online World Health Organization information page on pneumococcal vaccines: http://www.who.int/vaccines/en/pneumococcus.shtml United States Centers for Disease Control and Prevention factsheet on pneumococcal vaccine: http://www.cdc.gov/nip/publications/VIS/vis-PneumoConjugate.pdf Health Canada information on pneumococcal vaccine: http://www.hc-sc.gc.ca/english/iyh/medical/pneumococcal.html Information for health-care providers from the United Kingdom Nation-al Health Service: http://www.prodigy.nhs.uk/guidance.asp?gt=Immunizations%20-%20pneumococcal PneumoADIP Web page on childhood pneumococcal disease: http://www.pneumoadip.com/	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545206.xml
514707	Treatment of Retinopathy of Prematurity with topical ketorolac tromethamine: a preliminary study	Background Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress to blindness from retinal detachments or severe retinal scar formation. This is a preliminary report of the effectiveness and safety of a new and original use of topical ketorolac in preterm newborn to prevent the progression of ROP to the more severe forms of this disease. Methods From January 2001 to December 2002, all fifty nine preterm newborns with birthweight less than 1250 grams or gestational age less than 30 weeks of gestational age admitted to neonatal intensive care were eligible for treatment with topical ketorolac (0.25 milligrams every 8 hours in each eye). The historical comparison group included all 53 preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. Results Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing enterocolitis. The duration of oxygen therapy was significantly longer in the control group. In the ketorolac group, among 43 children that were alive at discharge, one (2.3%) developed threshold ROP and cryotherapy was necessary. In the comparison group 35 children survived, and six child (17%) needed cryotherapy (Relative Risk 0.14, 95%CI 0.00 to 0.80, p = 0.041). Adjusting by duration of oxygen therapy did not significantly change these results. Adverse effects attributable to ketorolac were not detected. Conclusions This preliminary report suggests that ketorolac in the form of an ophthalmic solution can reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size.	Background Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress in some infants to partial vision loss or blindness from retinal detachments or severe retinal scar formation [ 1 ]. ROP remains as one of the most frequent cause of blindness in children, in particular in countries with infant mortality rates between 10 and 60/1000 [ 2 , 3 ]. Among 177 students attending schools for children with visual impairment in the city were this study was conducted, 107 (60.5%) had ROP [ 4 ]. The incidence of both any acute ROP, and of the more severe stages, varies inversely with gestational age at birth. ROP is unusual (except in the mildest forms) in infants of greater than 31 weeks gestation, and severe complications such as retinal detachment occur in less than one half of one percent of infants greater than 31 weeks gestation. However, more than 80% of infants less than 28 weeks gestation develop some ROP, and around 10% develop "threshold ROP. In threshold ROP more that 40% of the cases progresses to retina folds or detachment, with its consequent blindness. In this stage, ablative surgery (cryotherapy or laser photocoagulation) to the peripheral avascular retina is recommended to reduce the risk of disease progression to retinal detachment [ 5 - 7 ]. Pre-threshold stage has been linked to bad results in the visual function: reduction of visual acuity, short-sightedness, amblyopic, etc. [ 8 , 9 ]. A number of strategies have been developed to try to diminish the progress of ROP, but with limited success. These strategies include antioxidants such as vitamin E [ 10 ], D penicillamine [ 11 ] and allopurinol [ 12 ], reduction of exposure to light [ 13 ] and supplementation with oxygen [ 14 ]. The active disease appears in the premature about 4 to 8 weeks after birth. In this period, the levels of vascular endothelial growth factor (VEGF) increase in the retina, as well as other chemical mediators of inflammation such as platelets activator factor (PAF), prostaglandins (PGs), and eicosanoids which would put again under way the process of vascularization that had stopped in the period of oxidative injury. This vascularization is now degenerated and invasive [ 15 - 17 ]. In models of animal experimentation it was possible to diminish the degree of retinal neovascularization with use of indometacin [ 18 ], dexamethasone [ 19 ], rofecoxib [ 20 ], and bucillamine [ 21 ], and increased activity of cyclooxygenase 2 (COX2) was also demonstrated in vessels of neo-proliferation in retina and its inhibition decreases the neovascularization in 37% [ 20 ]. Ketorolac is a non-steroid anti-inflammatory drug (NSAID) derived from indometacin. Its mechanism of action is developed through the interruption of the synthesis of prostanoids, inhibiting the way of COX 1 and 2 in arachidonic acid metabolism; in this way the tissue levels of prostaglandin F2alfa and thromboxane B2 decrease. Its adverse effects are linked predominantly to their inhibitory action of platelet aggregation. It does not alter either the platelet count, or factors of clotting. High digestive hemorrhage is the principal adverse reaction. Nervous and cardiovascular systems are not generally affected by the use of ketorolac to habitual doses [ 22 - 24 ]. Ketorolac administered as conjunctival topical diminishes prostaglandin E2 concentration in aqueous humor, without modifying the intraocular pressure [ 24 ]. In addition it is effective in uveitis induced by tumors necrosis factor (TNF) [ 25 ]. Ketorolac is unquantifiable in plasma when administered in ophthalmic drops [ 24 ]. Ketorolac ophthalmic solution is usually used in older adults with retinal disorders. It is used to diminish the Cystoid Macular Edema that complicates the surgery of cataracts. In this pathology ketorolac has proved to be effective in diminishing the macular edema and improving the visual acuity, providing evidence that their conjunctival instillation produce effects on the most internal layers of the eye. The ophthalmic use of ketorolac only reports occasional episodes of discomfort and ocular burning [ 26 - 28 ]. The use of ketorolac ophthalmic solution in pediatrics is frequent as an analgesic in corneal abrasions, and in allergic and post surgical conjunctivitis. The FDA recognizes its indication for allergic conjunctivitis, ocular pain, post surgical ocular inflammation, ocular pruritus and photophobia [ 24 , 29 ]. On the basis of experimental evidence and physio-pathogenic rationality, we treated preterm newborns admitted to neonatal intensive care unit (NICU) of our hospital with ketorolac in ophthalmic drops with the aim of decreasing the progression and severity of ROP. This study compares this group of children treated with ketorolac with historic controls to assess the impact of this treatment on the incidence of severe ROP and adverse effects. Methods This is a preliminary report, that compares a cohort of children treated with ketorolac with historic controls that did not received such treatment. From January 2001 to December 2002 all preterm newborns with birthweight less than 1250 g or gestational age less than 30 weeks of gestational age, admitted in the NICU of the University Hospital of Maternity and Neonatology of the city of Córdoba, Argentina, were eligible for treatment with topical ketorolac. The comparison group included all preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. None of these newborns received treatment with ketorolac. There were no differences either in treatment guidelines, equipment or in the number of physicians and nurses that took care of the patients in the two periods. All the children were examined by the same group of ophthalmologists, which have many years of experience in treating patients with ROP. The international classification for ROP [ 30 ] was used to define the stages of the disease and verified by more than one observer. During the period in which ketorolac tromethamine was used, when risk signs for ROP were identified (zone I incomplete vascularization, vessels only in zone of transition I-II or anomalous ramification and equatorial incurvation of vessels in the avascular – vascular junction) [ 31 ] treatment was started with a drop of ketorolac tromethamine (0.25 mgrs.) every 8 hours in each eye. The treatment continued until they presented signs of threshold ROP and cryotherapy was indicated, or till the resolution of the condition. Parents of these children gave their consent so that their children could receive treatment. The variables considered to assess the comparability among both groups were: birthweight, Apgar score at birth less than 6 at 5 minutes, duration of oxygen therapy, peri-intraventricular hemorrhage (PIVH) equal or greater than 3 degrees, necrotizing enterocolitis (NEC) equal of greater than II degrees, and late sepsis. The analyses included only those children that were alive at discharge, but the number of deaths was reported for both study periods. The presence of undesirable effects of ketorolac such as hemorrhages, oliguresis, local manifestations of intolerance, and conjunctival infection were analyzed. Fischer exact test was used to obtain two tailed p-values. Relative risks (RR) with 95% confidence intervals where also calculated. Adjusted analyses were performed by using Poisson regression with robust estimates [ 32 ]. Statistical analysis was carried out using the statistical software SPSS version 8.0, Stats Direct, and STATA version 8.0. Results During the analyzed period 112 eligible preterm newborns were admitted, 53 between 1999 and 2000 (historic controls) and 59 between 2001 and 2002 (ketorolac group). The number of newborns that died before discharge was similar between groups (see table 1 ). Table 1 presents the characteristic of those alive at discharge. Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing Enterocolitis (see table 1 ). The duration of oxygen therapy was significantly longer in the control group (see table 1 ). Table 1 Characteristics of newborns treated with ketorolac and historic controls. Historic controls Ketorolac p** Year 1999 – 2000 Year 2001–2002 N = 35† N = 43† n(%) n(%) Dead before discharge* 18 (34.0) 16 (27.1) 0.54 Birthweight (g) 0.28 <= 750 4 (11.4) 8 (18.6) 751–1000 13 (37.1) 9 (20.9) 1001–1250 18 (51.4) 26 (60.59 Apgar Score < 6 at 5 minutes 10 (28.6) 11 (25.5) 0.80 Oxygen (days) 0.01 0–10 9 (25.7) 21 (48.8) 11–27 10 (28.6) 15 (34.9) >= 28 16 (45.7) 7 (16.3) Sepsis 16 (45.7) 24 (55.8) 0.50 Peri–intraventricular hemorrhage > grade III 8 (22.9) 6 (14.0) 0.38 Necrotizing Enterocolitis >= II degree 5 (14.3) 9 (20.9) 0.56 † Excludes newborns that died before discharge. * N = 53 in historic controls group and N = 59 in ketorolac group, that includes all eligible live newborn during the study period. ** P-value Fisher exact test. In the ketorolac group 45 children were alive at the first ophthalmic control, two died before discharge due to late sepsis, so 43 received treatment with ketorolac. Nineteen children were discharged from NICU with treatment indication, and continued with ketorolac up to 44 weeks of gestational age, when the ophthalmologists considered that the risk for developing ROP was very small. The incidence of threshold ROP in newborns treated with ketorolac was significantly lower (Relative Risk Reduction 86%) than in the control group (see table 2 ). Adjusting for duration of oxygen therapy did not significantly change these results (see table 2 ). Further exploration of this relationship showed that duration of oxygen therapy equal or higher than 28 days was not associated with severe ROP in this data (Relative Risk 1.04, 95%CI 0.25 to 4.51, p = 0.28). This finding explains why duration of oxygen therapy does not act as a confounder. Hemorrhages were not observed in the vitreous after treatment with ketorolac. In four cases hemorrhages in the vitreous were already present at the beginning of therapy and they disappeared after 14 days of treatment. No signs of local intolerance, or conjunctival infection were observed. We did not find hemorrhages in other organs attributable to the drug, or signs of renal failure. Treatment was not suspended in none of the cases and all preterm babies received ketorolac until its interruption due to the resolution of ROP or the indication of cryotherapy. Table 2 Incidence of threshold retinopathy of prematurity in newborns treated with ketorolac and historic controls. Only those alive at discharged included in the analyses. Historic Controls Ketorolac Relative Risk (95%CI) P-value n/N(%) n/N(%) Crude Adjusted* Threshold retinopathy of prematurity 6/35 (17.1) 1/43 (2.3) 0.14 (0.00 to 0.80) 0.041** 0.12 (0.02 to 0.92) 0.04 * Adjusted by oxygen administration using robust Poisson Regression. **Fisher exact test Discussion In this preliminary study we have shown that the incidence of severe ROP was significantly lower in very preterm newborns treated with ketorolac, compared with historic controls not receiving such treatment. These results suggest that administration of ketorolac as an ophthalmic solution might be an effective preventive strategy in patient at risk of developing severe ROP. The study was conducted on non-concurrent patient groups, and changes in the subjects risk profile or quality of care between the two study periods might have an impact in the risk of developing severe ROP. The magnitude of the effect of ketorolac on the incidence of severe ROP found in this study (from 17% in the control group to 2.3% in the ketorolac group) is large and in our opinion unlikely to be explained completely by confounding factors, although this possibility cannot be completely ruled out. To our knowledge, no significant change in the standard of care took place between the study periods, but mortality was lower during the period were ketorolac was used, although the difference was not statistically significant. The groups were comparable in terms of their birthweight distribution, Apgar score, and indicators of severe morbidity. A significant difference was found in duration of oxygen therapy, but in our data there was no association between duration of oxygen therapy and severe ROP, and adjusting for oxygen did not change the results. The principal factor involved in the genesis and severity of ROP is the injury due to O2 radicals; there were no substantial changes in the management of O2 supply to the children of our unit in both periods, the equipment used to measured O2 saturation and to administer the oxygen. The prevalence of severe ROP in our maternity was relatively high, considering that for 1998 the Vermont-Oxford Network reported an incidence of 9.5% for severe ROP and of 57.2% for ROP of any degree [ 33 ], but similar to the one reported by other units in our country and in others countries with similar development [ 34 - 36 ] Ketorolac was apparently safe. No patients in the group that received ketorolac presented oliguric, or with biochemical signs of renal failure during treatment. There were no hemorrhagic manifestations that could be attributed to ketorolac; hemorrhages observed in vitreous evolved favorably with collyrium. Neither local intolerance episodes to the drops nor purulent conjunctivitis among the treated cases were observed. The effect on ROP we have observed is compatible with an impact of Ketorolac in the active phase of neovascularization, a fact that is also observed in numerous reports in animal models of ROP with NSAIDs and steroid drugs administered systemically. The hypothesis of an inflammatory component in active ROP has been quite recently recognized by experts, extensive leukocyte adhesion was observed at the leading edge of pathological neovascularization [ 17 ]. Recent research work with inhibitors of COX2 shows that anti-inflammatory drugs apparently have an impact on the evolution of ROP [ 20 ]. Neufeld and collaborators found high plasmatic profiles of TNF and other cytokines during the phase of installation of the pre-threshold stage for ROP [ 37 ]. COX has a strong angiogenic action in the normal development of retina. The inhibition of COX2 diminishes the angiogenesis in cancer and rheumatoid arthritis [ 38 ]. The ganglion cells of retina secrete PGs that would interact with angiogenic substances as VEGF and Insulin like growth factor. The neo-vessels express a greater concentration of COX2 and inhibitors of COX2 stop the angiogenesis mediated by VEFG, which is the principal factor involved in the neovascularization and which would be regulated by the PGs secreted by ganglion cells of retina and endothelial cells [ 20 ]. The hyperoxia induces liberation of TNF with a powerful inflammatory action; dexamethasone interferes with TNF production attenuating the manifestations of retinopathy by hyperoxia [ 19 ]. Topical ketorolac is very effective in neutralizing uveitis generated by experimental infection with monoclonal TNF or bacterial endotoxins [ 25 , 39 , 40 ]. ROP is a problem that modern medicine has generated due to the survival of very small preterm children and which has been unable to solve so far. About 2% of the children with birthweight less than 1500 grams are blind because of this pathology. Current preventive interventions can prevent blindness in nearly 70% of the cases. Earlier treatment using ablation of the avascular retina in pre-threshold ROP has been proposed [ 41 ], but a preventive measure that could avoid the need of ablation would be much better. Conclusions This preliminary report provides evidence suggesting that ketorolac in the form of an ophthalmic solution can be used to reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. We cannot exclude the possibility of bias been responsible of the observed effect. On the other hand, if confirmed, these results can have important public health implications. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size to provide a definite answer to this research question. Competing interest None declared. Authors' contributions MAV and RM conceived the study. MAV, MLC, EB and RM design the study. MF carried out the revision of the clinical histories. MS carries out the ophthalmologic exams of all the cases. RM verified the ophthalmologic finds. EB carried out the statistical analysis. MAV and EB drafted the article. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514707.xml
423154	Ecology Drives the Global Distribution of Human Diseases	null	It's no surprise that the Amazonian rainforest contains far more species than, say, the Siberian tundra. Over 50% of the world's species live in tropical rainforests, which cover just 6% to 7% of the earth's terrestrial surface. That the number of marine and terrestrial species declines with distance from the equator is a well-documented phenomenon called the latitudinal species diversity gradient. What's proven challenging, however, is figuring out what drives this pattern. Over 30 hypotheses have been proposed in the past two decades, but only four have garnered serious attention. These four focus on variables relating to area and energy factors, geographic constraints, and habitat diversity. Understanding the factors—both contemporary and ancient—responsible for the diversity gradient could help answer one of the fundamental questions in evolutionary ecology: what regulates species diversity? But teasing out the likely mechanisms behind this diversity has practical implications as well: mounting evidence suggests that ecological and climatic conditions influence the emergence, spread, and recurrence of infectious diseases. Global climate change is likely to aggravate climate-sensitive diseases in unpredictable ways. The number of pathogen species increases towards the equator Increasingly, public health programs aimed at preventing and controlling disease outbreaks are considering aspects of the ecology of infectious diseases—how hosts, vectors, and parasites interact with each other and their environment. The hope is that by understanding how ecological factors impact the global distribution of parasitic and infectious diseases, public health officials can predict and contain future outbreaks. Even though parasitic and infectious organisms account for a major fraction of the biological diversity on the planet, few studies have analyzed the factors affecting the spatial distribution of these organisms or attempted to quantify their contribution to biodiversity. In this issue, Vanina Guernier, Michael Hochberg, and Jean-François Guégan address the influence of ecological factors on the biological diversity and distribution of parasitic and infectious diseases and find that climatic factors are the most important determinant of the global distribution of human pathogens. The current understanding of human disease and availability of complete datasets on many parasitic and infectious diseases, the researchers explain, present a unique opportunity to explore the relationship between parasitic and infectious disease species richness (defined in their study as total number of pathogens within a given country's borders) and latitude. This information, in turn, can help identify potential factors that affect diversity gradients. After compiling epidemiological data on 332 different human pathogens across 224 countries, Guernier et al. used sophisticated statistical modeling methods to identify and characterize the influence of a number of potential contributing factors on species richness. After adjusting the model to control for cofactors that might influence the relationship between latitude and species richness indirectly rather than directly (cofactors such as the size of countries and demographic, economic, and environmental variables), the researchers confirmed that, on average (seven times out of ten), tropical areas harbor a larger number of pathogen species than more temperate areas. In other words, the species richness of human pathogens follows the same pattern seen in other species. These results, Guernier et al. argue, suggest that the latitudinal species diversity gradient “might be generated in large part by biotic interactions.” This in turn indicates that current estimates of species diversity, which ignore parasites and infectious organisms, are “substantially underestimated.” The authors went on to explore groupings of individual pathogen species within larger parasitic and infectious disease communities along the gradient and found that species present at northern latitudes are a subset of those present in equatorial areas, rather than a different set of species (a phenomenon called “nestedness”). Since nestedness is strongly associated with latitude, which is typically used as a proxy for a range of climatic factors, the researchers investigated the relationship between various climatic variables and pathogen diversity. The climatic variable most strongly correlated with diversity was the maximum range of precipitation of a region. The finding that climatic factors are largely responsible for the spatial distribution of human pathogens has important implications for predicting and managing future infectious disease outbreaks. These results counter the conventional assumption that socioeconomic conditions are the most important factor in controlling disease, indicating that global climate change could have far more significant effects on global patterns of disease, with diseases once relegated to the tropics migrating to temperate zones, for example. Identifying the links between ecology and disease, however, could lay the foundation for effective preventive strategies.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC423154.xml
529445	A common genetic factor underlies hypertension and other cardiovascular disorders	Background Certain conditions characterised by blood vessel occlusion or vascular spasm have been found to cluster together in epidemiological studies. However the biological causes for these associations remain controversial. This study used a classical twin design to examine whether these conditions are linked through shared environmental exposures or by a common underlying genetic propensity to vasospasm. Methods We investigated the association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease in twins from a national register. Phenotype status was determined using a questionnaire and the genetic and environmental association between phenotypes was estimated through variance components analysis. Results Responses were obtained from 2,204 individuals comprising 525 monozygotic and 577 dizygotic pairs. There was a significant genetic contribution to all four traits with heritabilities ranging from 0.34 to 0.64. Multivariate model-fitting demonstrated that a single common genetic factor underlies the four conditions. Conclusions We have confirmed an association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease, and shown that a single genetic factor underlies them. The demonstration of a shared genetic factor explains the association between them and adds weight to the theory of an inherited predisposition to vasospasm.	Background A number of conditions characterised by blood vessel occlusion and/or vascular spasm have been found to be associated in both clinical and epidemiological studies. These include hypertension (HPT), Raynaud's phenomenon (RP), migraine (MIG) and coronary artery disease (CAD) [ 1 - 5 ]. Whether these associations are the consequences of a shared environmental risk factor or represent an underlying propensity to develop the conditions through a common biological mechanism remains controversial. The exploration of the genetic and environmental relationships underlying these conditions is one approach to resolving the biological basis for the association. We have examined the association between these phenotypes in a classical twin study conducted in a large sample. This approach allows us to assess whether the association between HPT, RP, MIG and CAD is explained by a common genetic or environmental aetiology. Methods Subjects and methods Subjects for this study were twins enlisted with the St Thomas' UK Adult Twin Registry [ 6 ]. These monozygotic (MZ) and dizygotic (DZ) twin volunteers have been recruited since 1992 using twin registers and successive national media campaigns. For historical reasons most enrolled twins are female. This well studied population is sent regular questionnaires for self-completion concerning a wide range of health issues. Questions relating to HPT, MIG, RP and CAD respectively were contained within large questionnaires sent to the twins in 1998 and 2000. The questions were non-consecutive and respondents were unaware of the hypothesis being tested. The questionnaires also included standard questions relating to zygosity assignment [ 7 ]. In addition, fifty-four percent of the respondents had their zygosity assigned with certainty by multiplex DNA fingerprinting using variable tandem repeats on samples taken on previous attendances at the Twin Unit. Classification of HPT, RP, MIG AND CAD Classification of the traits HPT, RP, MIG and CAD was based on standard, validated criteria where possible. HPT was classified by asking about a doctor's diagnosis of "high blood pressure" when not pregnant. Questions to determine presence of migraine were based on the UCSD Migraine Questionnaire [ 8 ] (including at least five episodes of unilateral, pulsating headache over the previous year, duration four to seventy-two hours, noise and light sensitivity). RP was classified by confirming digital sensitivity to cold and required the respondent to have had recurrent episodes of colour change involving at least two colours [ 9 ]. CAD was classified either by respondents having a doctor's diagnosis of heart disease or angina, a previous heart attack or heart operation; or by answering affirmatively to questions taken from the Rose Angina questionnaire [ 10 ]. Analysis Traits were defined categorically as present or absent using the definitions listed above. Phenotypic associations between the four traits were assessed using odds ratios and 95% confidence intervals (CI) corrected to take into account the paired nature of the data. The odds ratios were adjusted for possible confounding by age, smoking and body mass index (BMI). Evidence for a genetic contribution to each trait was examined by estimating casewise concordance [ 11 ]. This measures the probability that the co-twin of an affected twin also expresses the trait. Higher casewise concordance in MZ pairs compared to DZ twin pairs indicates a genetic effect. Where data suggested a genetic influence, a quantitative measure of genetic contribution was estimated using structural equation modelling (Mx software [ 12 ]). This standard approach to twin analysis assumes an underlying model in which the observed phenotypic correlation among twins is explained by latent additive genetic influences (A) (which have a correlation of 1 in MZ twins and 0.5 in DZ twins), common environmental influences (C) (having a correlation of 1 in both MZ and DZ twins) and the random environment (E) (uncorrelated among twins). Analysis proceeds on the assumption that the observed categorical phenotype is accounted for by a continuous underlying liability to the trait [ 13 ]. Correlation in underlying liability is used as the basis for measuring the association between variables in the modeling. The significance of shared genetic and environmental factors was tested by stepwise deletion in a sequence of models containing the variance components (A, C and E) and assessing the deterioration in chi-squared for the fit of the model. Heritability was estimated from the size of the additive genetic contribution to the final selected model. Modeling was then extended to consider the genetic and environmental associations between all four variables simultaneously [ 13 ]. Three multivariate models were considered:- (1) the Cholesky decomposition (Figure 1 ) which includes four independent genetic and environmental factors. The first factor loads on all the traits, the second factor loads on all traits except the first, the third loads on all traits except the first two etc. This provides the fullest potential explanation of the data Figure 1 The Cholesky AE model. Diagram representing the Cholesky AE model, in which additive genetic (A) effects are shown loading on to the four traits: the unique environment (E) would load similarly (2) the independent pathway model (Figure 2 ) is a submodel of the Cholesky model and considers the data to be explained by a single shared genetic and shared environmental factor Figure 2 The independent AE pathway model. Diagram of the independent AE pathway, in which a single genetic and single unique environment factor loads on to each of the four traits, as well as factors specific to each trait (3) the common pathway model (Figure 3 ) which considers a single shared latent phenotype, determined in turn by latent genetic and environment factors. Figure 3 The common pathway model. Diagram of the common factor pathway in which single genetic and unique environment factors load on to the traits via a phenotypic latent variable The suitability of the common and independent pathway models may be determined by comparing the model's Akaike information criterion (AIC) with that of the fullest model, the Cholesky. The AIC represents the balance between model fit and the number of parameters (parsimony), with lower values of AIC indicating the most suitable model. Parameter estimates from the most appropriate model were used to calculate the genetic and environmental correlation between variables. Results Complete data for analysis were available from 1,102 pairs of female twins, comprising 525 MZ and 577 DZ pairs. The mean (± SD) age of the twins was 48.5 (± 8.2) years. The prevalence of each trait is shown in Table 1 . Table 1 Prevalence of the traits by zygosity and the p value of the difference between zygosities trait MZ prevalence (%) n = 1050 DZ prevalence (%) n = 1154 p HPT 131 (12.5) 181 (15.7) 0.052 RP 111 (10.6) 131 (11.4) 0.584 MIG 264 (25.1) 266 (23.1) 0.290 CAD 71 (6.8) 76 (6.6) 0.873 n represents number of twins, MZ monozygotic, DZ dizygotic, HPT hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease Four of the six combinations of traits showed significant phenotypic association after adjusting for age, smoking and BMI (odds ratios, Table 2 ). HPT was clearly not associated with RP but there was a suggestion of association of HPT with migraine (significantly so if adjusted for age and smoking only, data not shown). Since smoking status, age and BMI did not influence the size of the odds ratios, these variables were not included in the multivariate modeling. Concordance data and the results of univariate modeling confirmed a significant heritable component to all 4 traits (Table 3 ). Multivariate model fitting showed that for each of the three models tested, a model containing genetic (A) and unique environmental (E) factors provided the best explanation of the data (Table 4 , in bold): incorporation of a shared environmental factor (C) offered no significant improvement in the fit of any model. The independent AE pathway model (Figure 2 ) offered the best explanation of the data, suggesting that a single common genetic factor loads on the four traits HPT, RP, migraine and CAD. A unique environmental factor loads similarly on the traits. Using this model, genetic and unique environmental correlations between the four traits were calculated (Table 5 ). Overall, the genetic correlations were greater than the environmental correlations, suggesting a greater role for the genetic factor than the unique environmental factor. Table 2 Odds ratios (95% confidence interval) of unadjusted and adjusted (for age, smoking and BMI) cross-trait associations HPT RP MIG CAD HPT adj RP 0.88 (0.59–1.31) adj 0.99 (0.64–1.54) MIG 1.31 (0.99–1.73) 1.62 (1.21–2.17) adj 1.31 (0.95–1.80) 1.68 (1.23–2.3) CAD 2.85 (1.96–4.16) 2.03 (1.29–3.17) 2.19 (1.53–3.13) adj 2.41 (1.56–3.73) 2.27 (1.42–3.65) 1.77 (1.19–2.63) Calculation of odds ratios included a correction factor for familial clustering. Unadjusted odds ratios are shown above and adjusted below, for each combination of phenotypes. BMI represents body mass index, HPT hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease. Table 3 Casewise concordance rates by zygosity and heritability estimates of the four traits twin type total pairs discordant pairs (+/-) concordant pairs with trait (+/+) casewise concordance (95% CI) heritability (95% CI) HPT MZ 525 73 29 0.44 (0.34–0.55) 0.64 (0.49–0.79) DZ 577 135 23 0.25 (0.17–0.34) RP MZ 525 75 18 0.32 (0.21–0.44) 0.46 (0.30–0.63) DZ 577 111 10 0.15 (0.07–0.24) MIG MZ 525 144 60 0.46 (0.38–0.53) 0.43 (0.30–0.55) DZ 577 186 40 0.30 (0.23–0.37) CAD MZ 525 55 8 0.23 (0.10–0.35) 0.34 (0.13–0.55) DZ 577 72 2 0.05 (0.00–0.12) Casewise concordance rates (95% confidence intervals, CI) are shown for each trait by zygosity. An estimate of trait heritability (95% CI) is also given. HPT represents hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease. Table 4 Results of the model fitting: comparison of the 3 models model chi 2 df p AIC CHOLESKY ACE 32.95 30 0.33 -27.05 AE 33.35 40 0.76 -46.65 CE 65.82 40 0.01 -14.18 INDEPENDENT ACE 38.98 36 0.34 -33.02 AE 40.82 44 0.61 -47.18 CE 72.03 44 0.01 -15.97 COMMON FACTOR ACE 49.68 42 0.19 -34.32 AE 49.68 47 0.38 -44.32 CE 81.49 47 0.00 -12.51 For each submodel, the chi 2 statistic, the degrees of freedom (df) the probability (p) and the Akaike information criterion (AIC) are shown. AIC is used to evaluate the fit, with the best fitting submodel shown in bold in each case. Table 5 Genetic correlations (bold type, below in table) and unique environmental correlations (above) of the traits HPT RP MIG CAD HPT 0.29 0.01 0.17 RP 0.20 0.00 0.05 MIG 0.23 0.36 0.00 CAD 0.35 0.56 0.65 HPT represents hypertension, RP Raynaud's phenomenon, MIG migraine, CAD coronary artery disease Discussion This is the first study to examine the role of genetic and environmental factors in explaining the association between HPT, RP, migraine and CAD. The results suggest that all four variables share a heritable basis. These conditions have been shown to be associated with one another and individually each is known to have a genetic basis. The data presented here confirm these previous findings and suggest that shared environmental factors such as diet and lifestyle do not contribute to their expression. In view of the nature of these phenotypes, we speculate that the shared genetic component leads to a predisposition to vasospasm. Indeed, a 'vasospastic phenotype' to account for their co-occurrence has been postulated by others [ 14 ]. The demonstration of a single genetic component lends weight to such a theory. A number of considerations should to be taken into account when interpreting these results. Self administered questionnaires were used for trait ascertainment, introducing the possibility of recall bias. In the present study efforts were taken to minimise recall bias: when surveyed, the twins were unaware of the hypothesis being tested; the questions relating to vascular phenotypes were included non-sequentially and were amongst many other questions in two questionnaires mailed at different times; and the twins completed questionnaires separately with no knowledge of their co-twin's responses. Furthermore there is no reason to suspect differential rates of recall in MZ when compared to DZ twins, hence any effects of recall should not have biased our estimates of genetic influence. Some conditions, such as RP, are notoriously difficult to diagnose regardless of the method employed [ 15 ] and it is possible that some subjects have cardiac valve rather than coronary artery disease. No attempt was made to differentiate primary RP and essential HPT from their secondary forms. However, questionnaire diagnoses were based on standard methods [ 8 - 10 ]. Despite these limitations, the traits' prevalences are in keeping with the findings of others for RP (9.6%[ 3 ], 5% and 16.8% according to geographical area sampled [ 16 ]), migraine (20% [ 17 ]) and CAD (8% [ 18 ]). In addition, heritability estimates are consistent with published findings for MIG (49–58% [ 19 ]), blood pressure (40–70% [ 20 ], 57% [ 21 ]) and CAD (15–30% [ 18 ]). Taken together, these observations suggest that our twins are representative of the adult female population and do not simply reflect a 'healthy volunteer' sample. The assumptions underpinning twin studies themselves may be questioned. Unequal sharing of the family environment by MZ and DZ twins has been raised as a concern, but this has been shown not to be the case [ 22 ]. In addition, twins from this cohort have been shown to be similar to singletons with respect to anti-hypertensive drug use, blood pressure and other phenotypes [ 23 ]. The proposed inverse relationship between birth weight and HPT [ 24 ] and CAD [ 25 ] could potentially bias a study of twins and cardiovascular disease. The explanation for this relationship is still debated, but maternal environment has been suggested to be the main influence on adult blood pressure [ 26 ]. As we have demonstrated that the shared environment makes no significant contribution to the vasospastic phenotype, this is not a likely source of error. It is clear that genetic factors play an important role in all four conditions. The demonstration that they are heritable is consistent with numerous reports of clustering in twin and family studies conducted in a range of settings, including a Dutch kindred with an autosomal dominant condition characterised by vascular retinopathy, migraine and Raynaud's phenomenon [ 27 ]. The detection of a common, genetically determined mechanism that contributes to these conditions is important because it points to an intermediate phenotype, vasospasm, and provides a possible focus for future studies. As with all chronic diseases and traits, the difficulty is in establishing which genes are responsible. Vascular tone is controlled at many levels, including local soluble mediators and neurotransmitters. Genes proposed through the study of the individual phenotypes include the beta2-adrenergic receptor gene in hypertension [ 28 ]; the muscle acetylcholine- and serotonin receptor genes in RP [ 29 ]; and a G protein subunit polymorphism and endothelial nitric oxide gene polymorphism in CAD [ 30 ]. A gain of function mutation in a vasoconstrictor or a loss of function mutation in a vasodilator may predispose to vasospasm: many of these genes deserve further consideration with the identification of a common genetic factor underlying HPT, migraine, RP and CAD. In summary, this twin study has identified phenotypic associations between four vascular conditions and shown that the association is explained by a single common genetic factor. These findings are consistent with the proposed 'vasospastic phenotype' and suggest that studies of genes controlling vascular tone will help to define the genetic basis of these conditions. Competing interests The author(s) declare that they have no competing interests. Authors' contributions FMKW analysed the data and drafted the manuscript. LFC designed the questionnaires, analysed the data and assisted with the manuscript. TDS collected the twins on the Twin Register and assisted with the manuscript. AJM conceived of the study, collected the twins on the Twin Register, analysed the data and co-drafted the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529445.xml
526762	Transmembrane carbonic anhydrase isozymes IX and XII in the female mouse reproductive organs	Background Carbonic anhydrase (CA) classically catalyses the reversible hydration of dissolved CO 2 to form bicarbonate ions and protons. The twelve active CA isozymes are thought to regulate a variety of cellular functions including several processes in the reproductive systems. Methods The present study was designed to investigate the expression of transmembrane CAs, CA IX and XII, in the mouse uterus, ovary and placenta. The expression of CA IX and XII was examined by immunoperoxidase staining method and western blotting. CA II and XIII served as positive controls since they are known to be present in the mouse reproductive tract. Results The data of our study indicated that CA XII is expressed in the mouse endometrium. Only very faint signal was observed in the corpus luteum of the ovary and the placenta remained mainly negative. CA IX showed weak reaction in the endometrial epithelium, while it was completely absent in the ovary and placenta. Conclusion The conservation of CA XII expression in both mouse and human endometrium suggests a role for this isozyme in reproductive physiology.	Background Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that are responsible for the reversible hydration of carbon dioxide in a reaction CO 2 + H 2 O ↔ H + + HCO 3 - . CAs are produced in a variety of tissues where they participate in several important biological processes such as acid-base balance, respiration, carbon dioxide and ion transport, bone resorption, ureagenesis, gluconeogenesis, lipogenesis and body fluid generation [ 1 , 2 ]. The mammalian α-CA gene family includes at least twelve enzymatically active isoforms with different structural and catalytic properties. CA I, II, III, VII and XIII are cytosolic enzymes [ 1 , 3 , 4 ]. CA VA and VB are mitochondrial proteins encoded by nuclear DNA [ 5 , 6 ]. CA VI is the only secretory form being present in saliva and milk [ 7 ]. The cluster of membrane-bound CAs includes four isozymes: CA IV, IX, XII, and XIV [ 8 - 11 ]. The other members of the CA gene family (CA VIII, X and XI) are inactive isoforms whose functions have not yet been described [ 3 , 12 , 13 ]. It has been previously suggested that CAs may play important roles in the uterine endometrium by maintaining the appropriate pH balance through the catalysis of the production of bicarbonate ions [ 14 ]. Indeed, the role of bicarbonate in fertilization has been demonstrated in a number of previous studies. It is functionally involved in some key processes such as sperm cell capacitation and regulation of sperm motility [ 15 - 17 ]. Similarly, CAs may have several functions also in the placenta. They can be active in intermediary metabolism and provide ions for exchange in transepithelial movement of ions and fluid [ 18 ]. CA activity has been studied in pig, horse, cow, mink, rat and human placentas, and the results show considerable heterogeneity among different species [ 18 ]. Previous immunochemical studies have shown evidence for expression of CA II but not CA I or III in the bovine placenta [ 19 ]. Both CA I and II are expressed in the human syncytiotrophoblasts [ 20 - 22 ] and, especially CA II, in the fetal villous endothelium of mature placenta [ 22 ]. CA IV-positive staining has been reported in the mouse placenta by Rosen and coauthors [ 23 ]. Their data showed strong CA IV immunoreactivity in the mouse trophoblasts and endodermal layer of the yolk sac. In the mouse genital tract, CA I, II and III have been reported by Ge and Spicer [ 24 ]. These isozymes were reported to be present in the theca interna cells in the mouse ovary, and CA I was found in the zona pellucida and cytoplasmic foci in follicular granulosa cells. In the mouse oviductal epithelium, CA II expression showed distinct variation. The reaction was absent in the infundibulum, whereas the ampulla and isthmus showed positive staining. CA XIII is the newest member of the CA enzyme family, which has been described in the mouse and human endometrium along with several other positive tissues [ 4 ]. As a cytosolic isozyme it may be one of the major proteins regulating the pH and bicarbonate homeostasis not only in the endometrial cells but also in the lumen of the uterus. These mechanisms are complex due to the presence of several isozymes, however, and may greatly differ between species. For example, the human endometrium contains CA II only in the capillaries, whereas this high activity isozyme is abundantly expressed in the epithelial cells of the mouse endometrium [ 4 , 24 ]. CA IX is expressed at the basolateral plasma membrane of the human, rat and mouse epithelial cells [ 25 , 26 ]. In a recent extensive study, Ivanov et al [ 27 ] analyzed a number of normal human tissues for the expression of CA IX. Among reproductive organs, they reported positive signal for CA IX mRNA and protein in the efferent ducts, rete testis, and rete ovarii. Human CA XII is expressed in several organs including colon, kidney, and pancreas [ 28 - 30 ]. In the human female reproductive tract, CA XII has been shown both in the glandular and surface epithelium of the endometrium, while it was only occasionally present in the cervix [ 14 ]. Ivanov et al [ 27 ] further confirmed CA XII expression in the glandular epithelium during the proliferative phase. In this report we studied the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. The studies were specially focused on CA IX and XII, which have been designated as tumor-associated isozymes [ 9 , 10 ]. In addition to some normal tissues, both isozymes are overexpressed in several carcinomas such as renal and colorectal cancers [ 9 , 27 , 31 , 32 ]. A previous study has also demonstrated CA IX and XII expression in a number of neoplasias derived from the female reproductive tract [ 27 ]. However, there have been no previous studies on these isozymes in the female murine reproductive organs. The conservation of CA XII expression in both mouse and human endometrium shown in the present paper suggests a role for this isozyme in reproductive physiology. Materials and methods Antibodies In the present study, we used the following antibodies which have been produced and characterized earlier: rabbit anti-mouse CA II [ 4 ], rabbit anti-mouse CA IX [ 26 ], rabbit anti-mouse CA XII [ 33 ], and rabbit anti-mouse CA XIII [ 4 ]. Collection of tissue samples Two adult Balb/c mice were sacrificed by CO 2 asphyxiation followed by decapitation. Uterus, ovary and placenta samples were collected from both animals. The samples were immersion-fixed overnight in Carnoy's fluid (ethanol, chloroform and acetic acid (6:3:1)). Then the specimens were treated with absolute ethanol for 30 min, with 1:1 mixture of ethanol and chloroform for 15 min, and finally with chloroform for 30 min. Paraffin embedding was performed in a vacuum oven for 2 h at +58°C. Paraffin wax was purchased from Fluka Chemie GmbH (Buchs, Schwitzerland). To collect a placenta sample, a mouse was sacrificed at 9 days of pregnancy. The ninth day was chosen since it represents the middle gestational phase. It is also the time when the most critical steps of organogenesis occur in mouse. For western blotting, uterus, kidney and colon were removed and rapidly frozen in liquid nitrogen. The tissue samples for western blot were homogenized with HEPES buffer. Total protein concentration was determined after homogenization using BCA Protein Assay Kit (Pierce, Rockford, IL) according to manufacturer's instructions. The study protocols were approved by the Animal Care Committee of Tampere University. Immunohistochemistry In the mouse tissues, the localization of CA IX and XII was examined by immunoperoxidase method. Antibodies against CA II and XIII were used as positive controls for the immunostaining. All experiments were performed in duplicate and included control staining with non-immune normal rabbit serum (NRS). NRS was obtained from a rabbit that was later immunized against mouse CA XIII. The tissue samples fixed in Carnoy's fluid and embedded in paraffin were cut at 5 μm sections and placed on microscope slides. The peroxidase-anti-peroxidase complex method included the following steps: a) pretreatment of the sections with undiluted cow colostral whey (Biotop, Oulu, Finland) for 40 min and rinsing in phosphate-buffered saline (PBS); b) incubation for 1 h with the primary antiserum (anti-mouse CA II, CA IX, CA XII or CA XIII) or NRS diluted 1:100 in PBS containing 1% bovine serum albumin (BSA) (BSA-PBS solution); c) treatment with undiluted cow colostral whey (40 min); d) incubation for 1 h with swine anti-rabbit IgG (Dakopatts, Copenhagen, Denmark) diluted 1:100 in 1% BSA-PBS; e) incubation for 30 min with peroxidase-anti-peroxidase rabbit conjugate (Dakopatts) diluted 1:500 in PBS; f) incubation for 2 min with 3,3'diaminobenzidine tetrahydrochloride (DAB) solution (6 mg DAB in 10 ml PBS plus 3.3 μl H 2 O 2 ) as chromogen. The sections were washed three times for 10 min in PBS after incubation steps b and d and four times for 5 min after incubation step e. All of the incubations and washings were carried out at room temperature. The sections were finally mounted in Neo-Mount (Merck, Darmstadt, Germany). The stained sections were examined and photographed with a Zeiss Axioskop 40 microscope (Carl Zeiss, Göttingen, Germany). Western blot The samples containing 50 μg of protein from mouse uterus, kidney and colon were analyzed by SDS-PAGE under reducing conditions [ 34 ]. All of the reagents and the protein standard (BenchMark™ Prestained Protein Ladder) for SDS-PAGE were purchased from Invitrogen (Carlsbad, CA) except Laemmli sample buffer that was obtained from Sigma (St. Louis, MO). Electrophoresis (200 V for 40 min) was performed in a Novex Xcell II mini cell electrophoresis unit (Invitrogen) with a 10% Bis-Tris gel (Invitrogen). The separated proteins were transferred electrophoretically from the gel to a polyvinylidene fluoride (PVDF) membrane (Invitrogen) in a Novex Xcell II blot module (Invitrogen). The transfer buffer (NuPAGE Transfer Buffer™) was purchased from Invitrogen. The blot module was filled with the transfer buffer until the gel/membrane assembly was covered. The outer buffer chamber was filled with 650 ml deionized water. The protein transfer was performed using a constant voltage of 36 V for 1 h 20 min. After the transblotting, the sample lines were detected by ECL western blotting detection reagents and analysis system (Amersham Biosciences, Buckinghamshire, UK) according to the manufacturer's instructions. First, the sample lines were incubated with TBST buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0,3 % Tween 20) containing 10 % cow colostral whey for 25 min and then the first antibodies diluted 1:2000 (anti-CA II, anti-CA IX, anti-CA XII, NRS) or 1:1000 (anti-CA XIII) in TBST buffer for 1 h. The PVDF membranes were washed five times for 5 min with TBST buffer and incubated for 1 h with peroxidase-linked ECL Anti-Rabbit IgG (Amersham Biosciences) diluted 1:25 000 in TBST buffer. After washing four times 5 min in TBST buffer, the polypeptides were visualized by a chemiluminescence substrate (ECL detection reagents 1 + 2, Amersham Biosciences). Kodak™ Biomax™ MS-1 films (Amersham Biosciences) were exposed to the chemiluminescence for 5 min (CA IX and XII) or 1 min (CA II and CA XIII). All the steps were carried out at room temperature. The western blotting experiments were performed in triplicate to confirm the reproducibility of the results. Results Immunohistochemistry All the studied CA isozymes showed positive immunostaining in the epithelial cells of the mouse endometrium (Fig. 1 ). CA II and XII showed a somewhat reciprocal distribution pattern in that CA II was confined to the surface epithelial cells (Fig. 1C ), while CA XII was more intensely stained in the deeper endometrial glands (Fig. 1A ). It is noteworthy, however, that CA XII was clearly expressed also in the surface epithelial cells, but the staining intensity was weaker compared to the glands. As expected, the strongest reaction for CA XII was associated with the basolateral plasma membrane, and unexpectedly, also CA II immunoreaction was most intense at the plasma membrane. CA IX and XIII showed weak reactions in both surface and glandular epithelia (Fig. 1B,1D ). The control immunostaining with NRS was negative (Fig. 4A ). Figure 1 Immunohistochemical staining of CA XII (A), CA IX (B), CA II (C), and CA XIII (D) in the mouse endometrium. All the studied CA isozymes show positive immuostaining, although the staining intensity varies between different isozymes. CA XII shows stronger reaction in the deep endometrial glands compared to the surface epithelium. This pattern is inversed with CA II showing high reaction in the surface epithelium. Insert in panel A shows that the CA XII immunostaining is most abundant in the basolateral plasma membrane of the epithelial cells. Insert in panel C demonstrates that CA II immunoreactivity is also closely associated with the plasma membrane. CA IX and XIII show faint immunoreactions in both the surface and glandular epithelia. Arrows = endometrial glands, arrowheads = surface epithelium. Original magnifications: × 400. Figure 4 Control immunostaining of mouse uterus, ovary and placenta with normal rabbit serum. No immunoreaction is seen. Oringinal magnifications: × 400. In the ovary, immunoreactions for different CA isozymes were negligible (Fig. 2 ). In fact, only CA XII showed occasional positive cells in the corpus luteum (Fig. 2A ). No staining for these isozymes was observed in the developing follicles. No immunoreaction was obtained with NRS (Fig. 4B ) Figure 2 Immunolocalization of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse corpus luteum (A,C,E,G) and follicle (B,D,F,H). Only faint positive reaction for CA XII can be seen in occasional cells of the corpus luteum that is indicated in the insert of the panel A (arrows). Original magnifications: × 200 (A,C,E,G), × 400 (B,D,F,H). In the 9-days-old mouse placenta, the immunostaining reactions for CA isozymes remained quite weak or absent (Fig. 3 ). CA II was located to the endothelium of the placenta blood vessels and erythrocytes (Fig. 3E ), and it was also present in the amnionic epithelium (Fig. 3F ). The amnionic epithelium showed no or very weak staining for CA XII, whereas the decidual glands were strongly labeled (Fig. 3B ). The control staining again showed no positive signal (Fig. 4C ). Figure 3 Immunohistochemical staining of CA XII (A,B), CA IX (C,D), CA II (E,F), and CA XIII (G,H) in the mouse placenta (A,C,E,G) and amnionic epithelium (B,D,F,H). CA II is located in the endothelium of the blood vessels and erythrocytes (arrows in the panel E). It is also expressed in the amnionic epithelium (arrows in the panel F). Insert of the panel B shows that CA XII is highly expressed in the decidual glands, while the amnionic epithelium is negative. DE = Decidua, P = placenta. Original magnifications: × 400. Western blot Western blotting was performed for the mouse uterine protein to evaluate the specificity of the immunoreactions. Mouse kidney and colon samples were used as positive control tissues, since they are known to express CA II, XII and XIII [ 4 , 33 , 35 ], and the colon contains CA IX [ 36 ]. CA II and XIII were positive in all tissue specimens (Fig. 5 ). Both CA IX and XII showed weak positive reactions in the mouse uterus. The molecular weights for these isozymes were 51 and 46 kDa, respectively. Based on the western blotting the expression of CA XII was weaker in the uterus than in the colon or kidney. On the other hand, CA IX showed the strongest signal in the colon. It is notable that anti-mouse CA XII serum cross-reacted with 30 kDa polypeptide in the western blotting. Previous immunostaining of gastric mucosa with the same anti-CA XII and anti-CA II antibodies has clearly indicated that anti-CA XII serum does not recognize CA II which has a molecular mass of 30 kDa in western blot [ 35 ]. Even though gastric epithelial cells contain high levels of CA II, no immunoreaction was obtained with anti-CA XII serum in those cells. Furthermore, no staining has been obtained by anti-CA XII antibody in the red cells which contain high levels of CA I and II, nor in the brain which expresses high levels of CA II and XIII (data not shown). Figure 5 Western blotting of total homogenate from mouse uterus, kidney and colon for CA II, IX, XII and XIII. Normal non-immune rabbit serum (NRS) was used instead of the first antibodies as a negative control. Both CA IX and XII show weak positive reactions for the uterine proteins (arrowheads). The molecular weights for these isozymes are 51 and 46 kDa, respectively. The signal for CA XII is weaker in the uterus than in the colon or kidney. Note that anti-CA XII serum cross-reacts with a 30-kDa polypeptide. This cross-reaction is evident only in western blotting conditions as pointed out in the Results section. CA IX shows the strongest signal in the colon. CA II and XIII are positive in all tissue specimens. Discussion This study describes the expression of CA II, IX, XII and XIII in mouse female genital organs including uterus, ovary and placenta. CA II showed a very limited distribution pattern in the mouse placenta, being present only in the erythrocytes, endothelium of the blood vessels and amnionic epithelium. In previous studies, CA II has been detected by immunohistochemistry in the human villous syncytiotrophoblasts and in varying amounts in fetal villous endothelium [ 21 , 22 ]. Using a histochemical staining method, Ridderstråle et al [ 18 ] showed in several species that the highest CA activity located in the maternal capillaries, and the membrane-bound CA activity varied among different species. To date, CA IV is the only membrane-bound CA isozyme which has been detected in the mouse placenta [ 23 ]. In our study, CA IX and XII were not found in the mouse placental tissue except that CA XII showed a very weak reaction in the amnionic epithelium. Concluding from the results of the present and previous studies, CA I and II appear to represent the enzyme forms that are most relevant for the placental function [ 22 ], while CA IX and XII may play a role in other reproductive organs such as the male excurrent duct and female uterus [ 14 , 37 ]. It is known that CA activity facilitates transport of CO 2 across biological membranes by converting it to bicarbonate and hydrogen ions. These ions are then translocated across the plasma membrane through specific carrier proteins in a coordinated manner. It is of considerable interest that CA isozymes II and IV have been recently described to form active metabolon systems with ion exchanger proteins such as anion exhanger isoform 1 (AE1) and Na + /H + -exhanger isoform 1 (NHE1) [ 38 - 40 ]. Even though these associations have not yet been described in the placental tissue, it is possible that such metabolons play a role in facilitating placental ion transport processes. Previous studies have shown CA activity in the endometrium of several mammalian species [ 41 , 42 ]. Until now the only established isozymes in the human endometrium are CA XII [ 14 ] and CA XIII [ 4 ]. Interestingly, the high activity isoenzyme, CA II, is not expressed in the human endometrial epithelium [ 4 ]. In the present study, all the examined CA isozymes – including CA II – showed positive immunostaining in the epithelial cells of the mouse endometrium. To our knowledge, there are only a few examples of clear species-specific difference in CA expression. These include e.g. CA XII expression in the kidney (human principal cells versus mouse intercalated cells) [ 33 , 43 ] and CA XIII in the human and mouse testis [ 4 ]. What would be the physiological consequence of such variation between different species? Of course, there are marked differences in human and rodent reproductive physiology. Mouse is characterized by tremendous reproductive potential. Females generally have 5–10 litters per year if conditions are suitable. Gestation period is 19–21 days. Litters consist of 3–12 (generally 5 or 6) offspring, and the mice reach sexual maturity at 5–7 weeks. Even though our observations do not provide any clues whether CA expression could contribute to some of the described characteristics, these differences may have fundamental physiological effects that should be addressed in future investigations. In the present study, CA XII and II showed more intense staining in the surface endometrial epithelia than CA IX and XIII. CA XII was more intensely stained in the deeper endometrial glands, while CA II was confined to the surface epithelial cells. Interestingly, CA II showed positive immunoreaction not only in the cytoplasmic compartment but also at the plasma membrane of the cells that is quite surprising for a cytosolic enzyme. The same phenomenon is detectable in some other tissues including the human gallbladder [ 25 ] and gut [ 44 ]. The cell membrane reactivity may reflect a possible physical association between CA II and ion transport proteins, which has been demonstrated in cell cultures [ 38 - 40 ]. When CA XII was first discovered in the normal human endometrium, it was suggested to play a role in reproductive functions [ 14 ]. In the endometrium, pH and ion balance has to be tightly regulated to ensure normal fertilization. For example, the bicarbonate concentration has been implicated in the regulation of sperm motility, capacitation, and acrosome reaction [ 15 , 17 , 45 ]. One major regulatory pathway includes a bicarbonate-sensitive adenylate cyclase that is present in the plasma membrane of the sperm cell [ 46 ]. In the female genital tract, the endometrial and oviductal epithelium may produce an alkaline – bicarbonate rich – environment for maintaining the sperm motility. This suggestion is in agreement with the observations by Guerin et al. [ 47 ], that the sperm motility is improved by co-culture of human spermatozoa with either endometrial or oviductal epithelial cells. In the future studies, it will be important to investigate whether the hormonal status regulates the expression of different CA isozymes – particularly CA XII – in the endometrium. Another interesting line of investigations would be to analyze the fertilization capacity of CA XII knockout mice as soon as they become available. One could hypothesize that endometrial CA isozymes are important factors, contributing to the appropriate bicarbonate concentration and pH balance in the cervical and endometrial mucus needed for normal fertilization process. Based on our recent studies, CA IX-deficient mice showed no apparent phenotypic changes linked to fertility [ 26 ]. Even more interesting from this point of view is that CA XII may be an important isozyme present in the endometrium, and therefore, CA XII knockout mice will be very attractive targets for reproductive physiological studies. Conclusions The present paper demonstrates for the first time the expression of transmembrane carbonic anhydrase isozymes IX and XII in the female murine reproductive tract. The data indicates that the endometrial epithelium is a prominent site for CA XII expression. The conservation of CA XII expression in the endometrium of different species (mouse and human) suggests a role for this isozyme in reproductive physiology. Authors' contributions All authors participated in the design of the study. PH, JL and SP collected the tissue samples. PH, JL, ET, PK and SP drafted the manuscript. PH, JL and SP performed the western blotting. SPas, JP, AW and WSS provided the antibodies. PH, JL and SP participated in the immunohistochemical staining. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526762.xml
423140	A Gene Responsible for Hybrid Incompatibility in Drosophila	null	Nearly 150 years after Darwin published On the Origin of Species , biologists are still debating how new species emerge from old—and even the definition of species itself. Darwin demurred from offering a hard and fast definition, suggesting that such a thing was “undiscoverable.” One of the more enduring definitions characterizes organisms as distinct reproductive units and species as groups of individuals that can interbreed and produce viable, fertile offspring. The lack of genetic exchange between species, called reproductive isolation, lies at the heart of this definition. Environmental changes can create physical barriers between populations that preclude mating between the populations. Reproductive isolation can also involve changes at the genetic level, when molecular barriers prevent two recently diverged populations from producing viable or fertile offspring. Such factors limit gene flow between diverging species and allow the emergence of genetically novel yet sound populations—that is, new species. Multiple regions of Hmr show evidence for divergence driven by positive selection At the heart of reproductive isolation is a phenomenon called hybrid incompatibility, in which closely related species are capable of mating but produce inviable or sterile offspring. The classic example of hybrid incompatibility is the male donkey–female horse cross, which yields a sterile mule, but many other cases have been documented among mammals, and thousands of plant crosses produce infertile offspring. Much has been learned about the genetic architecture of hybrid incompatibility by studying the offspring of closely related, or “sibling,” fruitfly species in the lab. Sibling species are morphologically very similar, or even indistinguishable, but typically do not interbreed in nature. In the lab, their offspring are either sterile or inviable, a fate that varies depending on the gender of the offspring and species of the parents. To elucidate the molecular mechanisms of reproductive isolation, biologists must first identify candidate hybrid incompatibility genes. Species- or lineage-specific functional divergence is an essential trait of these genes. (That is, the genes evolve different functions after the species diverge from their common ancestor.) While several such candidate genes have been identified in the fruitfly Drosophila melanogaster , none has been shown to display this functional divergence. Now, working with D. melanogaster and its sibling species D. simulans and D. mauritiana , Daniel Barbash, Philip Awadalla, and Aaron Tarone establish the functional divergence of a candidate hybrid compatibility gene and confirm its status as a true speciation gene. Since the 1930s, investigations of reproductive isolation have been guided by the Dobzhansky-Muller model, which attributes hybrid incompatibility to the interactions between two or more genes that have evolved independently in two isolated populations. These independently evolving genes diverge functionally, and the interactions of these functionally divergent genes in a hybrid individual are responsible for the defective phenotypes observed (either inviability or sterility). If this is the case, the alleles, or versions, of the gene causing hybrid incompatibility should have distinct phenotypes in the two species. A corollary of the model says that the diverged allele (A) and not the ancestral allele (a) causes the incompatibility phenotype, which means that experimental manipulations of A but not a should affect the hybrid incompatibility phenotype. Barbash et al. tested the model's predictions by genetically manipulating the alleles of the Hybrid male rescue (Hmr) gene from each sibling species and observing the mutations' effects on the flies' hybrid offspring. In previous experiments the researchers had shown that loss-of-function mutations in the D. melanogaster Hmr gene “rescue” hybrid individuals from the hybrid incompatibility phenotype (male inviability) typically observed in the offspring of crosses between D. melanogaster and its sibling species, and that increased Hmr activity suppresses rescue and kills hybrids. If D. melanogaster Hmr has functionally diverged between the species, then transgenes containing Hmr from sibling species should not cause the hybrid incompatibility phenotype caused by the D. melanogaster Hmr . The researchers tested this hypothesis by introducing transgenic Hmr genes from sibling species into D. melanogaster . In all cases, the hybrid male offspring of D. melanogaster / D. mauritiana and D. melanogaster / D. simulans crosses “were at least as viable as their brothers without the transgene.” To examine this divergence at the genomic level, Barbash et al. compared the divergence of 250 genes in D. melanogaster and D. simulans and found that the Hmr gene was among the most rapidly evolving genes. By examining the frequency of mutations that have accumulated between D. melanogaster and sibling species relative to the number of mutations accumulated within species, the authors show that the mutations between species were by and large not neutral and that they occurred after D. melanogaster diverged from its sibling species, indicating that the gene has been under positive natural selection. Barbash et al. have not only identified a bona fide speciation gene by demonstrating its functional divergence, they've also created a platform for investigating the mechanisms through which such genes cause hybrid incompatibility and lay the groundwork for speciation.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC423140.xml
526776	The use of warfarin in veterans with atrial fibrillation	Background Warfarin therapy is effective for the prevention of stroke in patients with atrial fibrillation. However, warfarin therapy is underutilized even among ideal anticoagulation candidates. The purpose of this study was to examine the use of warfarin in both inpatients and outpatients with atrial fibrillation within a Veterans Affairs (VA) hospital system. Methods This retrospective medical record review included outpatients and inpatients with atrial fibrillation. The outpatient cohort included all patients seen in the outpatient clinics of the VA Connecticut Healthcare System during June 2000 with a diagnosis of atrial fibrillation. The inpatient cohort included all patients discharged from the VA Connecticut Healthcare System West Haven Medical Center with a diagnosis of atrial fibrillation during October 1999 – March 2000. The outcome measure was the rate of warfarin prescription in patients with atrial fibrillation. Results A total of 538 outpatients had a diagnosis of atrial fibrillation and 73 of these had a documented contraindication to anticoagulation. Among the 465 eligible outpatients, 455 (98%) were prescribed warfarin. For the inpatients, a total of 212 individual patients were discharged with a diagnosis of atrial fibrillation and 97 were not eligible for warfarin therapy. Among the 115 eligible inpatients, 106 (92%) were discharged on warfarin. Conclusions Ideal anticoagulation candidates with atrial fibrillation are being prescribed warfarin at very high rates within one VA system, in both the inpatient and outpatient settings; we found warfarin use within our VA was much higher than that observed for Medicare beneficiaries in our state.	Background Warfarin therapy is highly effective for the prevention of ischaemic stroke in atrial fibrillation [ 1 ]. Despite the accepted benefit of warfarin therapy, several reports have indicated that warfarin therapy is underutilized even in ideal anticoagulation candidates with atrial fibrillation. Most studies have reported rates of use between 13–60% [ 2 - 8 ]. For example, a national study of inpatient Medicare beneficiaries with atrial fibrillation demonstrated that approximately 55% of patients were discharged on warfarin [ 9 ]. Many of the previous studies about the use of warfarin in atrial fibrillation have focused on the prescription of warfarin on discharge from an acute hospitalization. Since some patients may be discharged from the hospital with a plan to begin warfarin therapy as an outpatient, these prior studies may have underestimated the use of warfarin for patients with atrial fibrillation. The Veterans Affairs (VA) Healthcare System is a useful setting for studying the use of warfarin therapy in both the inpatient and outpatient arenas because the electronic medical record contains prescription medication data as well as the inpatient and outpatient medical records (including progress notes, laboratory data, radiology reports, and other consult reports). The objective of this study was to examine the use of warfarin in both inpatients and outpatients with atrial fibrillation within a VA setting. Specifically, we used the same methodology as the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation [ 10 ], so that we could compare rates of warfarin use in ideal anticoagulation candidates with atrial fibrillation from one VA system to those in the private sector. Methods We assembled two retrospective cohorts of patients to evaluate both inpatients and outpatients with atrial fibrillation. The medical records of both the inpatients and the outpatients were reviewed to confirm the diagnosis of atrial fibrillation, to identify any exclusion criteria, and to determine if patients were being prescribed warfarin. Diagnosis of atrial fibrillation Using the criteria developed by the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation [ 10 ], a physician's documentation of the diagnosis of atrial fibrillation was required for inclusion (electrocardiogram data were not used to make the diagnosis of atrial fibrillation). For the outpatients, Physicians' Current Procedural Terminology (CPT) codes were used to identify potential patients with a diagnosis of atrial fibrillation. For the inpatients, the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM) discharge diagnosis code (427.31) was used to identify potential patients with a diagnosis of atrial fibrillation. Chart review was used to confirm the diagnosis for both the inpatients and the outpatients; a physician's documentation of atrial fibrillation in a progress note, a consult note, the discharge summary, or the problem list was needed for confirmation. Medical record abstraction was conducted by two of the authors (KR, SK) using standard definitions. All of the exclusions were reviewed by three of the authors (KR, SK, DMB), a sample of the charts of patients with an exclusion criteria was re-abstracted (DMB), and any disagreements were resolved by consensus. Cohort descriptions The outpatient cohort included all of the patients seen in the outpatient clinics of the VA Connecticut Health Care System during the month of June 2000 with a diagnosis of atrial fibrillation. Outpatient clinics include both primary care and subspecialty clinics. Some of these clinics have a particular interest in the care of patients with atrial fibrillation such as cardiology and anti-coagulation clinics, however, most of the clinics do not have such a special interest (e.g., mental health, physical therapy, dermatology, endocrinology). The inpatient cohort included all patients discharged from the VA Connecticut Health Care System West Haven Campus with any discharge diagnosis of atrial fibrillation (primary or secondary diagnosis) during the period of October 1, 1999 through March 31, 2000. Some of the patients in the inpatient cohort were readmitted during our study period; this report includes data from individual patients for their first hospital stay. Exclusion criteria The Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation project developed a set of exclusion criteria to identify ideal candidates for warfarin therapy; we used this exclusion criteria for the current study. Patients were excluded if they met one or more of the following: current sinus rhythm; bleeding disorder; endocarditis or pericarditis (within 6 months); seizures; intracranial hemorrhage; intracranial surgery or biopsy; lone atrial fibrillation; dual chamber pacemaker; alcohol or drug abuse; allergy to warfarin; hepatic failure; schizophrenia or active psychotic disorder; comfort care or terminal illness with life expectancy less than 6 months; un-repaired intracranial aneurysm; extensive metastatic cancer; brain cancer; malignant hypertension; peptic ulcer disease; hemorrhage; documentation that the patient refused warfarin therapy; prior complication or allergy related to past use of warfarin; or physician documentation of a rationale for not prescribing warfarin, including risk for bleeding, risk for falls, mental status impairment, liver disease, arthritis requiring non-steroidal anti-inflammatory medications or aspirin, pending surgery or other invasive procedure, terminal illness, patient's inability to obtain necessary blood work, or history of patient's non-adherence to warfarin [ 10 ]. Patients in intermittent or paroxysmal atrial fibrillation were included in the study, however, patients who were noted to be in current sinus rhythm and for whom atrial fibrillation was not a current problem were not included. For example, a patient with atrial fibrillation in the setting of an acute myocardial infarction or post-coronary bypass grafting for whom the atrial fibrillation was not a current medical problem was not included. Warfarin prescription For all of the patients, warfarin prescription was determined from the medical record. Patients receiving warfarin from the VA pharmacy were readily identified from the VA pharmacy component of the medical record. Patients receiving warfarin privately were identified from the progress notes. For inpatients, warfarin prescription was evaluated at the time of discharge from the hospital. For outpatients, warfarin prescription was evaluated at the time of examination of the medical record. Each outpatient medical record was examined in detail to determine the presence or absence of warfarin prescription. For those outpatients patients in whom this determination was difficult or if the data collector had a question about the patient, then the medical record was examined again by three of the authors to determine the presence or absence of warfarin prescription. This study received Institutional Review Board approval. Statistical analysis Student's t-tests were used to compare dimensional variables, and Fisher Exact and chi-square tests were used to assess binary variables. Two-sided p-values <0.05 were considered to be statistically significant. Exact binomial 95% confidence interval were calculated for the proportions of patients using warfarin in the inpatient and outpatient cohorts. The SAS System software release 6.12 (Cary, N.C.) was used for data analysis. Results Outpatient cohort A total of 538 patients from the VA Connecticut outpatient clinics were identified as having a diagnosis of atrial fibrillation (age: 74.0 years mean ± 8.3 standard deviation; 529 [98%] men). Of these, 73 patients had one or more contraindication to anticoagulation (Table 1 ). Of the 465 eligible patients, 455 (98%; 95%CI 96–99%) were prescribed warfarin. Table 1 Exclusion Criteria* Characteristic Inpatients Outpatients N = 212 N = 538 N (%)† N (%)† Sinus rhythm 32 (15) 42 (8) Death 21 (10) 1 (0.2) History of gastrointestinal hemorrhage 18 (8) 6 (1) Fall risk 14 (7) 4 (0.7) Pacemaker 4 (2) 9 (2) Lone atrial fibrillation 4 (2) 1 (0.2) Terminal illness 4 (2) 0 (0) Patient refused 3 (1) 6 (1) History of intracranial hemorrhage 3 (1) 3 (0.6) Transfer to outside facility 3 (1) 0 (0) Multi-infarct dementia in comfort care patients 2 (0.9) 0 (0) Warfarin held for procedure or surgery 2 (0.9) 0 (0) Warfarin allergy 1 (0.5) 0 (0) Failed to comply with warfarin protocol, warfarin stopped 1 (0.5) 1 (0.2) History of seizures 1 (0.5) 0 (0) Elective admission to begin sotalol and discontinue warfarin 1 (0.5) 0 (0) Previous bleeding on warfarin 0 (0) 1 (0.2) *These exclusion criteria were taken directly from the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation. 10 † Note: some patients had more than one reason for not being prescribed warfarin. The ten patients who were not prescribed warfarin did not differ from those who received warfarin with respect to age (mean age ± standard deviation; no warfarin: 76.6 ± 11.5, warfarin: 73.9 ± 8.1; p = 0.3). Among the ten patients who were not prescribed warfarin: one was a dialysis patient who received his medical care primarily from private physicians outside of the VA, he was eventually placed on warfarin; the medical record of one 90-years-old patient, who also received the majority of his health care from private physicians, indicated that his private physician had elected not to prescribe anticoagulation "because of age"; one patient had a history of alcohol use; and in the remaining 7 patients there was no documentation of a reason for why the warfarin had not been prescribed (3 of the 7 patients were receiving the majority of their medical care outside of the VA). Inpatient cohort A total of 212 individual patients were discharged with a diagnosis of atrial fibrillation (age: 72.9 years mean ± 9.9 standard deviation; 211 [99.5%] males). During the admission these 212 patients, 17 died during their hospitalization, 3 were transferred to a facility outside of the VA Connecticut Healthcare System, and 77 had one or more contraindication to anticoagulation. Of the 115 remaining eligible patients, 106 (92%; 95%CI 86–96%) were discharged on warfarin. The nine patients who were not prescribed warfarin did not differ from those who were prescribed warfarin with respect to age (mean age ± standard deviation; no warfarin: 76.4 ± 7.4, warfarin: 72.6 ± 7.9; p = 0.2). To determine if some of the eligible inpatients who were not discharged on warfarin later received warfarin in the outpatient setting, we examined the outpatient records of the nine inpatients who were not discharged on warfarin: 5 died; 3 no longer receive care at our medical center (and no medication data were available); and for 1 patient, the medical record stated that he was offered warfarin therapy but that he refused to accept it. Similarly, we evaluated a sample of 50 eligible patients who had been discharged on warfarin therapy and examined their warfarin use post-discharge: 7 no longer receive care at our medical center (and no medication data are available); 4 were taken off of warfarin (3 because they were cardioverted as outpatients, and for 1 patient the warfarin was discontinued after an episode of bright red blood per rectum); and 2 patients have died. Among the eligible inpatients 12/115 (10%) had history of prior stroke or transient ischemic attacks; 3/12 (25%) were not prescribed warfarin on discharge. No reasons were documented for why these patients were not given warfarin. Unique patients There was overlap between the inpatient and outpatient cohorts such that a total of 722 unique patients were identified among the 212 inpatients and the 538 outpatients. Discussion We found high rates of warfarin prescription in ideal anticoagulation candidates with atrial fibrillation treated within this VA system. A total of 561 of 580 ideal anticoagulation candidates (97%) were prescribed warfarin: 98% of ideal outpatient anticoagulation candidates and 92% of ideal inpatient anticoagulation candidates. These rates of warfarin use for atrial fibrillation are substantially higher than those reported previously from private sector academic and community hospitals. For example, as part of the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation, medical records were reviewed from a random sample of Medicare beneficiaries, hospitalized during the period 1998–1999, with any discharge diagnosis of atrial fibrillation from each state [ 9 , 10 ]. The exclusion criteria for the Medicare medical record review were the same as those used for the current study and were developed to select a cohort of atrial fibrillation patients who were "ideal" candidates for oral anticoagulation because they do not have any contraindications to oral anticoagulation [ 10 ]. Therefore, one would expect that the rates of warfarin use would be higher in ideal anticoagulation candidates than in a general population of patients with atrial fibrillation. Overall, the rate of warfarin prescription for ideal anticoagulation candidates with atrial fibrillation patients by state ranged from 31–65%, with a median of 55% in the Medicare study [ 9 ]. In Connecticut, 57% of eligible atrial fibrillation inpatients were discharged on warfarin [ 9 ]. Therefore, the inpatient rates observed in the current study of 90–92% are much higher than those observed for Medicare beneficiaries using similar methodology. We report the anticoagulation rates from one VA healthcare system, and our findings may not be generalizable to other VA healthcare systems or to non-VA hospitals. Specifically, our results may not be generalizable to women with atrial fibrillation. Furthermore, given that the out-patient cohort for this study was obtained from a one-month sample, we may have selected patients who are more likely to be seen at an out-patient clinic, and therefore, our findings may also have limited generalizability to atrial fibrillation patients who do not require or who do not have access to regular out-patient clinical care. Although we have found higher rates of warfarin use than most of the previous studies in this area [ 2 - 9 ], our findings are similar to those reported by Gottlieb and Salem-Schatz [ 11 ] who found that 78.8% of atrial fibrillation patients in an HMO setting were receiving warfarin. Our findings are also similar to those reported by Bradley et al. from another VA health care system [ 12 ]. Bradley et al. demonstrated that 89% of patients without a contraindication to anticoagulation were prescribed warfarin [ 12 ]. Several possible factors might account for the high use of warfarin in VA hospitals. First, the actual rates of warfarin prescription may be higher in VA facilities where anticoagulation clinics are well established, the electronic medical record ensures that all services (primary care and consult services) have access to a patient's medical record, the staff has academic affiliations, and the VA culture embraces quality improvement and medical error reduction initiatives. Within the VA Connecticut Health Care System, pharmacist-directed anticoagulation clinics are available at two sites, in West Haven and Newington, Connecticut. Veterans who choose to obtain warfarin from the VA pharmacy are usually followed at one of these two anti-coagulation clinics. Veterans can elect to purchase warfarin from private pharmacies and have their anticoagulation intensity monitored privately (usually by their private internist or private cardiologist). Second, the higher rate may result from data collection differences between studies. Specifically, given the comprehensive VA electronic medical record, VA-based researchers may be able to identify more contraindications for anticoagulation. No quality improvement projects to increase the use of warfarin for patients with atrial fibrillation in the VA Connecticut Healthcare System were initiated during or immediately prior to the study period. A limitation of the current study is that we were unable to determine the specific reasons for why such a high rate of warfarin use was observed. The retrospective nature of this study permitted us to evaluate clinical practices without altering physicians' behavior. However, this retrospective chart review may have limitations. First, we may not have identified those patients who are prescribed warfarin by private practitioners and obtain their warfarin from non-VA pharmacies. This would result in even higher rates of warfarin prescription than we have reported. Second, we assembled our cohort using diagnosis codes for atrial fibrillation and did not use electrocardiographic data. Those patients who had atrial fibrillation by electrocardiogram, but who were not identified as having atrial fibrillation by their clinicians, would not have been included in this study. Because such patients are unlikely to receive warfarin our estimates of warfarin use are higher than would have been observed if we had used electrocardiography to identify atrial fibrillation patients. While many studies of the use of warfarin for atrial fibrillation have also assembled cohorts using diagnosis codes and not electrocardiographic data, the VA-based study by Bradley et al. used electrocardiographic criteria and their findings are similar to ours [ 12 ]. Third, some may argue that we excluded patients who would benefit from anticoagulation. For example, patients with atrial fibrillation and numerous other risk factors for stroke might benefit from anticoagulation despite the presence of a contraindication to anticoagulation such as a risk for falls. We chose to use the exclusion criteria developed for the Medicare Health Care Quality Improvement Program's National Stroke Project – Atrial Fibrillation [ 10 ] so that we could compare the rates of warfarin prescription observed within one VA system to those seen for Medicare beneficiaries. Fourth, this study includes a total of 722 unique patients. Although some studies of warfarin use in patients with atrial fibrillation have included similar numbers of patients (e.g., N = 635 in the study of Medicare beneficiaries with ischemic stroke and atrial fibrillation by Brass, et al) [ 13 ], many studies have included much larger sample sizes (e.g., N = 11,699 in the study of Medicare beneficiaries with new-onset atrial fibrillation) [ 14 ]. Often, the studies with the largest sample sizes were secondary analyses of existing administrative datasets [ 14 ]. Future studies should be directed at evaluating the use of oral anticoagulation in veterans with atrial fibrillation using national VA data where both large sample sizes and nationally representative sampling are possible. Conclusion We conclude that high rates of adherence to treatment guidelines regarding the use of anticoagulation in patients with atrial fibrillation can be achieved. Our experience, and that of Bradley et al., indicates that high rates of warfarin use can be achieved across at least two VA settings [ 12 ]. Competing interests The authors declare that they have no competing interests. All of the authors are employed by the VA Connecticut Healthcare system. Dr. Bravata is supported by an Advanced Research Career Development Award from the Department of Veteran Affairs Health Services Research & Development Service. Authors' contributions All of the authors contributed to this manuscript, participated in the research design and manuscript preparation. Two of the authors (SK, KR) conduct the data collection. Three of the authors (DMB, SK, KR) reviewed the data and conducted the analyses. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526776.xml
555767	Pharmacokinetic-pharmacodynamic modelling of the cardiovascular effects of drugs – method development and application to magnesium in sheep	Background There have been few reports of pharmacokinetic models that have been linked to models of the cardiovascular system. Such models could predict the cardiovascular effects of a drug under a variety of circumstances. Limiting factors may be the lack of a suitably simple cardiovascular model, the difficulty in managing extensive cardiovascular data sets, and the lack of physiologically based pharmacokinetic models that can account for blood flow changes that may be caused by a drug. An approach for addressing these limitations is proposed, and illustrated using data on the cardiovascular effects of magnesium given intravenously to sheep. The cardiovascular model was based on compartments for venous and arterial blood. Blood flowed from arterial to venous compartments via a passive flow through a systemic vascular resistance. Blood flowed from venous to arterial via a pump (the heart-lung system), the pumping rate was governed by the venous pressure (Frank-Starling mechanism). Heart rate was controlled via the difference between arterial blood pressure and a set point (Baroreceptor control). Constraints were made to pressure-volume relationships, pressure-stroke volume relationships, and physical limits were imposed to produce plausible cardiac function curves and baseline cardiovascular variables. "Cardiovascular radar plots" were developed for concisely displaying the cardiovascular status. A recirculatory kinetic model of magnesium was developed that could account for the large changes in cardiac output caused by this drug. Arterial concentrations predicted by the kinetic model were linked to the systemic vascular resistance and venous compliance terms of the cardiovascular model. The kinetic-dynamic model based on a training data set (30 mmol over 2 min) was used to predict the results for a separate validation data set (30 mmol over 5 min). Results The kinetic-dynamic model was able to describe the training data set. A recirculatory kinetic model was a good description of the acute kinetics of magnesium in sheep. The volume of distribution of magnesium in the lungs was 0.89 L, and in the body was 4.02 L. A permeability term (0.59 L min -1 ) described the distribution of magnesium into a deeper (probably intracellular) compartment. The final kinetic-dynamic model was able to predict the validation data set. The mean prediction error for the arterial magnesium concentrations, cardiac output and mean arterial blood pressure for the validation data set were 0.02, 3.0 and 6.1%, respectively. Conclusion The combination of a recirculatory model and a simple two-compartment cardiovascular model was able to describe and predict the kinetics and cardiovascular effects of magnesium in sheep.	Background The effective use of some drugs can be limited by their adverse effects on the cardiovascular system, particularly when they are used intravenously in relatively high doses. There have been many studies documenting the cardio-vascular effects of drugs. Similarly, many mathematical models of the cardiovascular system, of varying complexity, have been presented in the literature [ 1 , 2 ]. In pioneering work, models of the cardiovascular system have been linked to pharmacokinetic models of volatile anaesthetic disposition [ 3 - 5 ]. These kinetic-dynamic models have since been developed into mannequin based anaesthesia simulators, which now have a pivotal role in the training of anaesthetists. This approach has been facilitated by the fact that models of volatile anaesthetic disposition have traditionally been physiologically based (e.g. using representations of tissue:blood partition coefficients and blood flows for individual organs or groups of organs). It is therefore possible to equate blood flow in the cardiovascular model to blood flow in the pharmacokinetic model. Nevertheless, a limiting factor in the implementation of this approach is the availability of experimental data on concentration-effect relationships [ 5 ]. In contrast, for traditional ("non-volatile") drugs, there have been very few instances in which kinetic models of a drug have been linked to cardiovascular pharmacodynamic models. The work of Francheteau et al. is an important exception [ 6 ], but even this early work was restricted to analysis of only one aspect of the cardiovascular system (i.e. accounting for heart rate mediated control of blood pressure but not Frank-Starling control of cardiac output). However, it is clear this approach has the potential to provide a more rational basis for designing dose regimens of cardio-active drugs, and could provide insight into strategies for controlling their cardio-vascular effects. It maybe possible to predict a priori the cardiovascular consequences of, for example, a change in dose regimen of a drug. There are a number of difficulties in implementing this approach for traditional drugs. One problem is that most drugs do not cause changes in one single cardiovascular variable (such as blood pressure) that can be described in the usual manner using a simple semi-empirical dynamic model (e.g. an E max model). Rather, a number of cardiovascular variables may be altered simultaneously in a manner that is complex and interrelated. Thus, any dynamic model used must account for these intrinsic relationships between cardiovascular variables. Another problem is that changes in the cardiovascular system (in particular blood flow distribution) invariably alter the kinetics of the drug under study. Therefore, the kinetic model of the drug must be able to account for the effects of blood flow changes on the disposition of the drug. This requires the kinetic model to have a physiological basis, and importantly excludes the widely used mamillary compartmental pharmacokinetic model. The general aims of this study were threefold. First, to develop a simple dynamic model of the cardiovascular system that was of sufficient complexity to account for the major mechanisms by which drugs can alter cardiovascular variables. Second, to examine whether recirculatory kinetic models [ 7 ] have sufficient physiological basis to account for drug related blood flow changes. Third, to examine approaches for identifying the important concentrations (and their sites in the body) that can be used to link the kinetic and dynamic models. The specific aim was to use previously published data collected using a chronically instrumented sheep preparation [ 8 , 9 ] to develop a kinetic-dynamic model for the cardiovascular effects of magnesium. Magnesium is given intravenously to treat a number of diseases, including pre-eclampsia. It relaxes smooth muscles in blood vessels thereby lowering systemic vascular resistance, with a consequent decrease of mean arterial blood pressure and increase in cardiac output. It provides a useful drug for initial analysis as its kinetics and dynamics are relatively simple and well understood. The overall hypothesis of this work is that it is possible to construct a faithful model of the cardiovascular effects of drugs such as magnesium. While doing so requires more assumptions and estimates of parameter values than normally associated with semi-empirical pharmacokinetic-pharmacodynamic modelling, a physiological approach greatly increases the utility of the resulting models. It is proposed that the general methods presented here could be applied to the development of similar models for other drugs with acute cardiovascular effects. Methods General rationale With respect to devising a pharmacodynamic model of the cardiovascular system, the important steps are: 1. Identifying which cardiovascular variables (e.g. heart rate, blood pressure) are important. This depends on the drug and the intended use of the model, but it is proposed that there is a minimum set of variables that is needed for a basic description of cardiovascular status. 2. Devising a way of conveniently presenting the output of the dynamic model for a range of cardiovascular variables for comparison with data. 3. Identifying a cardiovascular model of the appropriate complexity. Ideally the model must be of the minimum complexity that includes the cardiovascular variables identified above, and the major sites of action of the drugs. 4. Identifying which parameters of the cardiovascular model can be estimated by curve-fitting, and which require prior estimates or measurements of physiological values. Most cardiovascular models are stiff numerical systems with many parameters, and only a small number can be estimated by curve-fitting the data in the traditional way. With respect to the pharmacokinetic model, there remains one crucial step: 5. Constructing a kinetic model with a physiological basis that is sufficiently realistic to describe and predict the concentration of the drug in the key target organs controlling the cardiovascular system. On first principles, these could be expected to include: a. the myocardial concentrations when the drug has a direct myocardial effect (e.g. causes myocardial depression); b. the CNS concentrations when the drug affects the cardio-respiratory control centre of the brain; c. the arterial blood concentration when the drug affects baro-receptors or smooth muscle in the walls of the arterial vascular system; d. the venous blood concentration when the drug affects smooth muscle in the wall of the capacitance vessels of the venous vascular system. It is known that these concentrations can follow different time-courses, particularly after bolus administration or a change of infusion rate [ 10 , 11 ]. However, it may not be necessary to know the time-course of these concentrations for every drug, depending on its mechanism of action. Data sets and software The data used to construct the model were collected in the same laboratory using a conscious chronically instrumented sheep preparation and have been published previously [ 8 , 9 ]. This facilitated the model building process, as the effect of differences in species and measurement methods could be discounted. Data set 1 [ 9 ] (for model development) was a detailed set of cardiovascular measurements made after the administration of 30 mmol of magnesium over 2 min to 5 sheep. Measurements included arterial and coronary sinus (effluent from the heart) magnesium concentration, cardiac output, mean arterial blood pressure, heart rate, an index of myocardial contractility (Maximum positive rate of change of left ventricular Pressure, dp/dt) and an index of filling pressure (Left ventricular end diastolic pressure) and myocardial blood flow. These were made until 25 min after the start of administration. Data set 2 [ 8 ] (for model validation) was a less comprehensive set of cardiovascular measurements made after the administration of 30 mmol of magnesium over 5 min to 5 sheep (not the same sheep as Data set 1). Measurements included arterial magnesium concentration, cardiac output, and mean arterial blood pressure, and were made until 25 min after the start of administration. The blood pressure data for one animal in this data set was excluded, as it was idiosyncratically low. The time-course of the data averaged across sheep were used for all modelling – the resultant model therefore represents the response of the average sheep. Inter- and intra-animal variability were not considered, although it is noted that the final model may provide insight into sources of kinetic and dynamic variability for later study. The software used was the Scientist for Windows program (Version 2.01, Micromath, Salt Lake City, Utah, USA), predominantly for curve-fitting. The R language, Version 1.9.0, [ 12 ] was used for graphical data analysis, data handling and simulations. Coding the same model in the two different programs provided a useful check for errors. For the least squares curve-fitting, the best fit was determined as that with the highest Model Selection Criteria (MSC) and without non-identifiable parameters. The MSC is essentially an inverse Akaike Information Criterion scaled to compensate for data sets of different magnitudes (Scientist for Windows manual, Micromath, Salt Lake City, Utah, USA), and is calculated as follows where w i is a weighting term, p is the number of parameters and n is the number of data points: All data points were weighted equally. A parameter was arbitrarily defined as non-identifiable if the standard deviation of the parameter returned by the fitting program was greater than the parameter estimate (i.e. the coefficient of variation was greater than 100%). A model with non-identifiable parameters means that the data do not contain sufficient information to estimate the parameter with precision. The symbols used throughout have been based on standards for the pharmacokinetic literature. Unfortunately, the use of C for both concentration (in pharmacokinetics) and compliance (in cardiovascular physiology) creates of conflict for this paper. To avoid confusion, CPL will be used for compliance here. Pharmacodynamic model of the cardiovascular system Identification of important cardiovascular variables The choice of the cardiovascular variables used in the model is clearly dependent on the drug under study and the intended purpose of the model. However, we propose that a minimum of 7 fundamental cardiovascular variables is sufficient for most pharmacological purposes. These variables are shown with their default unit of measurement in the model: Central venous pressure (CVP, mmHg), Myocardial contractility (CNT, L mmHg -1 ), Stroke volume (SV, L), Heart rate (HR, min -1 ), Cardiac output (CO, L min -1 ), Systemic vascular resistance (SVR, Resistance units, RU) and mean arterial blood pressure (MAP, mmHg). This choice of variables requires several assumptions: Assumption 1 All variables are time averaged in that beat to beat variation is ignored (e.g. mean arterial blood pressure is used rather than systolic and diastolic blood pressures). Assumption 2 That the function of the left and right side of the heart is the same, and there are no abnormalities in the pulmonary vasculature so that the heart-lung system can be treated as one pump. Assumption 3 Long time-scale events such as fluid shifts and renal mechanisms controlling blood pressure are ignored. Furthermore, this choice of variables is dictated by several fundamental relationships between the variables. Firstly, that myocardial contractility is a proportionality constant between CVP pressure and stroke volume (the volume of blood pumped with each beat of the heart). CVP * CNT ≈ SV ...(2) Mathematically, CNT must therefore have the units of volume / pressure. However, contractility is difficult to measure in vivo, and that there are a number of surrogate measures including dp/dt. These can also be used with appropriate scaling factors. Assumption 4 That there are no factors affecting the relationship between myocardial fibre length (the true determinant of stroke volume) and central venous pressure (e.g. changes in myocardial compliance). CVP is therefore used as an easily measured index for myocardial fibre length – the assumption is that the two are related using a scaling factor. Left ventricular end diastolic pressure is also as an alternative index when data are presented as percent change from baseline. The second relationship is that between stroke volume and heart rate to give the cardiac output (the volume of blood pumped by the heart per unit time): SV * HR = CO ...(3) The last relationship is that between cardiac output and systemic vascular resistance to approximate the mean arterial blood pressure. CO/SVR ≈ MAP ...(4) This is because MAP usually greatly exceeds the CVP: CO/SVR = MAP-CVP ...(5) SVR therefore has the units of pressure over flow. In this paper, the resistance units (RU) are therefore mmHg L -1 min. Presentation of relationships between cardiovascular variables The effect of a drug on one or two variables can usually be summarised on a plot of the variable (drug effect) against time. However, it is more difficult to summarise the dynamic effect of a drug on the cardiovascular system for the following reasons: First, the large number of variables required in the summary, where the seven described above could be considered a minimum. Second, the fixed inter-relationships (e.g. Eqns. 2–4) between these variables that should be revealed by the summary (e.g. if SV increased by 25% and all else remains the same, then CO should also increase by 25% (Eqn. 3)). Third, usually an analysis requires comparing one cardiovascular state (e.g. pre-drug) with another (e.g. post-drug), or examining the time-course of drug effects. It is proposed that a "cardiovascular radar plot" with a modified logarithmic, normalised scale is an efficient means of limiting these problems. An example radar plot is shown and described in Fig. 1 . Radar plots are particularly useful for visually testing whether a model of the cardiovascular system behaves appropriately for all 7 key cardiovascular variables when challenged with a particular drug or physiological circumstance. It is particularly useful to see if the pattern of changes is internally consistent. For example, in Fig. 1 it is clear that magnesium dropped SVR, but the drop in MAP was not great as expected because there was a baroreceptor mediated increase in heart rate. Figure 1 An example of a cardiovascular radar plot. A cardiovascular radar plot of the effect of magnesium (n = 5 sheep) on the cardiovascular system under baseline conditions and for a number of time-points until 25 min after the intravenous administration of magnesium. A scale for each of the 7 key cardiovascular variables radiates from the centre of the plot. LVEDP (left ventricular end diastolic pressure) is a surrogate for CVP; both should change proportionally. The data are transformed and the scale constructed so that 3 is the baseline (pre-drug) value. Thus, the blue line for the baseline data is a ring passing through 3 for each variable. Baseline conditions therefore have a characteristic equilateral 7 sided shape. The full scale is structured as follows: 1 one quarter baseline 2 half baseline 3 baseline 4 twice baseline 5 4 times baseline This scale has the property that for an equivalent increase or decrease in a cardiovascular variable compared to baseline, the line will move an equal distance in or out from the baseline value. It can be seen that following magnesium there was a drop in SVR with a baroreflex increase in HR to compensate for the drop in blood pressure. LVEDP also dropped, but with minimal change in contractility. Drugs that affect the cardiovascular system via different mechanisms produce plots with characteristic shapes, which can be recognised with experience. Note: The order of the variables on the radar plot has been chosen to account for key relationships between the variables, with CVP (as given by LVEDP) as the most fundamental variable at the top: In an anti-clockwise direction the following relationships or approximations hold: CVP * CNT ≈ SV SV * HR = CO CO/SVR ≈ MAP Cardiovascular model – Structure and parameter estimation There are many published models of the cardiovascular system of various levels of complexity and intended for various tasks [ 1 ]. However, in this paper, the cardiovascular model was constructed progressively from first principles, with adaptations and increases in complexity as dictated by the requirements of the modelling process and the data. This ensured the model was the minimum that was needed for the task at hand. In vivo, the cardiovascular system has two major control systems; control of cardiac output via the Frank-Starling mechanism, and control of blood pressure via baroreceptor control of heart rate. These were added progressively to the model. A simple Frank-Starling model A simple model of the Frank-Starling mechanism was developed (Fig. 2 ) assuming the blood is predominately in two pools – the arterial and venous vasculature. The two pools are connected by a pump (the heart) moving blood from the venous to arterial side for which the rate of pumping is proportional to the venous pressure. Blood flows from the arterial to venous side through a passive resistance (the SVR). The pressure in each pool is a function of the compliance in the pool. Compliance (CPL) governs the relationship between volume and pressure: Figure 2 A simple Frank-Starling model of the cardiovascular system. A simple two compartment model of the circulation, with control of the cardiac output via the Frank-Starling mechanism. When the heart is not pumping, the pressures on the venous (P v ) and arterial (P a ) sides of the circulation are equal (the mean circulatory pressure (MCP = P v = P a ) is approximately 7 mmHg). The unstressed volumes of the venous (V v 0) and arterial sides (V a 0) are governed by the relative compliance of the venous and arterial pools (CPL v and CPL a , respectively). If the pumping action of the heart is initiated, a fraction of the blood (dV) moves from the venous to the arterial side thereby increasing arterial pressure and decreasing venous pressure. The pressure gradient causes blood to flow from the arterial side to the venous side (at a rate given by the venous return, CO R ). This depends on the pressure gradient (P a -P v ) and the systemic vascular resistance (SVR). Pressure = Volume/Compliance ...(6) The solution to the simple Frank-Starling model can be found algebraically, but for consistency is shown in Additional File 1 as differential equations. Central to the Frank-Starling model is the concept of cardiac function curves – usually given as the pressure in each pool as cardiac function (contractility) is increased from zero to a normal value. These curves are useful for finding appropriate initial estimates for blood volume, arterial and venous compliance, and systemic vascular resistance. To achieve the physiologically plausible cardiac function curve shown in Fig. 3 , blood volume was set at 3.5 L [ 13 ]. Given that in a normal (50 kg) sheep the baseline cardiac output is approximately 6 L/min and mean arterial blood pressure is 100 mmHg (Table 1 ), baseline systemic vascular resistance is therefore 100/6 ≈ 17 RU. The remaining unknowns of this system (kc, CPL a , CPL v ) were chosen to duplicate the following behaviour (Fig. 3 ) which is consistent with measurements in this species: When CNT is zero (i.e. the heart is not pumping) then dV is zero and the mean circulatory pressure (MCP) is approximately 7 mm Hg. When CNT is such that the cardiac output is approximately 6 L min -1 , then MAP and CVP are approximately 100 and 2 mmHg, respectively (Table 1 ). In practice, it was found easier to express the arterial compliance (CPL a ) as the ratio of arterial compliance to venous compliance (C ratio ). Figure 3 Cardiac function curves for the Frank-Starling model. A summary of the behaviour of the simple Frank-Starling model. The relationship between cardiac function (contractility) and the arterial and venous pressures matches well that reported in many textbooks. The venous compliance CPL v was 0.45, and the ratio of CPL v /CPL a was 15. Table 1 Baseline (pre-drug) cardiovascular variables. A set of target values that was representative of the sheep studied in our laboratory was compiled from previous measurements and literature values as indicated. A set of parameter values for the final (Constrained-Frank-Starling-Baroreceptor) was derived (Table 2) that produced an internally consistent model that closely replicated these target values (also shown for comparison). Variable Name Target Value Target value origin Model derived Value Units V blood Blood volume 3.5 literature [13] 3.5 L CVP Central venous pressure 2.00 unpublished previous measurements and literature [20] 2.00 mmHg CPL v Venous compliance 0.45 inferred from V blood & CVP 0.46 L mmHg -1 MAP Mean arterial pressure 100 previous measurements [21] 100.9 mmHg SVR Systemic vascular resistance 17.00 calculated from CO & MAP 17.0 RU CO Cardiac output 6 previous measurements [21] 5.8 L min -1 HR Heart rate 100 previous measurements [21] 98.3 beats min -1 SV Stroke volume 0.06 calculated from CO & HR 0.059 L CNT Contractility 3000 previous measurements [9, 21] 3000 mmHg sec -1 S1 Sympathetic tone – chronotropy 1 scaling factor only 1 dimensionless S2 Sympathetic tone – Contractility 1 scaling factor only 1 dimensionless Frank-Starling and Baroreceptor model The control of arterial blood pressure via baroreceptor control of heart rate was added to the simple Frank-Starling model, as shown in Fig. 4 . The arterial pressure set point (MAPset) was used to calculate the difference between the actual and set pressure (MAPdelta). This pressure difference was used to change heart rate with a gain given by "HRgain". When HRgain is zero, the model reduces to the simple Frank-Starling model. As HRgain is increased, the more heart rate is adjusted to defend changes in arterial pressure. A value of 3 was initially used for HRgain. The resultant cardiac function curve for this model is shown in Fig. 5 , and the equations for the model are shown in Additional File 2 . Figure 4 A Frank-Starling-Baroreceptor model of the cardiovascular system. The Frank-Starling model of the circulation from Fig. 2 combined with baroreceptor control of arterial blood pressure (P a ) via changes in heart rate (HR). MAP set is the set point of the control system, and HR gain is the gain of the control system that operates on the difference between the actual and set arterial blood pressures (MAP delta ; Eqn. 7). The right side cardiac output term is expanded to include the role of myocardial contractility (CNT), stroke volume (SV) and heart rate (HR). "kc" is a conversion factor to adjust for the index used to measure myocardial contractility (Eqn. 8). Strictly, myocardial contractility is the proportionality factor between Pv and stroke volume (SV = CO R /HR). However, it is often quantified using indirect indices, such as maximum positive change of ventricular pressure (dP/dt max ). The value of kc will depend of what index of contractility is used (see Eqn. 8). Figure 5 Cardiac function curves for the Frank-Starling-Baroreceptor model. A summary of the behaviour of the Frank-Starling-Baroreceptor model. The relationship between cardiac function (contractility) and the arterial and venous pressures matches well that reported in many textbooks. However, the vascular volumes show the majority of the blood in the arterial compartment, which is at odds with the fact that the majority of the blood under baseline conditions is in the venous vessels. Furthermore, no constraints have been placed on the model so that unrealistic values (e.g. large negative pressures) can be achieved in some circumstances. Constraining the model to increase physiological plausibility The final version of the model introduced a number of constraints to increase its physiological plausibility. These were: 1) Assuming that under baseline conditions that approximately 1/3 of the total blood pool is in the arterial system. 2) That the intercepts of the pressure-volume "curves" for the venous and arterial compartments were linear such that both curves gave the mean circulatory pressure (MCP) at the unstressed volumes (V v 0 and V a 0, see Fig. 6 ). 3) That the venous pressure could not be less than zero, and that the arterial pressure could not be less than the MCP. 4) That heart rate was constrained to be between 0 and 220. 5) That the venous pressure – stroke volume relationship was non-linear and reached a plateau consistent with the finite pumping capacity of the heart (Fig. 6 ). 6) For convenience, two additional parameters were introduced (S1 and S2) representing the state of the sympathetic nervous system. These gave the capacity to adjust the proportionality term between blood pressure and heart rate (HRgain) and between CVP and stroke volume (kc). This allowed these scaling constants to be separated into a constant term that is solely used to convert measurement units (HRgain or kc) and another term (S1 or S2) that represents changes in underlying physiology for use when fitting data. Their normal values were 1 in each case (giving no effect for baseline conditions) and their function is summarised in the following equations: Figure 6 Effect of introducing constraints on the model. Top: Venous pressure – volume curves. Venous volume starts at V v 0 when the heart is not pumping, at which point the venous pressure is the mean circulatory pressure (MCP). With increased pumping, the venous volume and venous pressure is reduced. In the simple (Frank-Starling-Baroreceptor) model, the CVP – volume relationship was linear, with an intercept of zero. In the constrained model, a lower intercept was used which was necessary to produce realistic venous volumes under baseline conditions. The multiple curves in the plot show the effect of changing venous compliance (CPL v ). Middle: Arterial pressure – volume curves. Arterial volume starts at Va0 when the heart is not pumping, at which point the arterial pressure is the mean circulatory pressure (MCP). With increased pumping, the arterial volume and arterial pressure are increased. In the simple model, the MAP – volume relationship was linear, with an intercept of zero. In the constrained model, a lower intercept was the used, which was necessary to produce realistic arterial volumes under baseline conditions. The multiple curves in the plot show the effect of changing compliance (CPL a ). Bottom: "Cardiac output curves" In this case cardiac output is given by stroke volume, which is plotted against central venous pressure (CVP). In the simple model, this relationship was linear. In the constrained model the relationship was given by a logistic equation which rose to a limit. The left-hand side of the curves are pseudo-linear, and the slope of the lines increase with increasing contractility. This behaviour mimics the "Cardiac output curves" found in many cardiovascular textbooks (e.g. Guyton [19]). HR = MAPset + MAPdelta(HRgainS1) ...(7) The resultant cardiac function curves for this model are shown in Fig. 7 , and the equations for the model are shown in Additional File 3 . Figure 7 Cardiac function curves for the Constained-Frank-Starling-Baroreceptor model. A summary of the behaviour of the final cardiovascular dynamic model. Baseline values for the cardiovascular model For convenience, the target cardiovascular variables of the final constrained model discussed above are summarised in Table 1 with references to their origins. The parameter set that produced cardiovascular variables similar to the target values is summarised in Table 2 . This was derived semi-empirically by inspection of cardiovascular function curves (Fig. 7 ) and pressure-volume relationships (Fig. 6 ) with incremental adjustment of parameter values. Note that some variables are also listed as parameters – this is purely for convenience. The distinction between variables (time-dependent) and parameters (time-independent) is semantic and depends on the proposed use of the model. Table 2 Baseline model parameters The parameters chosen as those producing representative baseline (pre-drug) cardiovascular variables (Table 1). The co-efficient of variation (CV (%)) of these parameter values as determined by the Monte-Carlo sensitivity analysis is also shown. Parameter Name Value Units CV (%) CPL v Venous compliance 0.45 L mmHg -1 12.7 CPL ratio Ratio of venous over arterial compliance 20 dimensionless 17.7 V blood Blood volume 3.5 L 17.9 SVR Systemic vascular resistance 17 RU 5.5 MAPset Mean arterial pressure set point 100 mmHg 4.2 HRgain Gain for heart rate control 1.8 bpm a mmHg -1 23.6 CNT Contractility 3000 mmHg sec -1 4.9 a bpm = beats per minute The sensitivity of the baseline cardio-vascular model to changes in parameter values was determined via Monte-Carlo simulation [ 14 ]. Multi-variate normally distributed noise was added to the parameter values for a series of 10,000 simulations of the resulting cardiovascular variables. Those parameter sets that produced a set of cardiovascular variables within 10% of the target set were selected and analysed for with respect to parameter variability and correlation. Fitting the cardiovascular model to the magnesium data Changes in cardiovascular variables with the administration of magnesium were analysed as percentage change from baseline. This removed the contribution of inter-animal variability in baseline cardiovascular variables (which was nevertheless minor [ 8 , 9 ]) to variability in the cardiovascular effects of magnesium. The analysis involved fitting cardiovascular radar plots to the measured magnesium data (Data set 1) for key time-points (1, 2, 4, 10 and 25 min) during and after magnesium administration. The cardiovascular model was parameterised in terms of primary cardiovascular variables that could be directly influenced by magnesium. These were SVR, CPL v , CPL ratio , CNT, S1 and S2. V blood could also be considered a primary variable, but it was considered unlikely that magnesium could change the blood volume. The remaining cardiovascular variables were considered secondary in that they would change in response to changes in the primary variables as given by Eqns 2 to 4. Initially, the only primary parameter fitted to the data for each time point was SVR while the other parameters were held constant. This was based on the prior knowledge that this was the primary mechanism of action of magnesium. If the MSC was low and the cardiovascular radar plot showed a poor fit between model predictions and the data, an additional parameter was fitted one at a time from the remaining parameters listed above. A parameter was removed from the fit if it produced an undefined estimate. The parameter was kept in the fit if it improved the MSC and the fidelity of observed vs. predicted plots on the cardiovascular radar. By this process, the values of the primary cardiovascular parameter at each key time point required to describe the observed data were determined. Recirculatory pharmacokinetic model of magnesium disposition Conventional mamillary pharmacokinetic models are essentially empirical and do not include parameters (other than clearance) that represent defined physiological processes. This is problematic when drugs affect the cardiovascular system, or it is necessary to predict the kinetics of the drug when the underlying physiology has changed. This was the case for magnesium, which affected cardiac output significantly (Figs. 1 & 10 ). Full physiological pharmacokinetic (PBPK) models are an alternative, but often require extensive data sets for their parameterisation. Recirculatory models have been used [ 7 , 15 ] as an alternative that retain the key physiological descriptions of important organs, but have lumped descriptions of the less important organs. Often, they can be parameterised by fitting blood concentrations alone. Figure 10 Best fits for the recirculatory pharmacokinetic model for magnesium. Top: The observed changes in cardiac output for Data set 1 (symbols). Also shown is the line of best fit for the empirical forcing function used for development of the kinetic model. The large and consistent increase in cardiac output illustrates why it was necessary to use a kinetic model that could account for the significant flow changes caused by magnesium. Middle: The observed arterial concentrations of magnesium for Data set 1 (symbols). Also shown is the line of best fit for the final kinetic model (not linked to the cardiovascular model) based on the parameter values given in Table 4. Bottom: A sensitivity analysis of the final kinetic model with respect to cardiac output when used to simulate the dose regimen used for Data set 1. Cardiac output was given values of 2, 4, 6, 8 or 10 L min -1 while the other parameters were fixed at the values given in Table 4. This illustrates how the cardiac output changes caused by magnesium can influence its own kinetics. This feedback process was inherent in the structure of the final kinetic-dynamic model. The magnesium concentration data from Data set 1 were used to develop a recirculatory model of magnesium kinetics that could account for the observed cardiac output changes. The processed used was similar to that described by the authors for other drugs [ 15 ]. The final form of the model is shown in Fig. 9 . Figure 9 Final recirculatory pharmacokinetic model for magnesium. A pictorial representation of the model. Parameter names are given in Table 4. Key points during the model development process were: 1) The representation of the lungs as a single compartment. 2) The representation of the cardiac output change as an empirical forcing function (see Fig. 10 , this would later be replaced by the predictions of the final cardiovascular model). 3) The representation of the body as extracellular and intracellular spaces connected by a permeability term, in keeping with the known slow cellular uptake of magnesium. 4) The clearance of magnesium is renal, but it can be reabsorbed or excreted in the tubules, as dictated by homeostatic requirements [ 16 ]. Thus, renal clearance may be variable. To confirm that the kinetics of magnesium were cardiac output dependent, the final kinetic model was subjected to a sensitivity analysis for this parameter. Cardiac output was assigned values of 2, 4, 6, 8 or 10 L min -1 while the other parameters were fixed at their best fit value. The time-course of the arterial magnesium concentration was recorded in each case. Linking the pharmacokinetic and pharmacodynamic models The relationship between the key cardiovascular parameters (effects) and the concentrations of magnesium in arterial and coronary sinus blood were examined using hysteresis plots (effect vs. concentration). A concentration-effect relationship was considered plausible if produced a predictable relationship with minimal hysteresis that was consistent with the known mechanisms of action of the drug. By these criteria, it was found that the arterial concentrations were the better predictor of the fitted cardiovascular parameters shown in Table 3 . The concentration – effect relationships are summarised in Fig. 11 . The major effect of magnesium was to drop systemic vascular resistance (SVR). SVR was related to the arterial magnesium concentration by a link model based on a linear relationship with a threshold (Fig. 11A ): Table 3 The fitted primary cardiovascular parameters for Magnesium data set 1 Units are as for Table 2. The parameter estimates are given with the standard deviation returned by the curve-fitting program. S1 could not be reliably fitted to the data. 0 min 1 min 2 min 4 min 10 min 25 min Fitted parameter (baseline) estimate (sd) estimate (sd) estimate (sd) estimate (sd) estimate (sd) MSC n/a 3.88 2.96 4.75 1.67 4.74 CPL v 0.45 0.490 (0.0028) 0.495 (0.0075) 0.497 (0.0019) 0.505 (0.0049) 0.492 (0.0007) SVR 17 11.99 (0.12) 9.84 (0.26) 11.84 (0.084) 14.64 (0.27) 16.74 (0.077) CNT 3000 2988 (41) 3031 (104) 3388 (32) 3108 (80) 3180 (23) S2 1 0.978 (0.023) 0.972 (0.055) 0.877 (0.014) 1.22 (0.06) 1.25 (0.017) Figure 11 Link models for concentration-effect relationships. A: The systemic vascular resistance (SVR) parameter (symbols, obtained by the fitting process that gave the radar plots shown in Fig. 8 and summarised in Table 3) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (Eqn. 9; line) based on a linear relationship with a threshold is also shown. B: The venous compliance (CPL v ) parameter (symbols, via Fig. 8) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (Eqn 10; line) based on a simple threshold that switches between two states of venous compliance is also shown. This is plausible if it is considered that magnesium, even at relatively low concentrations, causes maximal dilation of the venous capacitance vessels. C: The contractility (CNT) parameter (symbols, via Fig. 8) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (line) was based on the assumption that contractility was unaffected by magnesium (i.e parameter value was fixed). D: The sympathetic tone coefficient for contractility (S2) parameter (symbols, via Fig. 8) plotted against the concurrent exogenous arterial magnesium concentrations. The final link model (line) was based on the assumption that S2 was unaffected by magnesium (i.e parameter value was fixed). if C art < 2.66 then SVR = 17 else SVR = -1.759*C art + 21.68 ...(9) Magnesium also raised venous compliance (CPL v ). This was related to the arterial concentration using a simple threshold (Fig. 11B ): if C art < 2 then CPL v = 0.45 else CPL v = 0.50 ...(10) Magnesium had little effect on myocardial contractility (Fig. 11C ), and the linking function assumed that CNT remained at baseline values. Magnesium appeared to increase the sympathetic tone coefficient for contractility (S2) by approximately 25% at between concentrations of 2 and 4 mmol L -1 (Fig. 11D ). However, this rise in S2 only occurred late in the study (Table 3 ). It indicates subtle changes in the relationship between the filling pressure index (LVEDP) and the contractility index (dp/dt). This may reflect measurement error in these variables, non-stationarity in the experimental preparation or subtle delayed changes in myocardial compliance caused by magnesium. However, it was found that a link function assuming S2 remained at baseline values (Fig. 11D ) was an adequate account of the data and did not compromise the predictive power of the model in the validation stage. The final kinetic-dynamic model therefore consisted of the kinetic model shown in Fig. 9 linked to the Constrained-Baroreceptor-Frank-Starling cardiovascular dynamic model (Figs. 4 & 7 ) via the link Equations 9 and 10. This is summarised in Fig. 12 . The equations for the model are shown in Additional file 4 . Figure 12 Overview of the kinetic-dynamic model linking process. A schematic representation of how the final model was derived from Data set 1. The pharmacokinetic (PK) component of the model was developed by fitting the observed arterial magnesium concentrations (Fig. 10, middle). As cardiac output was a parameter of the recirculatory model, the magnesium induced changes in cardiac output were represented as a forcing function during fitting (Fig. 10, top). In the final model, this forcing function was replaced by the cardiac output predicted by the cardiovascular (CV) model. For the CV model, target baseline cardiovascular variables were derived from previous measurements and the literature (Table 1). A unique parameter set for the CV model was found that reproduced these values (Table 2). To account for the changes in cardiovascular variables from baseline following magnesium, four parameters (SVR, CPL v , CNT and S2) were fitted to the observed magnesium CV data (expressed as change from baseline) at selected time-points (Fig. 8; Table 3). Of these, two parameters (SVR, CPLv) showed concentration dependent changes that could be related via link functions to the time-course of magnesium concentrations (Fig. 11). The other parameters of the CV model were fixed at their baseline values. The final model was able to predict the concentrations and CV effects of magnesium for a different dose regimen (Data set 2, Fig. 13). Validation of the final model The final kinetic-dynamic model developed using Data set 1 was used to predict the arterial magnesium concentrations, cardiac output and mean arterial blood pressure for Data set 2. Data set 2 differed from Data set 1 in that the dose of magnesium was given over 5 min instead of 2 min. Consequently, although the dose was the same, the cardiovascular effects were less pronounced. For example, the lowest blood mean arterial pressure for Data set 1 was 76% of baseline, while for Data set 2 this was 86% of baseline. The only change made to the parameters of the final model was to alter the duration of infusion of the magnesium. Results Parameter sensitivity of cardiovascular model (baseline conditions) The baseline cardiovascular variables and the parameters that produced them are summarised in Tables 1 and 2 , respectively. Of the 10,000 random parameter sets examined in the Monte-Carlo sensitivity analysis, only 37 produced a set of cardiovascular variables that was within 10% of the target cardiovascular variables. The variability of these successful parameter values was low (Table 2 ), and the spread of each parameter showed a unimodal, approximately normal distribution. This suggests that there was a unique set of parameter values for the model that was consistent with normal baseline physiology. Visual inspection showed no obvious correlation between parameter values, except for CPL v and CPL ratio (correlation coefficient = 0.83). This suggests that specifying the value for one of these parameters significantly constrains the value that can be taken for the other, as would be expected on physiological grounds. It can be concluded that each parameter had an important role to play in the model, and that each could only take a limited range of values to be consistent with the required baseline physiology. By extension, the assumptions regarding the values of these parameters are likely to be appropriate. Furthermore, the changes in these parameters observed following magnesium administration therefore reflect the effects of this drug rather than uncertainty in the parameter space of the model. Parameter estimates – cardiovascular data The method of estimating cardiovascular model parameters from cardiovascular data for individual time points was effective. Thus, it was possible to find a parameter set at each time point (Table 3 ) that produced a fitted cardiovascular radar plot that closely matched the observed plot (Fig. 8 ). In general, the parameter estimates were precise. The most obvious effect of magnesium was a drop in systemic vascular resistance and a rapid and sustained increase in venous compliance. The changes in the other cardiovascular variables (e.g. HR and MAP) simply reflected reflex changes in response to these primary drug effects. Figure 8 Best fit cardiovascular radar plots for each key time point. Cardiovascular radar plots of the observed data (blue) and the best fit of the final cardiovascular model (red). Note that the shape of the radar plot changes with time, indicating the evolving effects of magnesium on the circulation. For each time-point, the fit was an adequate account of the data (Table 3). Parameter estimates – pharmacokinetic data The recirculatory pharmacokinetic model was able to fit the observed concentrations with adequate fidelity (Fig. 10 , middle) and produce precise parameter estimates (Table 4 ). As the clearance of magnesium was low, it would be expected that the permeability term into the deep compartment governed the rate of decline of the magnesium concentration rather than its clearance from the body. Table 4 The fitted pharmacokinetic parameters for the Magnesium data set 1 The parameter estimates are given with the standard deviation returned by the curve-fitting program. Fitted variable Value Units MSC 3.13 V lung 0.887 (0.221) L CL 0.0021 (0.1286) L min -1 V body 4.023 (0.486) L PS 0.589 (0.227) L min -1 V deep 8.63 (5.39) L A feature of recirculatory pharmacokinetic models is that their initial kinetics are governed by first-pass passage of drug through the lungs, and the dilution of the injected drug with the cardiac output [ 7 ]. The cardiac output sensitivity analysis for the model confirmed this behaviour for magnesium (Fig. 10 , bottom). This reinforces the need for a common cardiac output term for the cardiovascular and recirculatory kinetic model (Fig. 12 ). The resultant final model therefore accounts for the fact that by altering cardiac output, magnesium alters its own kinetics. Link functions Relating the estimated cardiovascular parameters in Table 3 to the concurrent arterial concentrations produced the concentration-effect curves shown in Fig. 11 . Link functions were established for SVR and CPLv, but not CNT or S2. The overall role of the link functions is summarised in Fig. 12 . Model validation – pharmacokinetic component The recirculatory model of magnesium disposition was able to accurately predict the time-course of the arterial magnesium concentrations observed for the validation Data set 2, despite the large change in cardiac output produced by magnesium (Fig. 13 ). The mean prediction error was 0.02% Figure 13 Observed and predicted results for Data set 2. The final kinetic-dynamic model developed using Data set 1 was used to predict the exogenous arterial magnesium concentrations (C art,x ), cardiac output (CO) and mean arterial pressure (MAP) for Data Set 2. The only change in the model between the two data sets was to increase the duration of the infusion from 2 to 5 min. The observed data are shown as symbols, together with the upper and lower 95% confidence intervals of the data (dotted lines). The predictions of the model are shown by the solid lines. Model validation – pharmacodynamic component The final pharmacodynamic model was able to accurately predict the time-course of the cardiac output changes observed for the validation Data set 2 (Fig. 13 ). The mean prediction error was 3.0%. The dynamic model captured the general trend of the mean arterial blood pressure for the validation data (Fig. 13 ), but some systematic deviations were evident. The model was accurate until the end of the infusion, but thereafter slightly over-estimated the rate of recovery of blood pressure. However, the model did predict that the drop in blood pressure would be considerably less for a 5 min versus 2 min infusion, and the overall magnitude of the changes in blood pressure for the 5 min infusion were small (less than 10% change). The mean prediction error was 6.1%. Discussion Concentration-effect relationships and recirculatory models In this paper, all cardiovascular effects were related to the arterial concentration of magnesium. As covered in the introduction, there may be other sites in the body that have a theoretical claim to being the most appropriate link concentration for certain cardiovascular dynamic effects. For example, the reductions in myocardial contractility caused by thiopental have been shown to have a better temporal relationship to the thiopental concentrations in the myocardium itself rather than in arterial blood [ 17 ]. This consistent with a direct thiopental effect on the myocardium. In recirculatory models, it is possible to add a "target organ" to represent organs such as the heart [ 18 ]. The fact that this was not necessary for magnesium may be the exception rather than the rule. As magnesium has small volumes of distribution, there is little difference in the time-course of the arterial and regional venous concentrations. Furthermore, the predominant effects of magnesium were directly on blood vessels (arterioles for SVR and large veins for capacitance) in direct equilibrium with blood rather than organs such as the heart or brain. Thus, a "systemic" recirculatory model was sufficient for magnesium. As other drugs are studied using this method, data on target organ kinetics and their incorporation into the kinetic model may be necessary. Limitations There are a number of limitations of this modelling approach, many of which are inherent in the assumptions made in the construction of the model. Other limitations may become apparent if the model is used outside of the range of the data used to develop the model. For example, the CL term in the kinetic model was very low (Table 4 ). This may reflect extensive tubular re-absorption, but may also reflect the fact that the concentrations were followed for only 25 min in the original paper (the time by which most cardiovascular variables had returned to baseline). Studies of a longer duration would help to define this clearance term better. The cardiovascular model also assumes an instantaneous baroreceptor response. While it is relatively easy (in modelling terms) to add a delay to this response, this was not supported by the data. However, if the model is extended to situations with very rapid blood pressure changes (e.g. orthostatic hypotension) this deficiency may become significant. Constructing physiologically based models, even of the simplicity presented here, requires crossing many decisions points where a choice must be made from multiple options – sometimes the choices are data driven, sometimes theory driven, sometimes the subjective experience of the model maker must be called upon. While a "wrong" model is evident because it does no match the data, there is clearly no "right" model of the cardiovascular system. It is anticipated that more limitations of the cardiovascular dynamic model will become apparent when model is rigorously compared to data for other drugs, and for other cardiovascular scenarios. It is should be expected that the model will continue to evolve as these data are collected and analysed. Conclusion The combination of the recirculatory kinetic model and the simple cardiovascular dynamic model was able to describe and predict the concentrations and cardiovascular effects of magnesium in sheep. It is proposed that the general methods used here could be applied to other drugs with cardiovascular effects. The authors are currently applying the method to intravenous anaesthetics. Abbreviations Cardiovascular term Description Frank-Starling model V blood Blood volume CVP = P v Central venous pressure MAP = P a Mean arterial pressure MCP Mean circulatory pressure V a Volume of blood in arterial compartment V v Volume of blood in venous compartment V a 0 Volume of blood in arterial compartment at MCP V v 0 Volume of blood in venous compartment at MCP CPL a Arterial compliance CPL v Venous compliance CPL ratio Ratio of venous over arterial compliance SVR Systemic vascular resistance CO Cardiac output CO L Cardiac output (left side) CO R Cardiac output (right side) HR Heart rate SV Stroke volume kc unit conversion factor – contractility CNT Contractility additional for Frank-Starling-Baroreceptor model MAPset Mean arterial pressure set point HRgain Gain for heart rate control additional for Constrained-Frank-Starling-Baroreceptor model P a S Pressure in arterial compartment when stressed P v S Pressure in venous compartment when stressed V a S Volume in arterial compartment when stressed V v S Volume in venous compartment when stressed slopeMAP slope for arterial pressure-volume relationship intMAP intercept for arterial pressure-volume relationship slopeCVP slope for venous pressure-volume relationship intCVP intercept for venous pressure-volume relationship S1 Sympathetic tone coefficient – Chronotropy S2 Sympathetic tone coefficient – Contractility SV max maximum for stroke volume-CVP relationship SV 50 half-volume for stroke volume-CVP relationship nSV "Hill factor" for stroke volume-CVP relationship Pharmacokinetic term Description Description Recirculatory model R 0 doserate of zero order infusion tau duration of zero order infusion C art Arterial magnesium concentration (total) C ven Venous magnesium concentration (total) C art,e Arterial magnesium concentration (endogenous) C ven,e Venous magnesium concentration (endogenous) C art,x Arterial magnesium concentration (exogenous) C ven,x Venous magnesium concentration (exogenous) V lung Apparent distribution volume of the lung CL Clearance V body Apparent distribution volume of the body compartment PS Permeability-surface area product of deep compartment V deep Apparent distribution volume of the deep compartment Authors' contributions RNU participated in the original magnesium studies and performed the modelling. GLL participated in the original magnesium studies and acted as a resource for cardiovascular theory. Both authors contributed to the manuscript. Supplementary Material Additional File 1 The simple Frank-Starling model. The simple Frank-Starling model written in pseudo-code to generate cardiac function curves. The code is intended to run in the "Scientist" differential equation solving program. Click here for file Additional File 2 The Frank-Starling-Baroreceptor model. The equations for the Frank-Starling-Baroreceptor model with baroreceptor control written in pseudo-code to generate cardiac function curves. Click here for file Additional File 3 The Constrained Frank-Starling-Baroreceptor model. The equations used for the final cardiovascular model written in pseudo-code to generate cardiac function curves. Click here for file Additional File 4 The final pharmacokinetic-pharmacodynamic model. The equations used for the final model written in pseudo-code. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555767.xml
544859	The involvement of survival signaling pathways in rubella-virus induced apoptosis	Rubella virus (RV) causes severe congenital defects when acquired during the first trimester of pregnancy. RV cytopathic effect has been shown to be due to caspase-dependent apoptosis in a number of susceptible cell lines, and it has been suggested that this apoptotic induction could be a causal factor in the development of such defects. Often the outcome of apoptotic stimuli is dependent on apoptotic, proliferative and survival signaling mechanisms in the cell. Therefore we investigated the role of phosphoinositide 3-kinase (PI3K)-Akt survival signaling and Ras-Raf-MEK-ERK proliferative signaling during RV-induced apoptosis in RK13 cells. Increasing levels of phosphorylated ERK, Akt and GSK3β were detected from 24–96 hours post-infection, concomitant with RV-induced apoptotic signals. Inhibition of PI3K-Akt signaling reduced cell viability, and increased the speed and magnitude of RV-induced apoptosis, suggesting that this pathway contributes to cell survival during RV infection. In contrast, inhibition of the Ras-Raf-MEK-ERK pathway impaired RV replication and growth and reduced RV-induced apoptosis, suggesting that the normal cellular growth is required for efficient virus production.	Introduction Rubella virus (RV) is the sole member of the Rubivirus genus of the Togaviridae . It has a positive-sense single stranded RNA genome that is 9762 nucleotides (nt) in length and contains two non-overlapping open-reading frames (ORFs). The 5' proximal ORF encodes the p200 polyprotein precursor for the nonstructural proteins (NSPs) p150 and p90 [ 1 , 2 ]. The 3' proximal ORF encodes the structural proteins: capsid (C), and glycoproteins E1 and E2 [ 3 , 4 ]. RV infection usually causes mild disease with few complications. However, infection during the first trimester of pregnancy results in fetal infection, and in more than 75% of cases this leads to the development of congenital abnormalities. These abnormalities include sensorineural deafness, mental retardation, and congenital heart defects, and are collectively termed congenital rubella syndrome (CRS) [ 5 ]. The cellular mechanisms activated by RV, which lead to the disruption of organogenesis, are not fully understood. However, in permissive cell cultures, the cytopathic effect (CPE) of RV has been shown to be due to caspase-dependent apoptosis [ 6 - 12 ]. Apoptosis is a key component of developmental processes in mammals, which functions to delete vestigial structures, control cell number and remodel tissues and organs [ 13 ]. Thus, it has been proposed that RV-induced apoptosis may cause irreparable damage to fetal tissues, resulting in the abnormalities observed in CRS [ 12 ]. However, the outcome of RV infection is likely to depend on multiple signaling events that control the balance between cell death and cell survival. Eukaryotic cells contain a large number of mitogen activated protein kinase (MAPK) signaling cascades that are activated in response to growth factors, cytokines and stress stimuli such as viral infection and UV irradiation. In common with apoptotic proteins, MAPKs are highly conserved and ubiquitously expressed [ 14 , 15 ]. These cascades integrate external stimuli and transmit signals to the nucleus resulting in the activation of transcription factors, which regulate expression of genes required for proliferation, differentiation, survival and apoptosis. Two well-studied mitogenic pathways are the phosphoinositide 3-kinase (PI3K)-Akt pathway and the Ras-Raf-MEK-ERK pathway, which are central to cell survival and proliferative signals respectively. PI3Ks phosphorylate plasma membrane inositol lipids at the 3' position of the inositol ring. These 3'phosphoinsoitides recruit proteins such as Akt and phosphoinositide dependent kinases 1 and 2 (PDK1/2) to the plasma membrane via their pleckstrin homology (PH) domains [ 16 , 17 ]. At the plasma membrane PDK1/2 activate Akt through phosphorylation at Ser 473 and Thr 308 . Activated Akt promotes cell survival by phosphorylating and inhibiting a number of pro-apoptotic proteins including BAD, caspase-9, GSK-3β and Forkhead transcription factors [ 18 , 19 ]. The Ras-Raf-MEK-ERK is a classical MAPK pathway where growth factor-receptor interactions trigger intracellular activation of the small G-protein Ras. Ras recruits and directly activates the MAPK kinase kinase (MAPKK) Raf, which phosphorylates and activates the MAPK kinase (MAPKK) MEK1/2, which in turn activate the MAPK ERK1/2. Activated ERK1/2 translocates to the nucleus where it can activate a number of transcription factors including c- myc , c- jun , and Elk-1 , which regulate cell cycle progression responses [ 20 ]. Activation of PI3K-Akt and Ras-Raf-MEK-ERK signaling cascades during virus infection is thought to play an important role not only in cellular growth and survival, but also in virus replication and growth during both acute and chronic virus infections [ 21 - 25 ]. This study was carried out to examine the role of PI3K-Akt and Ras-Raf-MEK-ERK signaling during RV infection in RK13 cells. The PI3K inhibitor LY294002 and the MEK inhibitor U0126 were used to investigate PI3K-Akt and Ras-Raf-MEK-ERK signaling respectively during RV replication, growth and induction of apoptosis. Apoptosis was measured in RV-infected cells by caspase activity and cell viability assays, DNA fragmentation analysis, and trypan blue exclusion staining. Involvement of PI3K-Akt and Raf-Raf-MEK-ERK signaling in RV-induced apoptosis was also examined by expression of constitutively active Akt and MEK in RV-infected cells. Results Phosphorylation of Akt, ERK1/2 and their downstream targets during RV infection The effect of RV infection on PI3K-Akt and Ras-Raf-MEK-ERK pathways was investigated by examining the expression and phosphorylation profiles of Akt, ERK1/2 and their downstream targets. Cell lysates from RV and mock infected RK13 cells were collected 12–96 hours post-infection (p.i.), separated by SDS-PAGE, and analyzed for total and phosphorylated Akt and ERK1/2 by Western blotting. Phosphorylated Akt and ERK1/2 could be detected in RV-infected cells from 48 hours p.i., and band intensity increased from 48–96 hours p.i. compared to total levels (Fig. 1A ). Phosphorylated Akt and ERK2 (but not ERK1) were detected in the mock-infected cells at 96 hours p.i. but not before, whereas total levels of Akt and ERK 1/2 were detectable at all time points (Fig. 1A ). Treatment of RV-infected cells with PI3K inhibitor LY294002 and MEK1/2 inhibitor U0126 completely inhibited activation of Akt and ERK1/2 respectively (data not shown). Figure 1 Kinase phosphorylation during RV infection. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i. of 4 PFU/cell. At indicated time points cell lysates were collected and proteins (30 μg/lane) were separated by SDS-PAGE, and analysed by Western blotting using phospho-specific antibodies. Blots were also probed with anti-tubulin antibody to demonstrate equal loading. A – Total and phosphorylated Akt and ERK (24–96 hours p.i.). B – Total and phosphorylated Akt, ERK, and p70S6K, and phosphorylated GSK-3β and c-myc. The data were consistently repeated in two independent experiments. The phosphorylation of Akt and ERK and their downstream targets p70S6K, GSK-3β, c- myc and BAD were also examined by Western blotting between 12–96 hours p.i. (Fig. 1B ). Phosphorylated Akt and ERK1/2 were detectable in RV-infected cells at 48 and 36 hours p.i. respectively. p70S6K is phosphorylated by FRAP/mTOR downstream of Akt at Thr 389 and at Thr 421 /Ser 42 , downstream of the Ras-Raf-MEK-ERK pathway. Phosphorylation at Thr 389 was observed at 12, 24, 60, 84 and 96 hours p.i. (Fig. 1B ). Phosphorylation of the Thr 421 /Ser 42 site was observed at all time points, although increases in band intensity could be seen at 12, 24, 60, 84 and 96 hours p.i., mirroring the phosphorylation at Thr 389 . Phosphorylation of Thr 421 /Ser 424 but not Thr 389 was observed in the mock-infected cells, albeit at a lower level than in RV-infected cells. The phosphorylation of GSK-3β, downstream of Akt, increased from 12 and 96 hours p.i. and was similar to that of Akt. Phosphorylation of BAD, another substrate for Akt, however, could not be detected in RV-infected or mock-infected cells. The phosphorylation of c- myc , a transcription factor activated by ERK1/2 phosphorylation, decreased between 12 and 96 hours p.i., in contrast to the phosphorylation profile of ERK1/2. GSK-3β and c- myc were also detectable in the mock-infected cells at 96 hours p.i. The effects of LY294002 and U0126 on cell viability in RV-infected cells RV induces apoptosis in RK13 cells with characteristic morphological and biochemical features [ 6 , 8 , 9 ]. The XTT assay was used to examine the effect of RV infection and LY29002 and U0126 treatment on cellular metabolism over time. XTT is a tetrazolium salt, which is cleaved by the succinate dehydrogenase system of mitochondria in metabolically active cells, to yield a soluble orange formazan product. A decrease in the intensity of formazan was used to monitor changes in cellular metabolism and cell viability in RV-infected cells by spectroscopy. Cellular viability during RV infection did not appear to be disrupted, supporting previous observations which reported that a large number of monolayer cells remain in tact and do not rapidly undergo apoptosis in RV infected cells [ 9 , 12 ] (Fig. 2 ). LY294002 treatment of RK13 cells reduced cell viability by 20%, which remained constant throughout the 12–96 hour period. Cell viability was reduced to 60% in the presence of both RV and LY294002. Thus the combined effect of PI3K inhibition and RV-infection caused a significant reduction in cell viability. Figure 2 The effect of PI3K and MEK1/2 inhibition on cell viability during RV infection. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). At indicated time points cell viability was determined by XTT assay. Tetrazolium salt (XTT) and electron coupling reagent were added directly to cells, and after 24 hours the absorbance at 405–690 nm was determined. Data represent mean ± S.E. from three independent experiments. As Ras-Raf-MEK-ERK signaling is crucial to the regulation of cell growth in many cell lines, inhibition of this pathway often has detrimental effects. A typical dose-response curve can be seen with MEK inhibitor U0126 in RK13 cells, with cell viability completely abolished by 60–72 hours p.i. (Fig. 2 ). With the addition of RV, the U0126 curve moved to the right, the effect of the drug was delayed by approximately 12 hours. Inhibition of PI3K results in an increase in the speed and magnitude of RV-induced apoptosis To evaluate the role of PI3K-dependent signaling during RV infection, the effects of PI3K inhibitor LY294002 on the development of RV-induced apoptosis were examined, 12–96 hours p.i., by caspase activity assay, trypan blue exclusion staining, DNA fragmentation and light microscopy. (Fig. 3A–D ). RV-induced apoptotic signaling has been reported to occur between 12–24 hours p.i., with peak caspase activity occurring around 72 hours p.i. at a multiplicity of infection (MOI) of 3 PFU/cell [ 6 ]. Fig. 3A shows that with a MOI of 4 PFU/cell the peak of RV-induced caspase activity occurs earlier at 60 hours p.i. When RV infection was carried out in the presence of LY294002, the maximum caspase activity increased by 53.9 % (P < 0.05) and occurred 12 hours earlier than with RV alone (Fig. 3A ). Figure 3 The effect of PI3K and MEK1/2 inhibition on RV-induced apoptosis. Serum-starved RK13 cells were mock infected or infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). Cells were harvested and analyzed for markers of apoptosis. A – At indicated time points, cell lysates were collected and incubated with artificial caspase substrate Ac-DEVD-pNA. Free pNA due to caspase cleavage was measured at an absorbance of 405 nm. Data represent mean ± S.E. from three experiments, P < 0.05. B – The number of measurable dead floating cells in the cell culture supernatant was determined by trypan blue exclusion staining at indicated time points. Data represent mean ± S.E. from three experiments, P < 0.05. C – Total DNA was extracted from detached and monolayer cells at 72 hours p.i. and apoptotic DNA fragments were resolved on a 1.5% agarose gel, stained with ethidium bromide, and visualized using UV transillumination. Molecular size markers were run in the left hand lane . D – Light microscopy photographs of cell monolayers at 72 hours p.i., at a magnification of 20X. This increase in speed and magnitude of RV-induced apoptosis is more strikingly observed in Fig. 3B , which shows the number of dead floating cells by trypan exclusion staining in the culture supernatant fluid of RV infected and LY294002 treated cells. LY294002 treatment doubles (and at 84 hours p.i. triples) the number of floating cells produced in RV-infected cells. Increases in the number of apoptotic floating cells are statistically significant at 84 and 96 hours p.i. (P < 0.05). Fragmented DNA patterns can be seen at 72 hours p.i. with both RV and RV in the presence of LY294002 (Fig. 3C ). However, the interesting feature of these apoptotic ladders is that in RV-infected cells, a significant proportion of genomic DNA is still intact, whereas when RV-infected cells are also exposed to LY294002, the majority of the genomic DNA is fragmented. The morphological changes caused by RV-infection and LY294002 were examined by light microscopy (Fig. 3D ). At 72 hours p.i. CPE and induction of apoptosis by RV can be clearly seen. RV-induced CPE is characterized in the earlier stages by clumps of apoptotic cells, surrounded by healthy cells. In the later stages the cell sheet is completely destroyed and the majority of cells have become apoptotic floaters [ 6 ]. In the presence of LY294002, RV-infected cells are almost all dead by 72 hours p.i., resembling the later stages of RV-induced CPE. LY294002-only treatment of RK13 cells did not induce apoptosis as evidenced by the lack of caspase activity (Fig. 3A ), DNA fragmentation (Fig. 3C ), and measurable floating cells (data not shown). Morphological examination of LY294002 treated RK13 cells show the cell monolayers were in tact with no visible cytotoxicity (Fig. 3D ). Inhibition of MEK1/2 reduces RV-induced apoptosis The role of Ras-Raf-MEK-ERK signaling in RV-induced apoptosis was investigated using MEK inhibitor U0126 as described above for LY294002 (Fig. 3A–D ). U0126 treatment reduced caspase activity in RV-infected cells by 51.9% (P < 0.05), with a low peak occurring at 48 hours p.i. (Fig. 3A ). The number of dead floating cells in RV and U0126-treated cells was slightly lower than in RV-infected cells at all time points (Fig. 3B ). DNA fragmentation was observed in both RV-infected cells and RV in the presence of U0126 (Fig. 3C ), although the presence of the drug also appeared to result in smearing of high molecular weight DNA, characteristic of necrosis [ 26 , 27 ]. The detrimental effect of U0126 on RK13 cell morphology is shown in Fig. 3D ; this correlates with the rapid decline in cell viability. Inhibition of MEK1/2 inhibits RV replication and growth To examine the effect of LY294002 and U0126 on RV replication and growth, RV-infected and drug-treated cell culture supernatants were tested for RV capsid gene expression by RT-PCR, and virus growth by TCID 50 assay 24–96 hours p.i.. The capsid gene is the first gene to be transcribed from the second ORF encoding the structural proteins. Therefore detection of capsid RNA by RT-PCR is a good measure of RV replication [ 1 , 28 ]. In RV-infected cells simultaneously treated with LY294002, levels of RV capsid RNA increased over time, as in RV-infected cells (Fig. 4A ). In the presence of U0126, however, levels of capsid RNA were reduced, and remained lower than that seen at 24 hours p.i. in RV-infected cells. Figure 4 The effect of PI3K and MEK1/2 inhibition on RV growth and replication. Serum-starved RK13 cells were infected with RV at an m.o.i of 4 PFU/cell with or without LY294002 (30 μM) or U0126 (15 μM). Cell culture supernatants were extracted from cells at indicated time points. A – RV RNA was extracted from virus-infected cell culture supernatants and the capsid gene was amplified by RT-PCR as described under 'Experimental Procedures'. B – Monolayers of RK13 cells in 96-well plates were infected with RV-infected cell culture supernatants, and virus titers were determined using the TCID 50 assay. Results are representative of a least two independent experiments. Both LY294002 and U0126 affected virus growth (Fig. 4B ). During RV-infection of RK13 cells with 4 PFU/cell of virus, virus titers reached 10 8 TCID 50 /ml by 96 hours p.i. However, in the presence of U0126 the titer was approximately 10 2 lower at 24 hours p.i., 10 3 lower at 48 hours p.i., and 10 4 lower at 72–96 hours p.i. LY294002 reduced virus growth to a similar extent, but unlike with U0126, by 96 hours p.i. the virus titer recovered slightly. Constitutively active Akt and MEK1/2 enhance RV-induced apoptosis To determine the importance of PI3K-Akt and Ras-Raf-MEK-ERK in the transduction of cell survival and proliferative mechanisms during RV-infection, RK13 cells were transiently transfected with constitutively active forms Akt and MEK. Significant expression of both proteins was seen after 24 hours (Fig. 5A ). Over-expression of both activated Akt and MEK enhanced RV-induced caspase activity (Fig. 5B ). RV infection in the presence of the empty pUSEamp(+) control vector slightly decreased caspase activity. Caspase activity following Lipofectamine treatment alone or pUSEamp(+) transfection was below that of the mock-infected cells (data not shown). Figure 5 Over-expression of Akt and MEK enhances RV-induced apoptosis. RK13 cells were transfected with eukaryotic expression vector pUSEamp(+) containing constitutively active HA-tagged MEK1 or myristoylated myc-tagged Akt1 under the control of a CMV promoter, or with an empty pUSEamp(+) control. A – Expression of MEK1 and Akt1 was determined by Western blotting. Cell lysates were collected 24 hours post-transfection and 30 μg protein separated by SDS-PAGE and transferred to nitrocellulose membranes. MEK1 and Akt1 were detected by anti-HA and anti-myc antibodies respectively. B – RK13 cells in 6-well plates were transfected with Akt, MEK or pUSEamp(+) control constructs for 24 hours and subsequently infected with RV or mock-infected. 24 hours later cell lysates were collected and tested for caspase activity using artificial caspase substrate Ac-DEVD-pNA. Discussion We have previously shown that RV induces caspase activation during the early stages of infection in vitro , prior to the appearance of morphological apoptotic changes [ 6 ]. In this study we demonstrated that, in common with other viruses such as Coxsackievirus B3 virus, human cytomegalovirus, influenza virus A, and respiratory syncitial virus (RSV) (Cooray, 2004; Johnson et al., 2001; Opavsky et al., 2001; Pleschka et al., 2001), signaling downstream of PI3K stimulates a survival response in the cell following RV infection and that signaling downstream of MEK1/2 is required for RV replication, growth and induction of apoptosis. Analysis of phosphorylation profiles during RV infection demonstrated that the presence of the virus stimulated an increase in the phosphorylation of ERK1/2, Akt, and Akt target GSK-3β over time. The presence of phosphorylated Akt (and occasionally ERK2) at 96 hours p.i. in the mock-infected cells, suggests that cell survival mechanisms may be activated in older uninfected cell cultures. The phosphorylation pattern of downstream target p70S6K did not follow that of Akt and ERK1/2. Apart from being phosphorylated by ERK1/2 and mTOR/FRAP downstream of Akt, p70S6K can be phosphorylated by an array of different proline-directed kinases, including PDK1, PKCζ, JNK and cdc2 which may explain this difference [ 29 - 33 ]. The phosphorylation of c- myc , a downstream target of ERK1/2, did not follow the same pattern. Levels of phosphorylated c- myc decreased as infection progressed, which was probably due to its targeted degradation or the action of cellular phosphatases. RV infection has been observed to slow cell cycle progression both in vivo and in vitro [ 12 , 34 ]. As c- myc is a transcription factor that stimulates cell cycle progression, its de-phosphorylation or degradation as RV infection progresses supports these observations. The expression and activity of c- myc and other downstream transcription factors in relation to the cell cycle during RV-infection requires further investigation. Phosphorylation of BAD, downstream of Akt, could not be detected in RV-infected cells (data not shown). However, BAD is not ubiquitously expressed and therefore may not be produced in the rabbit kidney epithelial cells (RK13) used [ 16 ]. Inhibition of PI3K signaling with LY294006 significantly increased the speed and magnitude of RV-induced apoptosis as shown by increased caspase activity, dead floating cells, apoptotic laddering of genomic DNA and decreased cell viability. Thus, RV-induced apoptotic signaling appears to be held in check by host cell survival signals downstream of PI3K. Although inhibition of PI3K did not affect RV replication, virus growth was affected. The speed of apoptotic monolayer death may have prevented production of optimal virus titers. The importance of PI3K survival signaling has been observed with other viruses. Recently phosphorylation of Akt, GKS3β and PKCζ (another downstream target of PI3K signaling), has been demonstrated in Vero E6 cells early during infection with severe acute respiratory syndrome (SARS)-associated corona virus (CoV) [ 35 ]. However, unlike in this study the survival response due to PI3K-Akt signaling was deemed to be weak, as LY294002 treatment did not result in an increase in apoptotic DNA laddering. PI3K, Akt and NFκB have also been shown to be activated prior to epithelial cell apoptosis in RSV-infected cells [ 36 ]. As with RV, inhibition of PI3K increased the speed and magnitude of RSV-induced apoptosis, although host-cell survival was suggested to occur prior to apoptotic signaling, as opposed to RV where caspase activation and Akt phosphorylation occur concomitantly [ 6 ]. PI3K-Akt signaling has also been shown to reduce coxsackievirus B3 (CVB3)-induced apoptosis. However, in contrast to RSV, the replication of CVB3 was also reduced, suggesting that PI3K-Akt survival signals may also serve as a defense mechanism against virus infection [ 37 ]. Inhibition of the MEK1/2 in RK13 cells by U0126 resulted in necrotic monolayer destruction and a significant decrease in cell viability. XTT assay and light microscopy demonstrated that RV infection appeared to delay the effect of U0126. As discussed above, RV infection stimulates ERK activity, downstream of MEK, and may therefore counteract the effect of the inhibitor. Despite this, U0126 impaired RV replication, growth, and induction of apoptosis. Therefore it appears that although RV infection slows the cell cycle progression, cells must be cycling and metabolizing normally for RV replication to occur. ERK1/2 phosphorylation has also been observed during infection with a number of other viruses, and inhibition of ERK1/2 signaling by U0126 has consistently been shown to be detrimental to virus growth. Infection of Jurkat cells with CVB3, for example, leads to up-regulation of ERK1/2 phosphorylation, and elevated levels of phosphorylated ERK1/2 have been observed in the myocardium of mice susceptible to CVB3-induced myocarditis [ 38 ]. Treatment of cultured cells with U0126 reduced CVB3 titers and inhibited the release of virus progeny [ 38 , 39 ]. Similarly, HCMV infection in human embryonic lung fibroblasts (HELs) has been shown to stimulate biphasic activation of MEK1/2 and ERK1/2, and treatment of infected cells with U0126 reduced viral DNA replication, protein production and virus titer [ 40 ]. Influenza A virus infection in vitro has also been shown to stimulate biphasic activation of MEK1/2 and ERK1/2, and U0126 treatment prevented export of ribonucleoprotein complexes from the nucleus and inhibited virus production [ 24 ]. Inhibition of MEK1/2 during HIV infection has been demonstrated to reduce infectivity, but unlike the other viruses mentioned herein, did not affect protein levels or virus production [ 25 ]. These findings, along with the results of this study, suggest that signaling downstream of MEK1/2 and ERK1/2 is important for viral infectivity and efficient virus replication and growth in vitro . Over-expression of Akt and MEK1/2 increased RV-induced caspase activity in RK13 cells. This response was not due to the transfection procedure, as the increase in caspase activity was not observed in the pUSEamp(+) or lipofectamine controls. Such a response is also seen in malignant cells, which are more readily killed by apoptotic stimuli. Thus, the over-expression of these mitogenic pathways may have resulted in a cell survival response whereby a negative feedback loop occurred that sensitized cells to RV-induced apoptosis. In order to study this further, it would be necessary to construct stable cell lines over-expressing active Akt and ERK1/2 as well as their dominant negative mutants and other signaling proteins. It is clear from the results of this and previous studies that the outcome of RV infection in vitro depends on numerous signaling events. It has been suggested that RV capsid protein, when anchored to the ER can independently induce apoptosis in culture (Duncan et. al, 2000). However this has not been confirmed by other groups and there is conflicting evidence that virus replication and the presence of the RV NSPs (which are necessary for replication) is required [ 10 , 12 , 41 ]. Interestingly the NSP p90 has been shown to interact with the retinoblastoma (pRB) cell cycle-regulatory protein and the cytokinesis regulatory protein citron-K kinase (CK), and it has been suggested that this may perturb the cell cycle [ 42 , 43 ]. How these interactions interfere with signaling pathways and modulate cellular responses, however, remains to be determined. In relation to CRS, study of the expression and localization of apoptotic and mitogen activated signaling proteins in RV-infected fetal tissues would be necessary to confirm the theory that the pathogenesis of the disease is related to perturbation of the cell cycle. However as CRS is now rare in the UK and work with fetal tissues is tightly regulated, such a study would be hard to carry out. In vivo studies are difficult, as a reliable animal model does not exist for CRS. However, it may be possible to extrapolate findings from cell culture systems. We used RK13 cells because they are the best cells in which to detect rubella-induced apoptosis; further studies are required to confirm our findings in primary human embryonic cells. Materials and methods Chemical Compounds Stock concentrations of PI3K inhibitor LY294002 [2-(4-Morpholinyl)-8-phenyl-1-4H-1-benzopyran-4-one] and MAPK/MEK inhibitor U0126 [1, 4-Diamino-2, 3-dicyano-1, 4-bis (2-aminophenylthio) butadiene] (Calbiochem, UK) were made up in dimethyl sulfoxide (DMSO). In all experiments LY294002 and U0126 were used at concentrations of 30 μM and 15 μM respectively. Cell Culture & Viral Infection Mycoplasma-free rabbit kidney epithelial (RK13) cells were obtained from the European Collection of Cell Cultures and cultured as previously described (3). RV (wild type strain RN) was propagated as previously described (3). For infection, cells were grown to confluence in minimal essential medium (MEM) supplemented with 15 mM L-glutamine and 5% FCS (v/v) (Invitrogen, UK) at 37°C in 5% CO 2 in air, and serum starved overnight. Cells were infected with RV at a MOI of 4 plaque forming units (PFU) per cell and maintained in MEM with 1% FCS until harvested at indicated time points. Where appropriate kinase inhibitors (LY294002 and U0126) were added to the media at the same time as the virus, and were present during subsequent incubation periods. Mock-infected cells were treated and harvested in the same manner as those infected, except that MEM without virus was used during the infection. RV titers, in the presence of inhibitors, were determined by TCID 50 assay in RK13 cells as the sample number was too large to perform plaque assays. Inhibitor, virus and serum concentrations were optimized to ensure that the effect of both the virus and the inhibitors could be monitored. Transfection Control and expression plasmids [pUSEamp(+), and constitutively active HA-Akt1 and Myc-MEK1 in pUSEamp(+)] were purchased from Upstate Biotechnology Inc. (UK). RK13 cells were grown to confluence in 25 cm 2 tissue culture flasks and transiently transfected with 0.25 μg of control or expression plasmids. Tranfections were carried out in serum-free MEM using Lipofectamine Plus (Invitrogen, UK), according to the manufacturer's instructions. For optimal transfection, cell monolayers were incubated with the DNA-liposome mixture for 5 hours at 37°C. Following transfection, the DNA liposome complexes were removed and replaced with fresh medium. After 24 hours, RV was added to cells, which were maintained on MEM with 1% serum (as above). After an additional 24 hours, cells were analyzed for protein expression by Western blot analysis, and for apoptosis by caspase activity assay. Western Blot Analysis Polyclonal anti-PI3K p85, anti-HA Tag, anti-myc Tag, and monoclonal anti-β-tubulin antibodies were from Upstate Biotechnology inc. (UK). Polyclonal anti-caspase-3 antibody was from Sigma (UK). All other primary antibodies were purchased from Cell Signaling Technology (UK). Cells were treated as described above and at indicated times post-infection (p.i.), washed in PBS and harvested in cell lysis buffer [50 mM Tris, 150 mM NaCl, 1% Triton-X-100, 2 mM EDTA, 2 mM EGTA, 100 μM protease inhibitor cocktail, and 100 μM each of phosphatase inhibitor cocktails 1 and 2 (Sigma, UK)]. Protein concentrations were determined using the BioRad assay (BioRad, Hemel Hemstead, UK), and equal protein loading was determined by Coomassie staining (Invitrogen, Paisley, Scotland). Lysates were electrophoresed on 12% Bis-Tris polyacrylamide gels (Invitrogen, UK) and transferred onto Hybond™ ECL nitrocellulose or PVDF membranes (Amersham Biosciences, UK). Membranes were blocked with 5% non-fat dried milk in PBS containing 0.1% Tween-20, and subsequently incubated with primary antibody (1:1000) overnight at 4°C. Specific antibody binding was detected using horseradish peroxidase conjugated anti-rabbit or anti-mouse IgG (1:2000) (Dako, UK), and immunoreactive bands were visualized using the ECL detection system according the manufacturer's instructions (Amersham Biosciences, UK). XTT Assay RK13 cells were grown to confluence in 96-well tissue culture plates at 37°C in 5% CO 2 in air. Cells were treated, in a final volume of 100 μl, with RV and kinase inhibitors as described above. At indicated times p.i., 50 μl of labeling mixture containing XTT (sodium 3'- [1-(phenylaminocarbonyl)-3, 4-tetrazolium]-bis (4-methoxy-6-nitro) and coupling reagent PMS (N-methyl dibenzopyrazine methyl sulphate) (Roche Applied Science, Mannheim, Germany) was added directly to the wells to give final concentrations of 0.3 mg/ml and 2.5 μg/ml respectively. Plates were incubated in a humidified atmosphere (37°C, 5% CO 2 ) for 24 hours. The absorbance of the formazan product was measured at a test wavelength of 450 nm and a reference wavelength of 690 nm. Caspase Activity Assay DEVD specific caspase activity assay (Promega, UK) was carried out as previously described (3). Briefly, RK13 cells were grown to confluence, and treated with RV, LY294002, and U0126 (as above). Cell lysates were collected at indicated times p.i. and stored at -70°C until required. For the assay, lysates were incubated with colorimetric substrate DEVD-p-NA for 4 hours at 37°C, and absorbance of free pNA cleaved by endogenous caspases-3 and -7 was measured at 405 nm. DNA Fragmentation Analysis Analysis of apoptotic DNA fragmentation was carried out as previously described (3). Briefly, RK13 cells in 6-well plates were treated with RV, LY294002 and U0126 as above, and harvested 72 hours p.i. Total cellular DNA was extracted from 2 × 10 6 cells according to the manufacturer's instructions (Calbiochem, Nottingham, UK). Nucleic acids were precipitated using 3 M sodium acetate, 2-propanol, and ethanol. DNA pellets were dried and re-suspended in 10 mM Tris pH 7.5, 1 mM EDTA. Ladder fragments were electrophoretically separated on 1.5% Tris-Acetate EDTA (TAE) agarose gels. Gels were stained in ethidium bromide solution (5 mg/ml) and fragmented DNA was visualized under UV light. Examination of floating cells Floating dead cells in the supernatant following infection with RV or drug treatment (as described above) were quantified by trypan blue exclusion staining. The morphological changes to the cells were examined by light microscopy using a Nikon Eclipse TS100 light microscope. Pictures of cells were taken at a magnification of 20X using a Nikon COOLPIX 4500 digital camera and processed with Adobe Photoshop 7.0 software. RV Capsid RT-PCR Total RNA was extracted from 100 μl tissue culture supernatants, collected at indicated times p.i., using a silica-guanidinium isothiocyanate method [44]. Prior to reverse transcription, RV RNA was heated to 95°C for 1 minute and kept on ice. RNA was transcribed to cDNA using Superscript III RNase H - reverse transcriptase (Invitrogen, UK). Reverse transcription was performed in 20 μl reaction volumes containing 200 U enzyme, 10 μl sample RNA, 0.5 mM of each dNTP, and 5 pmoles external reverse primer (5'-CCTGTACGTGGGGCCTTTAA-3'). RNA bound to cDNA in RNA-DNA hybrids was removed by incubation of the cDNA with RNase H (Roche Diagnostics, UK) for 20 minutes at 37°C. PCR amplification was carried out using a GC-Rich PCR System (Roche Diagnostics, UK). In the PCR reaction 10 μl cDNA was added to 40 μl of PCR reaction mix to give final concentrations of 1X GC-Rich PCR buffer, 1.5 mM MgCl 2 , 0.2 mM each dNTP, 0.5 M GC-rich resolution solution™, 0.5 pmole of forward and reverse primers (5'-TAGGAGGTGCCGCCATATCA-3' and 5'-CCTGTACGTGGGGCCTTTAA-3' respectively), and 2U Taq polymerase and a mixture of proof-reading polymerases. The cycling conditions, as recommended by the manufacturer were: 95°C for 3 minutes followed by 10 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute; and 25 cycles of 95°C for 30s, 57°C for 30s, 72°C for 1 minute (plus an additional 5 seconds per cycle), and a final extension of 72°C for 7 minutes. Amplified capsid product (1053 b.p.) was electrophoretically separated on 1% Tris-Borate (TBE) agarose gels, stained with ethidium bromide solution (5 mg/ml) and visualized under UV light. Authors' Contributions SC conceived of the study, carried out the virological and biochemical assays and drafted the manuscript. JL participated in the design of the study. JMB participated in design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544859.xml
549602	Why Blood Glucose Control Matters for the Kidney	null	One of the most common and most serious complications of both type 1 and type 2 diabetes is diabetic nephropathy. It occurs in around 30% of patients with type 1 diabetes and 10% to 40% of patients with type 2 diabetes. Diabetic nephropathy is the leading cause of renal failure in the developed world. The main effect of diabetic nephropathy is proteinuria, initially in very small amounts but which increases, leading to nephrotic syndrome and end-stage renal disease in most cases. Apoptotic renal tubular cells in diabetic nephropathy Various risk factors in individuals with diabetes are known to increase the chance of developing diabetic nephropathy, including South Asian or African background, male sex, long history of diabetes, poor blood sugar control, high blood pressure, and smoking. One early change associated with diabetic nephropathy is degeneration of the renal tubular epithelium, but the exact cause of this at the cellular level is unclear. Erwin Böttinger and colleagues have dissected out one key point in the progression to diabetic nephropathy. They looked at cell lines of renal tubular cells from humans and mice and kidney biopsies from patients with diabetic nephropathy, patients with non-diabetic renal disease, and mice with genetic and induced diabetes. In the human cell lines they showed that glucose induced the expression of CD36, a receptor known to have a role in adhesion and signal transduction (in addition to being the receptor for malaria-infected erythrocytes). They then went on to show that apoptosis of these cells occurred in the presence of glycated (glucose-modified) albumins or free fatty acids, which are present in increased amounts in patients with diabetes, and that CD36 was essential for the apoptosis to occur. They then examined how CD36 triggered apoptosis and found that it involved src kinase, p38 MAP kinase, and caspase 3. Comparing mice and humans, the researchers found that the two species are not alike: diabetic mice did not show an increase in tubular expression of CD36—even though the gene is present in mice—and had normal tubular epithelium and no tubular apoptosis. They confirmed this difference between humans and mice by showing that normal mouse epithelial cell lines were resistant to apoptosis caused by the glycated albumins; however, artificially expressing CD36 in these lines made them susceptible to apoptosis by these modified albumins. These results provide insight into one of the crucial steps in diabetic nephropathy and, in humans at least, might help to explain why high blood glucose is so damaging to the kidney, hence providing a good reason—if another is needed—for encouraging patients to control blood glucose as tightly as possible.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC549602.xml
529451	Pudendal nerve decompression in perineology : a case series	Background Perineodynia (vulvodynia, perineal pain, proctalgia), anal and urinary incontinence are the main symptoms of the pudendal canal syndrome (PCS) or entrapment of the pudendal nerve. The first aim of this study was to evaluate the effect of bilateral pudendal nerve decompression (PND) on the symptoms of the PCS, on three clinical signs (abnormal sensibility, painful Alcock's canal, painful "skin rolling test") and on two neurophysiological tests: electromyography (EMG) and pudendal nerve terminal motor latencies (PNTML). The second aim was to study the clinical value of the aforementioned clinical signs in the diagnosis of PCS. Methods In this retrospective analysis, the studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002. To accomplish the first aim, the patients sample was compared before and at least one year after surgery by means of descriptive statistics and hypothesis testing. The second aim was achieved by means of a statistical comparison between the patient's group before the operation and a control group of 82 women without any of the following signs: prolapse, anal incontinence, perineodynia, dyschesia and history of pelvi-perineal surgery. Results When bilateral PND was the only procedure done to treat the symptoms, the cure rates of perineodynia, anal incontinence and urinary incontinence were 8/14, 4/5 and 3/5, respectively. The frequency of the three clinical signs was significantly reduced. There was a significant reduction of anal and perineal PNTML and a significant increase of anal richness on EMG. The Odd Ratio of the three clinical signs in the diagnosis of PCS was 16,97 (95% CI = 4,68 – 61,51). Conclusion This study suggests that bilateral PND can treat perineodynia, anal and urinary incontinence. The three clinical signs of PCS seem to be efficient to suspect this diagnosis. There is a need for further studies to confirm these preliminary results.	Background The objective of perineology is to treat each defect of the perineum with the right procedure [ 1 - 3 ]. Pudendal nerve decompression (PND) is theoretically a basic procedure in perineology thanks to its ability to treat the defect "pudendal neuropathy". Before going into details of this procedure, it is necessary to remember the anatomy of the pudendal nerve. This anatomy is still controversial. While summarizing the data of the literature and the results of our dissections, the likeliest anatomy of the pudendal nerve presents itself as follows. The pudendal nerve is a mixed nerve carrying motor and sensory fibers. Its fibers are derived from the sacral roots S2, S3 and S4 [ 4 , 5 ]. Once the roots traverse the sacral foramen, they divide into autonomic branches forming the pelvic plexus (parasympathetic supply of the pelvic organs) and somatic branches merging to form the pudendal nerve travelling under the piriformis muscle. Near its formation point, it gives a levator branch running on the inner (upper) surface of the levator plate and providing the innervation of this muscle [ 4 ]. For Barber et al [ 6 ], this levator nerve originates directly from the S3, S4 or S5 roots. Some somatic fibers coming from S2 and S3 run close to the pelvic plexus to innervate the levator ani and the urethral sphincter [ 4 ]. Caudally, the pudendal nerve enters a small space ("clamp") between the sacro-spinal and sacro-tuberous ligaments very near the ischial spine. Just inferior to the ischial spine, the nerve gives its first branch, the dorsal nerve of the penis [ 4 ] or the clitoridal nerve. These nerves are separated from the main trunk by the pudendal vein and artery. Then, it enters the Alcock's canal formed by a division of the obturator muscle aponeurosis. In the canal the nerve crosses the sharp edge of the sacro-tuberous ligament (falciform process) [ 7 , 8 ]. Caudally, at the level of the anus, the nerve gives medially the inferior rectal nerves (usually two branches) which innervate the anal sphincter (and probably the pubo-rectalis) and the skin of the posterior perineum and anterolaterally the transversus perinei branch (for this muscle, for the ischiocavernosus muscle and maybe for the urethral sphincter) [ 4 ]. The remaining part of the nerve is usually called the perineal nerve. This nerve gives a bulbocavernosus branch and finally divides into a sphincteric branch (innervation of the urethra) and a branch which innervates the skin of the anterior perineum [ 9 ]. The pudendal canal syndrome (PCS) and its surgical treatment have been described by Shafik in 1991 [ 5 ]. This syndrome is induced by the compression or the stretching of the pudendal nerve in the Alcock's canal. The complete syndrome presents with anal incontinence, pain, hypo or hyperesthesia and urinary incontinence (and impotence in males). Some important studies were done earlier by Amarenco [ 10 ] and Robert [ 7 ] but these authors focused mainly on pain which is only a part of the syndrome. The cause of the PCS is not always clear but it is often possible to find a compression (biking, long time sitting, haematoma...) or a stretching (descending perineum, surgery, delivery....) of the pudendal nerve in the Alcock's canal [ 10 - 19 ] in the history of the patient. A change in the shape or orientation of the ischial spine induced by some athletic activities during the youth could also explain some cases [ 20 ]. The clinical signs and investigation results proposed by Shafik [ 5 ] to confirm the diagnosis of PCS before surgery are: tenderness over the pudendal canal in the ischio-rectal fossa, diminished perineal sensation, weak or absent anal reflex, reduced EMG activity of the external anal sphincter and increased PNTML. The surgical procedure described by this author (trans-perineal approach) consists in opening the Alcock's canal to give the pudendal nerve a sufficient length to be unstretched and/or to suppress compression. The trans-gluteal approach proposed by Robert to treat pudendal neuralgia aimed to open also the "clamp" between the sacro-spinal and the sacro-tuberous ligament by cutting one or two of them [ 8 ]. Since Shafik's study in 1991, some questions about the effect of PND on the PCS remained open. No peer reviewed publications confirming the efficacy of this surgery on anal incontinence or on urinary incontinence could be found. Even the existence of a genuine PCS has never been validated. The aim of this study was to answer the following questions: Is there any effect of the bilateral PND according to Shafik on: - three main symptoms of PCS ? - perineodynia (vulvodynia, perineal pain, proctalgia) [ 21 ] - anal incontinence - urinary incontinence - three clinical signs of PCS ? - abnormal sensibility - painful Alcock's canal - painful "skin rolling test" [ 22 ] - two neurophysiological tests ? - electromyography (EMG) of the anal sphincter and of the bulbocavernosus muscles. - pudendal nerve terminal motor latencies (PNTML) of the anal and perineal branches. What is the clinical value of the aforementioned three clinical signs in the diagnosis of PCS ? Methods Studied sample A retrospective analysis to study the effects of a bilateral PND. The studied sample comprised 74 female patients who underwent a bilateral PND between 1995 and 2002 done by the same surgeon. The average age was 56.1 years (range: 28 – 77). All these patients underwent a complete history and clinical examination following the three perineal axis (gynaecological, urological and colo-proctological) according to the concept of perineology [ 1 - 3 ]. The frequency of the 3 main symptoms of the PCS (anal incontinence, perineodynia, urinary incontinence) in the 74 patients before surgery is presented in Figure 1 . Figure 1 Frequency of the 3 main symptoms of the pudendal canal syndrome (perineodynia, anal incontinence, urinary incontinence) before surgery. Associated surgical procedures were performed at the same time as the PND to treat all the defects revealed by the clinical examination, and are presented in Table 1 . Table 1 Procedures associated with the bilateral pudendal nerve decompression Associated procedures Operated (n = 74) Reviewed after one year or more (n = 56) None 17 10 MVT according to Mouchel [48-50] 46 38 Correction of rectocele 49 42 Correction of cystocele 20 17 Vaginal hysterectomy 16 13 Levatorplasty according to Shafik [26] 14 13 Urethral meatotomy 4 3 Prepubien section [45] 2 1 Anal sphincteroplasty 2 2 Urethrolysis 1 1 Diagnostic tools for PCS The following variables were used: Three main symptoms of the PCS Perineodynia For perineodynia, four situations were encountered: no pain, proctalgia, unilateral pain, bilateral pain. The effect of surgery was estimated by the patient using one of the following proposals: cured, improved, unchanged or worsened. Anal incontinence For anal incontinence, a four levels ordinal scale was used: no incontinence, gas incontinence, liquid incontinence, solid incontinence. "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "solid" to "gas" incontinence. The patient was defined as "worsened" if there was a change of at least one level in the opposite direction. Urinary incontinence For urinary incontinence, a four levels ordinal scale was used: no incontinence, mild incontinence, moderate incontinence and severe incontinence. The two types of urinary incontinence, stress and urge incontinence, were evaluated separately even if both were present in the same patient (mixed incontinence). "Cured" was defined as "no incontinence". The patient was considered "improved" if there was a change of at least one level in the scale going from "severe" to "mild" incontinence. If the change observed was in the opposite direction the patient was considered "worsened". Three clinical signs of the PCS (the examinations were done in gynaecological position) Abnormal anal or vulvar sensibility Sensibility was tested with a needle comparing the left and the right sides of the vulva and of the skin 2 cm lateral to the anus. The interpretations of the results were done using a four levels ordinal scale: 0 = total anaesthesia, 1 = reduced sensibility, 2 = normal sensibility, 3 = hypersensibility. 0, 1 and 3 were considered as "abnormal sensibility". Painful Alcock's canal on rectal examination The pain induced by the palpation of the pudendal canal by rectal examination was evaluated using a seven levels ordinal scale : 0 = no pain, 1 = mild pain, 2 = mild pain with Tinel sign (irradiation of the pain), 3 = moderate pain, 4 = moderate pain with Tinel sign, 5 = severe pain, 6 = severe pain with Tinel sign. The Alcock's canal was considered "painful" if the pain was 4 or more. Painful "skin rolling test" Beginning from 5 cm behind the level of anus the skin was pinched and then rolled to the front until the skin fold was at the level of the clitoris. The skin rolling test was considered "painful" if it induced a severe pain at least at one level (Figure 2 ). Figure 2 Skin rolling test : the skin of the perineum is pinched just beneath the level of the anus and then rolled to the front searching for a sharp pain at one level. This sign is well known in the diagnosis of neuralgia. Two neurophysiological tests Concentric needle EMG Concentric needle EMG was done at rest and during voluntary contraction on both sides of the external anal sphincter and on each bulbocavernosus muscle. The richness of the EMG was grossly evaluated using a six levels scale: 1 = simple, 2 = poor, 3 = intermediate poor, 4 = intermediate, 5 = intermediate rich and 6 = rich. Anal and perineal PNTML Anal and perineal PNTML were measured before and after the operation using the St Marks electrode to stimulate the pudendal nerve by the rectal route just under the ischial spine. For anal PNTML the electrical potentials induced in the striated anal sphincter were collected using the ring of this electrode. For the perineal PNTML the method described by Kiff [ 23 ] and Snooks [ 24 ] was modified according to Amarenco [ 25 ]. The electrical potentials were collected with a concentric needle in the two bulbocavernosus muscles. In our laboratory the normal values were: less than 2.5 msec for the anal PNTML and less than 5 msec for the perineal PNTML. EMG and PNTML were done by the same physician before and at least one year after the operation. Minimal criteria for surgery At least one of the 3 following symptoms resistant to conservative treatments (physiotherapy, drugs, infiltrations, modification of diet or behaviour): a. Anal incontinence b. Perineodynia c. Urinary incontinence Associated with at least two of the five following criteria: a. increased anal or perineal PNTML b. pathological EMG of the anal sphincter or bulbocavernosus muscles (neurogenic trace, reduced activity: richness "poor" or "simple"). c. painful Alcock's canal on rectal examination (at least on one side) d. abnormal perineal sensibility (at least at one level) e. painful "skin rolling test" (at least on one side). Surgical procedure Surgical procedure as described by Shafik in 1991 [ 5 ]. The operations were done under spinal or general anaesthesia. The patients were installed in the gynaecological position. The different steps of the procedure were: - Vertical incision of the skin between the anus and the ischial tuberosity. - Opening of the ischio-rectal fossa with scissors. - The inferior rectal nerve is hooked under the finger and followed to the entrance of the Alcock's canal (Figures 3 and 4 ). Figure 3 Left Alcock's canal (showed by the tip of the forceps) viewed from the mid side on a female cadaver: on the left the pudendal nerve, on the right the inferior rectal nerve on the finger. Figure 4 Alcock's canal viewed from below like in the operating room (right side of a female cadaver): inferior rectal nerve (horizontal) showing the entrance of the canal. - Opening of this canal (without opening the clamp between the sacro-spinal and sacro-tuberous ligament). - Control of the haemostasis. - Self draining closing of the skin with nylon. Evaluation of PND The efficacy on the symptoms, on the clinical signs and on the neurophysiological tests was evaluated during a follow up consultation one year or more after the surgical procedure because the nerve healing can be very slow [ 5 ]. Statistical methods Firstly, the efficiency of the PND on the symptoms and clinical signs was studied by means of descriptive statistics. Tests of hypothesis were done to compare the mean values of the neurophysiological tests before versus after PND. Secondly, the diagnostic value of the clinical signs was evaluated in a "case control" setting. A subject belongs to the "controls group" when PCS is considered to be absent, namely if the patient does not present any of the following symptoms, signs or risk factors for PCS: perineodynia, anal incontinence, prolapse, previous surgery in the area, dyschesia. The clinical signs are not used to decide if a subject belongs to the controls or cases group. The statistical comparison was done between the patient's group before ("cases group") and after the operation, and the "controls group" of 82 women (average age: 48.8 years, extremes 27–76). Statistical analysis of differences was performed using chi-square testing for categorical variables and t-tests for continuous variables. Results Effects of surgery Effect on the symptoms of the PCS The effect of PND on the symptoms of PCS is presented in Table 2 . Table 2 Effect of PND on the 3 main symptoms of the PCS Parameters Perineodynia (pain) Perineodynia (pain) Anal incontinence Anal incontinence Stress urinary incontinence Stress urinary incontinence Urge incontinence Urge incontinence All Without: levat All Without: sphincteroplasty, levat, recto All Without: levat, mvt, cysto, prepubien, meato, urethrolysis All Without: levat, mvt, cysto, prepubien, meato, urethrolysis Number of cases studied 74 59 74 22 74 22 74 22 Number of pathological results 26 22 46 9 47 4 33 4 Follow up less than 1 year or lost 8 8 10 4 10 3 6 0 Follow up 1 year or more 18 14 36 5 37 1 27 4 Mean follow up in months (range) 22,2 (12–48) 24,5 (12–48) 26,4(12–70) 17,2 (12–26) 32 (12–96) 12 26,7(12–72) 18,5 (12–26) Cured (%) 11 (61,1%) 8 (57,1%) 23 (63,9%) 4 (80%) 26 (70%) 0 (0%) 17 (62,9%) 3 (75%) Improved (%) 3 (16,6%) 2 (14,3 %) 7 (19,4%) 1 (20%) 7 (18,9%) 1 (100 %) 6 (22,2%) 0 (0%) No change (%) 4 (22,2%) 4 (28,6%) 4 (11,1%) 0 (0%) 4 (10,8 %) 0 (0 %) 3 (11,1%) 0 (0%) Worse (%) 0 (0 %) 0 (0 %) 2 (5,5%) 0 (0%) 0 (0%) 0 (0 %) 1 (3,7%) 1 (25%) levat = levatorplasty, recto = cure of rectocele, cysto = cure of cystocele, prepubien = prepubien section, meato = meatotomy. In order to treat completely the patient, PND was frequently associated with other procedures which might have an effect on the symptom studied. Therefore, the results for each symptom were presented in two columns: the first corresponds to the entire sample ("all") and the second to the small group of patients in which the symptom was treated by PND only ("without"). Effect on perineodynia On the 26 patients with pain before the operation, 18 were reviewed 12 months or more after the operation. The pain had disappeared in 11 and was reduced or had another origin (painful puborectalis) in 3. The cure rate with a mean follow-up of 22,2 months was 61,1 % (77,7% cured or improved). As none of the surgical associated procedures used in this study were known to improve or cure perineodynia, the only procedure removed was levatorplasty. Theoretically this operation can reduce the stretching on the pudendal nerves by reducing the sagging of the levator plate. The results were similar in the group without levatorplasty. Effect on anal incontinence On the 46 patients with anal incontinence before the operation, 36 were reviewed 12 months or more after the operation. 23 of them were cured, 7 improved, 2 were worse and 4 reported no change. The cure rate with a mean follow-up of 26.4 months was 63.9 % (83,3% cured or improved). The results according to the severity of incontinence are presented in Table 3 . Table 3 Effect of PND on anal incontinence according to the incontinence level. Cured Improved Unchanged Worsened Solid (n = 5) 3 2 0 0 Liquid (n = 20) 12 5 3 0 Gas (n = 11) 8 0 1 2 To study the real impact of PND on anal incontinence, we removed the following cases in which PND was associated with anal sphincteroplasty, levatorplasty or cure of rectocele by fascia and perineal body restoration: - 2 patients had anal sphincteroplasty together with the PND: one was cured and the other improved. - Levatorplasty according to Shafik [ 26 ] was used in 8 patients who had a severe levator plate sagging. This procedure could have a "post-anal repair effect" [ 27 ] and therefore improve anal incontinence. - 30 cases had a cure of rectocele by fascia and perineal body restoration (Ayabaca and coll. [ 28 ] found an improvement of anal incontinence in 25 of their 34 patients. Nevertheless, none of them gained full continence post-operatively). In the small group of patients with PND only, 5 were reviewed one year or more after the operation: 4 were cured (3 incontinences for liquid and 1 for gas) and 1 improved (liquid incontinence). Anal ultrasound was done before the operation in 13 of the 36 patients reviewed. Only 4 were normal, 3 showed a rupture of the internal and external sphincters, and 6 presented a disruption of the external sphincter alone. In the 7 patients who had a rupture of the anal sphincter (5 external only, 2 internal and external) without anal sphincteroplasty and a follow up of more than 12 months (mean 18.5 months), 4 were cured and 3 were a failure. 5 patients who were still incontinent after 1 year follow up (2 incontinences for flatus and 3 for liquid) became continent 2 years after the operation. Effect on urinary incontinence Five patients presenting urinary incontinence (4 urge and 1 stress incontinence) had PND without any other procedure around the urethra. The mean follow up was 18,5 months for the 4 patients with urge incontinence. In this small group, 3 patients were cured and one was a failure. The patient with stress urinary incontinence was improved (the number of pads used per day was reduced from 9 to 4) one year after surgery. Effect on the clinical signs The effect of PND on the three clinical signs is described in Table 4 . Table 4 Effect of PND on the three clinical signs Parameters Abnormal sensibility (at least at one level) Abnormal sensibility (at least at one level) Painful Alcock's canal (at least on one side) Painful Alcock's canal (at least on one side) Painful skin rolling test (at least on one side) Painful skin rolling test (at least on one side) All Without: levat All Without: levat All Without: levat Number of tests before surgery 42 27 46 32 39 26 Follow up less than 1 year or lost 11 9 19 16 22 16 Follow up 1 year or more 31 18 27 16 17 10 Normal test before (reviewed 1 year or more after) 15 8 9 6 8 2 Abnormal test before (reviewed 1 year or more after) 16 10 18 10 9 8 Mean follow up (range) 27,7 (12–68) 32,2 (12–68) 28,1 (12–68) 32,8 (12–68) 28,7 (12–60) 33,7 (12–60) Normal before => Normal after 14 8 9 6 7 2 Normal before => Abnormal after (%) 1 0 0 0 1 0 Abnormal before => Normal after (%) 11 (68%) 6 (60%) 11 (61%) 7 (70%) 6 (66,6%) 5 (62,5) Abnormal before => Abnormal after 5 4 7 3 3 3 Because levatorplasty can theoretically reduce the stretching of the pudendal nerve, the evaluation of the effect on the clinical signs has been done for the entire sample ("all") and for the same group without the patients who had a levatorplasty ("without: levat"). The cure rate of the 3 clinical signs was between 60 and 70 % depending of the sign and of the type of sample studied. Effect on EMG and PNTML 38 patients underwent a complete EMG evaluation before and after surgery. A relevant comparison was possible in 35 patients. 3 patients were excluded because one of the two EMG explorations was insufficient for technical reasons. The average follow up was 16,9 months (range: 12 – 35,4 months). Left and right values for one parameter correspond to two different nerves. Therefore, these values were considered independent (maximum number of analysed cases: 35 × 2 = 70). The effect of PND on EMG and PNTML is presented in Table 5 . Table 5 Effect of PND on EMG and PNTML Anal Richness (range 1 to 6) BC Richness (range 1 to 6) Anal PNTML (msec) Perineal PNTML (msec) All subjects 74 74 74 74 Follow up less than 12 months or lost 39 39 39 39 Analysed cases(left and right) 70 61 70 51 Mean Before 2,70 2,23 3,38 5,63 Mean After 3,11 2,44 2,63 5,21 t-test p-value(one-tail) 0,00007 0,06989 0,00004 0,00816 Left and right values for each level (anal and perineal) were included in the same group. P-values at the bottom line correspond to one-tailed significance tests of the mean differences "before" versus "after". The "Anal Richness" on EMG after surgery was significantly higher than before. The mean "Bulbocavernosus Richness" after surgery was slightly higher than before but this difference was not significant. Both anal and perineal PNTML after PND were significantly reduced compared to values before. The box-plots of the 4 studied parameters are presented in Figures 5 and 6 . Figure 5 Effect of PND on anal and bulbocavernosus (BC) richness on EMG. The box is defined by the sample mean plus or minus one standard error of the sample mean. The probability to obtain a value in the box is 67 %. The whiskers represent the 95% confidence intervals of the population means. Figure 6 Effect of PND on anal and perineal PNTML. The box-plots definitions are the same as in Figure 5. Evaluation of the clinical signs We present here the results concerning the evaluation of the three clinical tests: abnormal sensibility, painful Alcock's canal and painful "skin rollling test" as diagnostic tests for PCS. The statistical analysis is based on the following contingency tables presented in Tables 6 to 9 . Table 6 "Abnormal sensibility" in the diagnosis of pudendal canal syndrome Cases Controls Abnormal sensibility Before Surgery After Surgery Abnormal 24 7 19 Normal 18 36 63 Total 42 43 82 Chi-square versus Controls 12,691 0,449 P-value < 0,001 0,503 At the bottom lines, p-values and chi-square test statistics correspond to the homogeneity tests comparing the "Cases Before Surgery" to "Controls". The results obtained for the homogeneity test comparing "Cases After Surgery" versus "Controls" are also reported in the tables, although this does not contribute to the sensibility/specificity analysis. Table 7 "Painful Alcock's canal" in the diagnosis of pudendal canal syndrome Cases Controls Painful Alcock's canal Before Surgery After Surgery Abnormal 32 10 24 Normal 14 30 58 Total 46 40 82 Chi-square versus Controls 17,842 0,0776 P-value < 0,001 0,781 Table 8 "Painful skin rolling test" in the diagnosis of pudendal canal syndrome. Cases Controls Painful skin rolling test Before Surgery After Surgery Abnormal 21 6 13 Normal 17 28 69 Total 38 34 82 Chi-square versus Controls 17,968 0,001 P-value < 0,001 0,97 Table 9 The three clinical signs in the diagnosis of pudendal canal syndrome Cases Controls The three clinical signs Before Surgery After Surgery Abnormal – All positive 13 3 6 Abnormal – Two positive 8 3 9 Abnormal – One positive 5 3 20 Normal – All negative 6 24 47 Total 32 33 82 Chi-square versus Controls 26,528 3,834 P-value < 0,001 0,280 The proportions of observations in the "Cases Before Surgery" and "Controls" columns of the contingency table vary significantly from row to row (p-values <0,001), whereas no significant difference is observed between the proportions of observations between "Cases After Surgery" and "Controls" (p-values >0,05). Estimated sensibility, specificity, predictive values (positive and negative) and odd ratio (estimated values and 95% confidence intervals) corresponding to each of the three clinical tests and combinations of all of them are presented in Tables 10 and 11 , respectively. The predictive values were calculated for a PCS prevalence of 20 %. All the indicators in Tables 10 and 11 are estimated from data in Tables 6 to 9 , columns "Cases Before Surgery" and "Controls". Table 10 Evaluation of each of the three clinical signs of pudendal canal syndrome Tests SE SP PPV NPV OR 95%IC(OR) Nb of Cases Before Nb of Controls Abnormal sensibility 0,57 0,77 0,38 0,88 4,42 1,99 – 9,82 42 82 Painful Alcock's canal 0,70 0,71 0,37 0,90 5,52 2,51 – 12,15 46 82 Painful skin rolling test 0,55 0,84 0,47 0,89 6,56 2,74 – 15,68 38 82 SE = sensibility, SP = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odd ratio, 95%IC (OR) = 95 % confidence interval of the odd ratio. PPV and NPV for a prevalence of 20 %. Table 11 Evaluation of different combinations of the three clinical signs of pudendal canal syndrome Test: All Three Clinical Signs SE SP PPV NPV OR 95%IC(OR) Nb of Cases Before Nb of Controls All positive vs. All negative 0,68 0,89 0,60 0,92 16,97 4,68 – 61,51 19 53 At least 2 positive vs. At least 2 negative 0,66 0,82 0,47 0,90 8,53 3,40 – 21,39 32 82 At least 1 positive vs. All negative 0,81 0,57 0,32 0,92 5,82 2,16 – 15,66 32 82 SE = sensibility, SP = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odd ratio, 95%IC (OR) = 95 % confidence interval of the odd ratio. PPV and NPV for a prevalence of 20 %. The most sensible test is the "Painful Alcock's canal" and the most specific is the "Skin rolling test". Using the three signs altogether, the most sensible combination is "At least 1 positive versus All negative" and the most specific combination is the "All positive versus All negative". Side effects During one operation a heavy bleeding coming from the pudendal artery just near the pudendal nerve was very difficult to treat (selective ligature). This patient had a blood transfusion but no long term side effect. Since the operation, one patient has presented sometimes a short lasting clitoridal pain. This patient had also an increase in her anal incontinence (gas incontinence became a liquid incontinence). Three patients had wound healing problems which resolved with simple disinfection. Prevalence In the literature there is no data available about the prevalence of the PCS. Therefore, it seems to be a rare event. In this study, we evaluated the prevalence of the PCS in an outpatient perineology clinic. By using three different methods during the last 24 months (percentage of pudendal nerve decompressions in the treatment of prolapse or incontinence : 13/78; percentage of anal incontinence and/or perineodynia in our outpatient consultation : 78/316 ; percentage of positive skin rolling tests : 9/55) the estimated prevalence should be around 20%. Discussion Effects of surgery Before discussing the results of surgery, the first important issue is about the interest of a bilateral decompression. The benefit – risk ratio must be studied. The results of Shafik were obtained after bilateral decompression [ 5 , 29 , 30 ]. For urinary and anal incontinence, it seems logical to treat both nerves because the sphincters have a bilateral innervation and if one nerve is suffering maybe there is a problem on the other. The EMG exploration is not very sensitive and doesn't study the sensory pathway, which could be very important for continence. For the same reason, bilateral decompression seems logical in the treatment of proctalgia. For unilateral pain, the dilemma is more important. The risk is to induce pain at the "normal" side. Until now we have been performing bilateral operations without such a side effect but a controlled randomised study would be necessary to conclude. The treatment of pain starts with a holistic approach of the woman (drugs, psychotherapy, relaxation...) with exclusion of other causes of pain: piriformis syndrome, coccygodynia, interstitial cystitis, endometriosis... The other neurological causes must be excluded by a complete electrophysiological study of the perineum (sacral latencies, PNTML, detection EMG and sensory evoked potentials) and imaging of the spinal cord [ 31 ]. If the diagnosis is confirmed an infiltration of the Alcock's canal under scanner control can be tried. This infiltration is successful in 57% in the short term but only in 15 % of the cases after one year [ 32 ]. It can be repeated maximum 3 times to avoid a nerve irritation. In the treatment of pain the results of this study are similar or better than those obtained in previous studies [ 32 - 34 ]. Even with the transgluteal approach where the "clamp" between the sacro-spinal and the sacro-tuberous ligament is opened by sectioning the sacro-spinal ligament, the cure rate remains around 50%. In the 4 cases of proctalgia fugax the results were better (3 cured and 1 improved). Shafik [ 5 ] had also very good outcomes with this type of pain (100% cured). In this study, the results were worse if the pain was bilateral. For Robert early diagnosis appears to be the determining factor in improving results [ 35 ]. He used the infiltration of the Alcock's canal with lidocaïne-corticoïds as a test before operating. According to him a sufficient pain relieve, lasting during a short period, is a good indication for surgery. Mauillon et al also thinks that complete disappearance of pain for at least two weeks after a nerve block repeated twice before surgery may be the best criterion to predict success [ 34 ]. In this study, the patients presenting with perineodynia only (n = 10; Figure 1 ) had an infiltration before surgery but the number of cases was not sufficient to give a relevant impression about the infiltration test. For anal incontinence, our results were in the same range as Shafik [ 29 ]. In a previous study we also had similar results [ 36 , 37 ]. The exclusion of the patients who had sphincteroplasty, levatorplasty (possible post-anal repair effect [ 27 , 28 ]) and/or a cure of rectocele did not change the cure rate. More interesting was the cure rate in the group of patients with a clear rupture of one or the two anal sphincters. The traumatic rupture of the anal sphincter (delivery, sphincterotomy...) usually induces an immediate anal incontinence. In the patients who remained continent the power of the broken muscles remain sufficient to avoid flatus or faeces leakage. In the long term the continence is probably maintained with the help of the fibrous tissue located between the two edges of the ruptured muscle which acts like a bridge and therefore enables the sphincter to be efficient during many years. The aging process of the muscle and the pudendal neuropathy reduce the power of the muscle (and probably the sensibility in the anal canal) with time and explain the appearance of an anal incontinence. Therefore it is logical to restore continence by improving the conduction in the pudendal nerve. This fact can also explain why the results of sphincteroplasty decrease with time especially in the non diagnosed or treated pudendal neuropathies [ 38 ]. The fact that 5 patients were cured from their anal incontinence only 2 years after surgery emphasized the importance of a long follow up period to obtain relevant cure rates. Surprisingly, the cure rate seems to be not dependant of the degree of anal incontinence but the number of solid incontinences (5 cases; 3 cured and 2 improved) was too small to validate this impression. The results of the pudendal nerve decompression seem to be equivalent to these of neuromodulation [ 39 ] and the procedure is far less expensive because there is no need for a special material. If this study is confirmed by others, the treatment of the neuropathy should be done before any trial of neuromodulation. In fact it is logical to repair the electric cable before enabling the current to pass. For urinary incontinence the number of cases is too small to give a relevant cure rate but there were enough cases to suggest that this surgery can treat some patients with stress or urge incontinence. In a previous study, 3 of the 7 patients presenting a stress urinary incontinence were cured by bilateral pudendal nerve decompression alone [ 36 , 37 ]. In Shafik's study 6 patients were cured from their stress urinary incontinence, 5 improved and one was a failure [ 30 ]. For this author, the efficacy of the pudendal nerve decompression on stress urinary incontinence is due to an increase of the external urethral sphincter EMG activity and to a decrease in the straining urethral reflex latency (time between the expiration involved with the cough and the first deflection of the reflex muscle action potential complex) and PNTML. For Shafik [ 40 ] the increase of urethral pressure during abdominal hyperpressure is not only passive but is induced by an active contraction of the urethral sphincter. After an injection of lidocaïne in the sphincter the urethral hyperpressure was suppressed. Thind & al. clearly demonstrated the role of the pudendal nerve in urinary continence. These authors showed a clear reduction of the maximum urethral pressure and a decrease in the adjunctive urethral closure forces during stress after bilateral pudendal blockade [ 41 , 42 ]. This is also in agreement with the study of Constantinou which demonstrated that a fast-acting contraction occurs in the distal third of the urethra 240 plus or minus 30 msec before the bladder hyperpressure [ 43 ]. Furthermore, Ko and Kim demonstrated that pudendal nerve block with a 7% phenol solution is very effective in the treatment of external urethral sphincter hypertonicity in patients with spinal cord injury [ 44 ]. This study is the first one dealing with a possible effect of the pudendal nerve decompression on urge incontinence. It is probably due to a better control of the urethral sphincter which can reduce urethral instability [ 45 ] and improve the inhibition of the detrusor activity. One weakness of this study is the rough evaluation of the symptoms. We did not use any scoring system, pad test, quality of life questionnaires or "visual analog pain scale". Furthermore the number of anal and urethro-vesical manometries done before and after PND was too small to give relevant results. The objective evaluation of PND was done using two neurophysiological tests and the clinical examination. Like Shafik [ 29 , 30 ] we found a significant increase in anal richness on EMG, a significant reduction of anal and perineal PNTML after surgery and a significant reduction in the frequency of the clinical signs. The skin rolling test was improved as much as the perineal sensibility and the Alcock's canal pain, thus showing its relevant link with the PCS. Evaluation of the clinical signs and minimal criteria needed for the diagnosis Shafik described many clinical signs of the PCS [ 5 , 29 , 30 ]. In this study only three signs were studied carefully. Shafik described two of them: abnormal perineal sensibility and pain during the palpation of the Alcock's canal by a rectal examination. The third one is the "skin rolling test" which is well known in the diagnosis of neuralgia in other parts of the body [ 22 ]. This study is the first one in which this test was utilized as a clinical sign of PCS. Compared to patients with negative clinical signs, those having positive clinical signs have a 4,42 ; 5,52 and 6,56 higher likelihood of PCS for "Abnormal sensibility", "Painful Alcock's canal" and "Painful skin rolling test", respectively (Table 10 ). When patients with all three signs positive are compared to patients with all three signs negative, the odd ratio is 16,97 (Table 11 ). All the estimated 95% confidence intervals for the odds ratios are significantly higher than 1, indicating that the clinical signs can be considered as valuable signs in the diagnosis of PCS. The most specific sign was the "Painful skin rolling test" and the most sensitive was the "Painful Alcock's canal". The association of the three positive tests had a very high specificity in the diagnosis of a PCS (89 %). This high specificity was confirmed by the low frequency of this association after the operation (return to the same level as the control group). Therefore, in some cases the clinical examination should be sufficient to prove the existence of a PCS. For example, a patient presenting anal incontinence, an intact sphincter proven by ultrasound and the three clinical signs positive has almost certainly a PCS. Of course, from a scientific point of view it is still interesting to perform a complete electrophysiological study and a precise neurological examination to exclude a central problem (multiple sclerosis, tumor...) or a polyneuropathy. However, making the diagnosis of PCS is not usually an easy task. Many times, there is a high degree of suspicion but, as in many illness, all the symptoms or signs are not present. In this study, we decided to operate when at least two of the five clinical and neurophysiological signs described in the methods section were associated with one or more of the 3 classical symptoms (perineodynia, anal incontinence and urinary incontinence). At the beginning of this study, it was usually "increased PNTML" and "painful Alcock's canal". With the introduction of the "skin rolling test" and of the "sensibility test", clinical examination became more important in the decision. The more symptoms (especially anal incontinence and perineodynia) and signs were present, the more confident we were in the diagnosis of PCS. Further studies are necessary to validate this and to define more precisely the minimal criteria needed for the PCS diagnosis. Side effects The pudendal nerve decompression by the perineal route is a blind procedure. The search for the inferior rectal nerve and the opening of the Alcock's canal are done under finger control. In our experience it is not easier with retractors. Therefore it is necessary to have a clear anatomical vision of this area before performing the operation. Maybe the use of a laparoscope would help [ 46 ] but the procedure will become more expensive and time consuming. To suppress the blind character of the procedure the transgluteal approach proposed by Robert [ 8 ] or the more recent transvaginal approach from Bautrant [ 47 ] could be other ways to treat the PCS. Until now the results on pain are the same as those obtained by the Shafik's approach but with the concurrent sections of one or two ligaments of the pelvis (sacro-spinal and/or sacro-tuberous ligaments). However, we should be aware that the long term effects of these sections on the stability of the pelvic region are until yet unknown. Therefore, if the "clamp" must be open efforts should be done to open it without cutting a ligament. Up to now no data are available about a potential effect of the transgluteal or transvaginal procedures on urinary or anal incontinence. Despite the blind character of the procedure we only had one heavy bleeding probably coming from the pudendal artery. One patient still presents with a mild intermittent clitoridal pain and a worsening of anal incontinence. Because the nerve of the clitoris leaves the pudendal nerve just before the entrance into the Alcock's canal this problem is probably the result of a too large dissection in the upper part of this canal. The two cases of anal incontinence worsening (gas incontinence becoming liquid incontinence), including the aforementioned patient, are difficult to explain. Maybe the neuropathy increased with the stretching involved in the procedure, the scarring process or a too large dissection. It could also be the result of the changes in the posterior level anatomy induced by concomitant procedures (easier expulsion of gas or faeces). For the 2 patients the EMG data and the clinical examination after the operation did not improve therefore showing that the neuropathy was not healing. Prevalence Because the roughly estimate prevalence of PCS is around 20%, this "defect" seems to be a very frequent problem in Perineology. Therefore it should be logical to search for it in each clinical examination of a patient presenting with prolapse, perineodynia, urinary or anal incontinence. Conclusions Pudendal neuropathy is probably a frequent "defect" in perineology. Pudendal nerve decompression seems to be the defect specific procedure indicated in such a problem. In fact it can treat perineodynia, anal and probably urinary incontinence. Anal incontinence can be cured by pudendal nerve decompression alone even in the presence of a clear disruption of the anal sphincter on anal ultrasound. Anal richness on EMG increases and PNTML decrease significantly after surgery proving an objective effect on the nerve. The frequency of abnormal puncture sensibility, painful Alcock's canal and painful "skin rolling test" are significantly reduced by the operation. This study suggests that the three clinical tests could be used in practice to confirm or suspect the diagnosis of pudendal neuropathy in case of pain, urinary and/or anal incontinence. However, further studies are necessary to confirm these preliminary results. Abbreviations PCS = pudendal canal syndrome PND = pudendal nerve decompression EMG = electromyography PNTML = pudendal nerve terminal motor latency Competing interests The authors declare that they have no competing interests. Authors' contribution JB conceived the study, carried out surgery and clinical follow up and drafted the manuscript. DC participated in the design of the study and performed the statistical analysis. MB carried out the neurophysiological examinations. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529451.xml
544871	WINPEPI (PEPI-for-Windows): computer programs for epidemiologists	Background The WINPEPI (PEPI-for-Windows) computer programs for epidemiologists are designed for use in practice and research in the health field and as learning or teaching aids. They aim to complement other statistics packages. The programs are free, and can be downloaded from the Internet. Implementation There are at present four WINPEPI programs: DESCRIBE, for use in descriptive epidemiology, COMPARE2, for use in comparisons of two independent groups or samples, PAIRSetc, for use in comparisons of paired and other matched observations, and WHATIS, a "ready reckoner" utility program. The programs contain 75 modules, each of which provides a number, sometimes a large number, of statistical procedures. The manuals explain the uses, limitations and applicability of specific procedures, and furnish formulae and references. Conclusions WINPEPI provides a wide variety of statistical routines commonly used by epidemiologists, and is a handy resource for many procedures that are not very commonly used or easily found. The programs are in general user-friendly, although some users may be confused by the large numbers of options and results provided. The main limitations are the inability to read data files and the fact that only one of the programs presents graphic results. WINPEPI has a considerable potential as a learning and teaching aid.	Background This paper describes the WINPEPI (PEPI-for-Windows) programs recently added to the PEPI suite of computer programs for epidemiologists, and discusses some of their uses and limitations. The programs were developed for use in practice and research in the health field and as learning or teaching aids. PEPI (an acronym for P rograms for EPI demiologists) grew from a set of programs for programmable pocket calculators that was published in 1983 to "make life easier for investigators, extend the use of appropriate analytic methods, and enable researchers to concentrate on substantive issues rather than on procedural technicalities" [ 1 ]. The first version of PEPI appeared in 1993 [ 2 ], and was followed by version 2 (where the name "PEPI" was first used, in 1995) [ 3 ], version 3 (in 1999) [ 4 ], and version 4 (containing 43 programs, in 2001) [ 5 ]. The original programs were DOS-based. The first WINPEPI program, WHATIS, was included in version 4 of the PEPI package, a review of which stated: "WHATIS, the only Windows program, is our pick for the best program in PEPI. If all the programs could be converted into the WHATIS type of format, PEPI will be a truly outstanding package!" [ 6 ]. Four WINPEPI programs, containing 75 modules, have so far been issued. They provide many procedures not offered by the DOS-based programs, but do not include all those provided by the latter (which can be run in Windows as DOS applications, complementing the WINPEPI programs). Implementation There are at present four WINPEPI programs, DESCRIBE, COMPARE2, PAIRSetc, and WHATIS. The programs are free, and can be downloaded from the Internet. New versions have been issued at frequent intervals. Comprehensive manuals are provided. These furnish full information about each module, including explanations of the uses, limitations and applicability of specific procedures, and formulae or references. DESCRIBE DESCRIBE has 14 modules for use in descriptive epidemiology. It can appraise rates or proportions and categorical or numerical data (including survival data), examine a sequence of rates or other values (including the appraisal of seasonal variation), perform direct and indirect standardization, estimate prevalence from a cluster or stratified sample or by the capture-recapture method, determine required sample sizes, and appraise screening or diagnostic tests (with procedures for use in meta-analyses of studies of these tests). COMPARE2 COMPARE2 has 28 modules for use in comparisons of two independent groups or samples, and may be used to analyze cross-sectional, cohort and case-control studies, and trials. It can compare proportions or odds, risks, rates, and categorical and numerical data (including survival data), appraise the effect of misclassification, and determine power and sample size for a variety of tests. The program can deal with stratified data, analyzing the combined strata as well as each stratum; it permits the control of possible confounding by the stratifying variable or variables, and the assessment of heterogeneity as an indication of effect modification. It can be used in meta-analyses, to compare study results and, if warranted, combine them. PAIRSetc PAIRSetc has 29 modules for use in comparisons of paired and other matched observations, such as matched-control trials and cohort studies, matched case-control studies, before-after studies, and reliability studies that compare replicate observations or methods of measurement. The "etc" in its name indicates the program's ability to deal with matched sets larger than pairs. The program can compare dichotomous, categorical and numerical data (including paired survival data), appraise the effect of misclassification, and determine power and sample size for a variety of tests and for measuring kappa or intraclass correlation coefficients. Like COMPARE2, PAIRSetc can deal with stratified data. WHATIS WHATIS is a "ready reckoner" utility program with four modules. It provides a calculator (expression evaluator) that stores values and formulae, enabling them to be recalled when needed, and it computes confidence intervals for a variety of statistics, P-values corresponding to given values of z , t , chi-square, or F , or vice versa, and time-spans. The modules Some of the modules have very specific purposes; for example, to determine the sample size required to perform a specific test with a given power or precision, or to appraise the effect of misclassification in a given situation by computing the "true" findings that would give rise to the observed findings. Other modules provide many statistical procedures, as is illustrated by the following summaries of what two of the richer modules do. A module may not only provide numerous tests and measures, it may also use alternative methods of estimation. Comparison of proportions or odds (module A of COMPARE2) After entry of a 2 × 2 table, this module provides exact one-tailed and two-tailed tests (Fisher's, mid-P, and Overall's continuity-corrected tests and Tocher's test), chi-square tests (with and without Yates's, Upton's, and Haber's corrections), an optional equivalence test, the ratio of proportions (with its standard error and 90%, 95% and 99% confidence intervals and Jewell's low-bias estimator), the difference between proportions (with its standard error and 90%, 95% and 99% confidence intervals computed by Fleiss's procedure and by Wilson's score method, without and with a continuity correction), the odds ratio (with 90%, 95% and 99% confidence intervals – Cornfield's and exact Fisher's and mid-P intervals – and Jewell's low-bias estimator), Yule's Q, phi , and lambda . Separate results are shown for studies in which inverse sampling was used. For stratified data, the combined analysis provides Fisher and mid-P exact and Mantel-Haenszel tests, an optional equivalence test, three estimators of the overall ratio of proportions and of the overall difference between proportions (precision-based, Mantel-Haenszel, and DerSimonian-Laird estimators, with 90%, 95%, and 99% confidence intervals), and four estimators of the overall odds ratio (conditional and unconditional maximum-likelihood estimators, a Mantel-Haenszel estimator, and a DerSimonian-Laird estimator, with 90%, 95%, and 99% Fisher's, mid-P, Mantel-Haenszel, Cornfield-Gart, and Dersimonian-Laird intervals), heterogeneity tests and measures ( H and I -squared, with their 95% confidence intervals), and (for meta-analysis) estimates of the fail-safe N and two tests for a skewed funnel plot (regression asymmetry and adjusted rank correlation tests). Appraisal of numerical data (module D of DESCRIBE) This module appraises a frequency distribution, and also appraises a sequence of numbers. It describes the frequency distribution in terms of its central tendency (the mean, with its standard error and 90%, 95% and 99% confidence intervals, three robust estimators of the mean, the geometric mean, and the median, with its 95% confidence interval) and dispersion (quantiles, standard deviation, variance, mean deviation from the mean, and median absolute deviation from the median), and it performs the Grubbs test for outliers. The shape of the frequency distribution is appraised in terms of symmetry or skewness (Bowley's quartiles-based skewness coefficient, Randles-Fligner-Policello-Wolfe test, Wilcoxon signed-rank test of symmetry around the sample median) and peakedness or flatness (Moors octiles-based kurtosis coefficient, Kolmogorov-Smirnov test for an even distribution). The shape of the frequency distribution is pictured in box-and-whisker diagrams, for both raw and log-transformed data. Two tests for normality (Lilliefors and D'Agostini-Pearson tests) are applied to the raw and log-transformed data. The median or mean can be compared with a hypothetical value (using a t -test and Wilcoxon's signed-ranks test), and the Poisson dispersion test for heterogeneity is done (appropriate only if the values that were entered are counts). If a sequence of numbers is entered, it is tested for randomness (two runs tests, an up-and-down-runs test, and the mean square successive difference test), trend (Mann-Kendall and Cox-Stuart tests – including a test controlling for seasonal variation), a change-point, and centrifugality. The module provides Sen's estimator of slope, parametric and nonparametric linear regression analyses, and Spearman, Kendall's, and Pearson's correlation coefficients, and it smooths the curve, using procedures based on running medians and on Fourier transforms. Regression lines, smoothed curves, and the change-point are shown in a graph. Operating the Programs There is no special installation procedure; the programs need only be put in a folder of the user's choice. The appropriate program and module must first be selected. As an aid, a Pepi Finder (a Windows help file, FINDER.HLP) is provided; it is called up by clicking on its icon, and can be printed for easy reference. The Pepi Finder is an alphabetical index that shows which programs and modules deal with a specified procedure, measure, or kind of study. As seen in the excerpt shown in Figure 1 , the four WINPEPI programs are colour-coded. The Finder may point to more than one module; the entry for "Case-control study, unmatched", for example, is "COMPARE2 C,G". When COMPARE2 is opened (Figure 2 ) it is clear that its module G is designed for a case-control study with more than two exposure categories. The index also includes procedures provided by PEPI DOS programs (shown in italics) but not by WINPEPI programs. Figure 1 PEPI FINDER: Excerpt. Figure 2 COMPARE2: Opening screen. Each program has an opening screen (Figure 2 ) that displays a main menu and a top menu. Except in WHATIS, data entry is possible only after a selection has been made; a data-entry screen then appears. As an example, if option F of COMPARE2 is selected, i.e. "Categorical data (2 × k table)" (see Figure 2 ), the opening screen is replaced by the data-entry screen shown in Figure 3 . Figure 3 COMPARE2: Data-entry screen (for 2 × k table). The programs do not read data files, but require the entry of data that have already been counted or summarized, either manually or by using statistical software that processes primary data. The data can be entered at the keyboard, or (in multiple-entry boxes for the entry of tables) can be "pasted" from a file in which they are available. Once entered, tabular data can be pasted to a text file for future re-use by pasting. Alternative forms of data are often accepted, e.g. numerators instead of rates or proportions, and either individual or grouped observations. Warning messages are shown if obvious errors are made when entering data or if essential items are omitted. Simple on-screen instructions are provided, using simple language. For example, dichotomous variables are referred to as "yes-no" variables, and metric-scale observations, continuous or discrete, as "numerical". The term "rate" is used both for rates that have person-time denominators (e.g. incidence density) and for measures whose denominators are numbers of individuals (e.g. prevalence and risk); when the distinction is important, this is indicated. The instructions make use of terms well-known to epidemiologists, such as "case-control study", "exposed" and "not exposed", and "risk factor". (If the programs are used outside an epidemiological context, allowance must be made for their epidemiological labels.) To simplify operation, the program generally performs and reports all the prescribed procedures that the data will permit, without requiring choices by the user. But some options may be offered. In Figure 2 , for example, three options are shown: the categories may be nominal or ordinal, the scores allotted to the categories can be changed, and there is an option for performing a very specific kind of follow-up study. If "nominal" is checked instead of "ordinal", the instructions change, and the only option is for the partitioning of chi-square. Clicking on an option may modify the procedures a module performs, the manner in which the computation is done (e.g. depending on whether number-of-individuals or person-time denominators are entered, or whether a normal distribution can be assumed), and the data requirement (e.g. monthly or weekly or daily data for the appraisal of seasonal variation). Choice of an option may also modify the output. For example, the module that does a meta-analysis of studies of screening or diagnostic tests and produces forest plots for sensitivity, etc., permits optional display or suppression of the detailed numerical results for all studies. Pop-up hints and help screens are provided. Results are shown in an output screen (Figure 4 ), from which it is easy to return to the main menu or the previous screen. Results automatically go to the Windows clipboard, from which they can be pasted to other files. Clicking on "View" in the top menu displays all results obtained in the current session. "Print" options are offered. By clicking on "Note" in the top menu, it is possible to add comments to the results, for pasting, printing, or saving. A "Repeat" button is provided, permitting repeated analyses of the same data with changed options. Figure 4 COMPARE2: Results screen (for 2 × k table). All results are saved in a disk file, unless the user changes this default. The WINPEPI package contains a utility program (JOINTEXT) that can merge result files. DESCRIBE (but no other WINPEPI program) displays graphs – box-and-whisker plots, survival curves, seasonal peaks, regression lines, smoothed curves, forest plots, scattergrams, summary ROC curves, and graphs showing required sample sizes under different conditions. In most of the graphs, numerical values can be read by mouse-clicking at any location, optionally after magnifying a segment (zooming). Specimen graphs are shown in Figures 5 to 8 Figure 5 shows the number of clusters required for a cluster-based prevalence study (with stipulated requirements) for a true prevalence ranging from 5 to 20 per 100; the number can be read by clicking on the graph. Figure 6 shows a series of numerical observations, with regression lines, smoothed curves, and the change-point. Figure 7 shows post-test probabilities and net gain for a diagnostic test with a given likelihood ratio, for a range of pretest probabilities. Figure 8 shows a comparison of ROC curves, for use in appraising the effect of a covariate on the accuracy of a diagnostic test. Figure 5 Number of clusters required for a cluster-based prevalence study. Figure 6 A series of numerical observations. The straight lines are simple linear and nonparametric regression lines; the curved lines represent smoothing by two different methods; the red triangle marks the first point at which there is a significant change. Figure 7 Post-test probabilities and net gain for a diagnostic test. Positive likelihood ratio = 10. The net gain is the absolute difference between pretest and post-test probabilities. Figure 8 Comparison of ROC curves. Documentation Comprehensive manuals are provided. These furnish full information about each module, including explanations of the uses, limitations, and applicability of specific procedures, and formulae or references. The Pepi Finder serves as an index to the manuals. Discussion Criteria for the appraisal of statistical software for epidemiology [ 7 ] include not only its capabilities, but also "smoothness of the installation, simplicity of the interface, ease of use, completeness and statistical quality of the documentation, completeness and appearance of statistical graphics, accuracy of statistical computations". The WINPEPI programs are easy to install and easy to use (with the reservations discussed below). Their documentation is very detailed and (at the price of repetitiveness) includes a separate self-contained description of each module. A regrettable shortcoming of WINPEPI is that only one of the programs, DESCRIBE, presents graphic results. This is because DESCRIBE is the only 32-bit program, and the graph unit used by WINPEPI [ 8 ] is appropriate only for 32-bit programs. As for accuracy, the programs have been tested extensively, and all errors found have been promptly corrected; but (to cite the PEPI manual), it unfortunately remains a truism that no computer software can be entirely problem-free. But the WINPEPI programs do not provide data management facilities, and some other software package must be used if the data require processing. An epidemiologist or student whose data have been stored and maybe processed in another package, and who is well versed in the use of that package, may therefore have no need for the WINPEPI programs, despite their ease of operation, except when these do analyses not done by the other package. The WINPEPI programs aim "to complement – not replace – other statistics packages" [ 5 ]. Also (unlike the DOS-based PEPI programs for multiple logistic and Poisson regression analyses), the WINPEPI programs do not read data files. Data must be entered each time a program is used. This drawback is partly overcome by the possibility of pasting tabular data into data-entry boxes. But data entry can be tiresome, and users accustomed to programs that use data files may find it particularly vexatious. On the other hand, for some purposes keyboard entry may be seen as a boon: "Although conventional statistical software packages are adequate when you have a data set to work with, they are not always helpful when you need to do keyboard entry of data and rapidly perform simple analyses. For instance, you may want to replicate some analyses from a journal article and compute a Mantel-Haenszel odds ratio, or you may want to compute the sample size for your study while writing a grant proposal. Maybe you want to demonstrate to your students the impact of increasing sample size on the confidence intervals of a proportion. Perhaps you are a student and would like to do your epidemiology or biostatistics homework with some easy-to-use analytical routines... It is in this niche area that PEPI scores!" [ 6 ]. A criticism of version 3 of PEPI as being insufficiently user-friendly [ 9 ] led to a major revision in version 4. In the WINPEPI programs, user-friendliness is maximized by the provision of the Pepi Finder, simple on-screen instructions, pop-up hints and help screens, and warning messages, by streamlined data-entry procedures, which accept alternative forms of data, by the automatic saving of results, by the ease with which results can be recalled, annotated, printed, and pasted, and sometimes by the provision (in the output screens) of comments on the applicability of specific results. Unfortunately the wide variety of statistical procedures that is offered makes the WINPEPI programs less convenient to use; versatility carries a price. Even the provision made for the entry of alternative forms of data, meant as a convenience, necessitates a decision and may hence be an inconvenience – for example, a simple comparison of two proportions (using module A of COMPARE2) requires a choice between entry of four frequencies, of numerators and denominators, or of proportions and denominators. The DOS-based PEPI package elicited the comments "there are so many modules that sometimes it is difficult to remember which one to use" [ 10 ] and, with less restraint, "it is comprised of a large number of separate modules, which can make it a pain to use" [ 11 ]. The Pepi Finder was introduced (in version 3 of the package) to mitigate this problem. The advent of the WINPEPI programs, with their added statistical procedures, increased the potential for confusion and hence the value of the Finder, both for finding what program and module to use, and as an index to the detailed descriptions supplied in the manuals. The possibility of confusion is of course much reduced by the fact that related modules – for example, those concerning comparisons of two independent samples – are concentrated in the same WINPEPI program. Having opened the appropriate program, the user need only click on the kind of analysis that is required. But even that may tax some users. In COMPARE2, for example, a choice between modules B and D (see Figure 2 ) requires an awareness of whether the denominators are number-of-individuals or person-time ones. A further penalty for WINPEPI's versatility is that users may be confused by the large number of results in the output, some of them of little or no obvious relevance. As described above, module A of COMPARE2 (for a 2 × 2 table), for example, provides numerous "exact" and chi-square tests, and three measures of association, with confidence limits computed by different methods, as well as other results, including some that are valid only if inverse sampling was used. Similarly, module D1 of PAIRSetc (for paired numerical observations) provides three tests, six intraclass coefficients and a number of other measures of agreement, appropriate for different purposes. For this reason, every WINPEPI manual carries the admonition: "This program offers more options than most users will ever need, and will usually display more results than are needed. Ignore the options and results you don't require". (This of course assumes that the user knows what he or she wants.) But while all the results cannot be of interest to an ordinary user, each of them may be of interest to some users. As pointed out in a review of epidemiological software [ 11 ], "what one person might call 'statistical clutter' might be desirable to other people or even to that person if the person learned about that statistic". A review of PEPI says "Will you need all the programs in PEPI? Probably not. We have, for example, never used the Jonckheere-Tepstra test for trend or the Kullback-Leibler distances. However, more is good..." [ 6 ]. If a user wishes only to compute kappa , it can do no harm if the output provides extra results that draw attention to the fact that kappa has a ceiling value, or that its value can be adjusted to avoid paradoxical results. The user may be stimulated to use some of the additional procedures, after (if necessary) learning more about them. The manuals carry the warning: "It is unwise to use a statistical procedure whose use one does not understand. This manual cannot supply this knowledge, and it is certainly no substitute for the basic understanding of statistics and epidemiological thinking that is essential for the wise choice of methods and the correct interpretation of their results". The provision of alternative tests, and estimators based on alternative methods, may of course be confusing, whatever explanatory comments may be offered in the output or the manuals. But it may permit a knowledgeable user to select the method most appropriate in a particular situation, and it serves as a reminder to the less knowledgeable user that different methods exist, based on different assumptions and using different models, most of them yielding approximations, and none of them having absolute validity for all purposes, and as a warning that caution is indicated if different methods lead to very different conclusions. "Exact" results computed in different ways differ, and "exact" probabilities and confidence intervals are not always preferable to probabilities and confidence intervals computed in other ways. The length of the list in the Pepi Finder testifies to the wide variety of statistical routines offered. "The programs cover an amazing array of applications", says one review [ 6 ]. PEPI has repeatedly been called a "Swiss army knife" of utilities for epidemiologists and biomedical researchers [ 6 , 12 , 13 ]. One reviewer added, "one will find here more analytic options for a simple 2 × 2 or 2 × K table than will probably be needed during an entire epidemiology career" [ 13 ]. Another compared several packages when estimating sample size for a matched case-control study, and "found that PEPI provides an output richer than others do. This feature is common to other programs in PEPI" [ 14 ]. PEPI is of course very far from being a complete compendium of statistical routines for epidemiologists. It does not, for example, provide Cox regression, log-linear analysis, multiple regression analysis, procedures for the study of disease clustering, and many other procedures of interest to epidemiologists [ 7 , 11 ], which must be sought elsewhere. But it is a handy resource for many routines that are not very commonly used or very easily found, such as those concerned with misclassification, meta-analysis, reliability studies, the appraisal of screening and diagnostic tests, the equivalence of two proportions or means, cluster samples, inverse sampling, capture-recapture studies, serial correlation of residuals, skewed funnel plots, direct standardization using age intervals as weights, smoothing of curves, generalized odds ratios for ordinal data, quantitative measures of heterogeneity, harmonic analysis in the study of seasonality, and bias-adjusted and prevalence-adjusted bias-adjusted estimates of kappa (a feature picked out as "unique" in one comparison of PEPI with other epidemiological software [ 11 ]). From the viewpoint of veterinary epidemiologists, a shortcoming of WINPEPI is its use of "person-time" and not "animal-time". But they are doubtless used to this. WINPEPI's potential as a learning and teaching aid is worth stressing. Students welcome the facts that the package is free and requires no special installation procedure, and that (unlike major general-purpose statistical packages) it uses epidemiological language and provides results that are meaningful to epidemiologists. They rapidly learn to use the Pepi Finder and the programs themselves. They find the programs easy to use, although they may at first be confused by the multiplicity of modules and results; but they rapidly learn to focus on the specific modules and results that interest them, and to disregard others. At the same time, the rich output may serve to acquaint the student with other measures and tests, and excite interest in them. The weight the programs give to measures of association and their confidence intervals may help to counteract the belief that significance testing is the be-all and end-all of an analysis. "PEPI facilitates a ready understanding of important epidemiologic concepts, unfettered by the complexities of statistical programming", says a reviewer [ 6 ]. With appropriate data, for example, the Mantel-Haenszel results provided by COMPARE2's module B can serve as an object lesson on the assessment of confounding and effect modification, the control of confounding, and appraisal of the defensibility of a summary odds or risk ratio. The student can concentrate on analysis and interpretation, with no need to get involved in data management, sorting and tabulation. A useful feature is that, by clicking on the "Repeat" button and making changes to the data or options, students can easily do "what if?" exercises [ 15 ]. For example, they can easily learn, by manipulating data, how differences in prevalence or the number of controls per case can alter the required sample size, or how consideration of cost can alter sample size decisions in stratified sampling, or how the sensitivity or specificity of measures can alter a prevalence estimate or an odds or risk ratio. The sensitivity analysis provided by a module in COMPARE2 can demonstrate how markedly a single aberrant result can affect the results of a meta-analysis. Using the "misclassification" modules, it may be a salutary experience for students – and possibly also for some more experienced epidemiologists – to learn that an observed prevalence of 120 per 1000, using a measure whose sensitivity and specificity are 90%, points to a true prevalence of only 25 per 1000, or to find how inaccurate their guesses about the effect of misclassification on an odds or risk ratio can be. A recent epidemiology textbook makes frequent use of PEPI in its exercises "to relieve students from some of the tedium and anxiety of hand calculation, while opening up possibilities of using advanced techniques that might not otherwise be available. It is time to familiarize even introductory students to these essential tools of the trade" [ 16 ]. Conclusions WINPEPI complements other statistics packages. It is versatile, providing a wide variety of statistical routines commonly used by epidemiologists, but is far from being a complete compendium of such routines. It is a handy source of many procedures that are not very commonly or easily found. The programs are in general user-friendly, although some users may be confused by the large numbers of options and results provided. The main limitation is the inability to read data files, but tabular data can be entered by pasting, and for some purposes keyboard entry of data is an advantage. Only one of the programs presents graphic results. WINPEPI has a considerable potential as a learning and teaching aid. Availability and requirements The current version (at the time of this writing) of the software is available for free download as an additional file (WINPEPI.ZIP) attached to this article. It includes the programs, their manuals, and the Pepi Finder. Subsequent versions will be available at http://www.brixtonhealth.com for free download. Information about the latest WINPEPI version can be found at http://www.sagebrushpress.com/pepibook.html, where the DOS-based programs are available for free download. The programs and manuals are copyrighted, but may be freely copied and distributed for personal use; they may not be exploited commercially without permission. COMPARE2, PAIRSetc, and WHATIS are 16-bit programs (written in Delphi version 1) that can be run in any version of Windows. DESCRIBE is a 32-bit program (written in Delphi version 5), and can be run in any version of Windows except Windows 3. The manuals for DESCRIBE, COMPARE2, and PAIRSetc are in PDF format, and can be read or printed with Adobe Acrobat. WHATIS is documented in the version 4 manual [ 5 ]. Competing interests The author wrote the WINPEPI programs and manuals and is co-author of the DOS-based programs and manual, and hence may be biased in their favour. Supplementary Material Additional File 1 WINPEPI package. WINPEPI programs, with manuals and Pepi Finder. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544871.xml
555571	Influence of antioxidant (L- ascorbic acid) on tolbutamide induced hypoglycaemia/antihyperglycaemia in normal and diabetic rats	Background Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Increased oxidative stress and decreased antioxidant levels are the leading cause of diabetes and diabetic complications. So it is felt that supplementation of antioxidants may be useful in controlling the glucose levels and to postpone the occurrence of diabetic complications. The objective of our study is to find the influence of antioxidant supplementation (L-ascorbic acid) on tolbutamide activity in normal and diabetic rats. Methods L- ascorbic acid/tolbutamide/L-ascorbic acid + tolbutamide were administered orally to 3 different groups of albino rats of either sex in normal and diabetic condition. Blood samples were collected from retro-orbital puncture at different time intervals and were analyzed for blood glucose by GOD-POD method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route. Results L-ascorbic acid/ tolbutamide produced hypoglycaemic activity in a dose dependant manner in normal and diabetic condition. In the presence of L-ascorbic acid, tolbuatmide produced early onset of action and maintained for longer period compared to tolbutamide matching control. Conclusion Supplementation of antioxidants like L-ascorbic acid was found to improve tolbutamide response in normal and diabetic rats.	Background Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. It requires life long treatment with drugs coupled with diet control and exercise. It may be due to decrease in the synthesis of insulin (Type-I diabetes) or due to decrease in the secretion of insulin from β-cells of islets of Langerhans of pancreas (Type-II diabetes). Insulin is the drug of choice in type – I diabetes and sulfonylureas are the drugs of choice in type II. Among sulfonylureas, tolbutamide is the drug of choice for geriatrics because of its short duration of action and lower incidence of hypoglycaemia in early hours of night. Diabetes is one of the stress related disorder. Diabetic subjects are shown to have increased oxidative stress and decreased antioxidant levels [ 1 - 3 ]. It was also shown that tight control of blood glucose is possible with decrease in oxidative stress [ 4 ]. Antioxidants are claimed to work as antistress agents by decreasing oxidative stress. L-ascorbic acid used in therapy for disorders like scurvy produces antioxidant activity. Earlier reports show that the relationship between scurvy and diabetes mellitus indicates the low levels of plasma ascorbic acid in diabetic rats compared to control rats [ 5 , 6 ]. Hence the present study was conducted to find the influence of L-ascorbic acid, a water soluble antioxidant and a free radical scavenger on the hypoglycaemic and antihyperglycaemic activities of tolbutamide in normal and diabetic rats. Methods All animal experiments were performed in accordance with our institutional animal ethics committee. Albino rats of either sex (Mahaveer Enterprises, Hyderabad) weighing between 125 – 175 g were used in the study. They were housed five per cage at temperature 22 ± 2°C and 12/12 h light/dark under controlled environment. Rats were fed with standard pellet diet. (Mahaveer Enterprises, Hyderabad) and water ad libitum . They were divided into 3 groups of five each. They were fasted for 18 h prior to the experiment allowing access to water only, and the water was withdrawn during the experiment. Blood samples were collected from the retro-orbital plexus of each rat at 0, 0.5, 1, 1.5, 2, 4, 6 hr after drug administration. Blood glucose levels were determined by using GOD – POD method [ 7 ]. Group I received L-ascorbic acid 60 mg / kg body weight, Group II received tolbutamide 20 mg/kg body weight and Group III was given L-ascorbic acid (60 mg / kg body weight) prior to the administration of tolbutamide 20 mg/kg body weight in normal rats. In clinical practice tolbutamide and vitamin C are administered orally hence in our study also this was administered orally. Induction of diabetes Albino rats of either sex weighing between 125 to 175 g were fasted overnight before injection with alloxan. Alloxan monohydrate was dissolved in saline solution and was administered at a dose of 100 mg/kg body weight intraperitonially. Animals were treated with 10% dextrose orally to combat the early phase of hypoglycaemia. Rats showing fasting blood glucose levels above 150 mg/dl were selected for the study. They were divided into 3 groups of five each. Group I received L-ascorbic acid 40 mg/kg body weight and Group II received tolbutamide 20 mg/kg body weight while Group III was given L-ascorbic acid 40 mg/kg prior to tolbutamide administration (20 mg/kg). L-ascorbic acid dose was fixed based on its response, which produced above 40%. Statistical analysis The significance of blood glucose reduction produced by L-ascorbic acid with tolbutamide compared to tolbutamide control was determined by applying students unpaired t-test and the significance is indicated by * mark. Results The presence of L-ascorbic acid upto 20 μg did not interfere with the blood glucose estimation when tested with different quantities in in vitro studies. In normal rats L-ascorbic acid at the dose of 60 mg/kg body weight administered orally produced 50.91% blood glucose reduction at 0.5 h and 20 mg/kg body weight of tolbutamide produced 33% at 4 h as peak effects. In the presence of L-ascorbic acid (60 mg/kg), the action of tolbutamide was early in onset and maintained for 6 h. In diabetic rats, oral administration of L-ascorbic acid alone at the dose of 40 mg/kg body weight produced 42.53% blood glucose reduction at 1.5 h and tolbutamide 20 mg/kg body weight produced 45.09 at 4 h. Administration of L-ascorbic acid 40 mg/kg body weight prior to tolbutamide produced antidiabetic activity at 0.5 h and was maintained for 6 h. The percent blood glucose reduction with L-ascorbic acid / tolbutamide/ L-ascorbic acid + tolbutamide in normal rats and diabetic rats were given in table 1 & 2 . Table 1 Percent blood glucose reduction (Mean ± SEM) with L-ascorbic acid/ tolbutamide / L-ascorbic acid + tolbutamide in normal rats (n= 5) Time in hours L-ascorbic acid 60 mg/kg bd.wt. Tolbutamide 20 mg/kg bd.wt. L-ascorbic acid 60 mg/kg bd.wt. + Tolbutamide 20 mg/kg bd.wt. 0 - - - 0.5 50.91 ± 0.49 10.08 ± 1.08 53.57 ± 2.01* 1 20.26 ± 3.14 15.56 ± 1.48 28.70 ± 1.75* 1.5 6.06 ± 1.3 18.75 ± 2.1 23.25 ± 1.72 2 1.65 ± 0.93 22.22 ± 2.13 29.78 ± 2.57 4 -2.83 ± 0.4 33.0 ± 0.69 45.21 ± 2.79 6 - 10.28 ± 1.02 12.54 ± 2.5 Significance * P < 0.001, P < 0.01 Table 2 Percent blood glucose reduction (Mean ± SEM) with L-ascorbic acid/ tolbutamide / L-ascorbic acid + tolbutamide in diabetic rats (n = 5) Time in hours L-ascorbic acid 40 mg/kg bd.wt. Tolbutamide 20 mg/kg bd.wt. L-ascorbic acid 40 mg/kg bd.wt. + Tolbutamide 20 mg/kg bd.wt. 0 - - - 0.5 18.23 ± 1.88 3.03 ± 0.8 23.55 ± 2.37* 1 35.45 ± 3.26 7.83 ± 1.84 35.59 ± 4.48* 1.5 42.53 ± 1.78 18.58 ± 2.49 57.49 ± 1.63 2 34.04 ± 2.22 37.97 ± 6.40 59.74 ± 1.22* 4 20.02 ± 3.32 45.09 ± 4.95 62.55 ± 0.64 6 - 21.6 ± 1.94 39.43 ± 2.15* Significance * P < 0.001, P < 0.01, *P < 0.05 Discussion The drug interaction studies are usually conducted in animal models to find out the mechanism before they are conducted in humans. We have selected rat as animal model since it is one of the animal, which synthesize ascorbic acid and can be maintained easily in the laboratory conditions. Reactive oxygen species (ROS) are thought to be implicated in the pathogenesis of diabetes as well as other diseases[ 8 ]. Reactive oxygen species usually comprise radicals that have the ability to oxidize and damage DNA, proteins and carbohydrates. Hyperglycaemia appears to induce oxidative stress on cells and this can cause an increase in the production of free radicals[ 9 ]. Human antioxidant enzymes are mobilized during hyperglycaemia, but they cannot meet the continued demand due to increased oxidative stress[ 10 ]. This problem is either due to decreased intake of needed precursors or an inability to synthesise the antioxidant enzymes[ 11 ]. Antioxidant supplementation may provide the only means to reverse this process[ 12 ]. Use of typical antioxidants alone or in combination may retard or even prevent the normal progression of diabetic complications. It was reported that L-ascorbic acid levels were decreased in diabetic patients and rats[ 13 ]. So it is felt that L-ascorbic acid supplementation may help in the treatment of diabetes mellitus. In the present study L-ascorbic acid and tolbutamide reduced blood glucose levels in normal & diabetic rats in a dose dependent manner. L-ascorbic acid when administered alone produced an early onset of action 0.5 & 1.5 h in normal and diabetic rats respectively. This early onset may be due to increase in the insulin secretion which support earlier reports that L-ascorbic acid supplementation increase the plasma insulin concentration[ 14 ]. Tolbutamide when administered in therapeutic dose produced the maximum effect at 4 h and was maintained up to 6 h in both normal and diabetic rats. In the presence of L-ascorbic acid the onset of action of tolbutamide was early and maintained for longer duration compared to tolbutamide control. Tolbutamide acts by stimulating insulin secretion (pancreatic)[ 15 ] and also by increasing tissue uptake of glucose (extra pancreatic)[ 16 ]. The early onset of action was noticed to be due to L-ascorbic acid, which was maintained later due to tolbutamide activity since both are reported to have influence on insulin secretion [ 14 , 15 ]. Conclusion The study indicates that additive action of L-ascorbic acid on pharmacodynamic response of tolbutamide may be useful to improve the tolbutamide activity in insulin resistant cases and to postpone the occurrence of diabetic complications. However further work on human patients is required to confirm the observation in diabetic condition and usefulness of L-ascorbic acid as supplemental agent for improved control of blood glucose levels when administered along with sulfonylureas. Competing interests The author(s) declare that they have no competing interests. Authors' contributions SS Conceived of the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript. EK Participated in the design and coordination and in the standardization of methods. RJ Carried out the study in normal rats. VA Carried out the study in diabetic rats. All authors read and approved the final manuscript. Figure 1 Percent Blood Glucose Reduction with L-ascorbic acid / tolbutamide / L-ascorbic acid + tolbutamide in normal rats (n = 5) Figure 2 Percent Blood Glucose Reduction with L-ascorbic acid / tolbutamide / L-ascorbic acid + tolbutamide in diabetic rats (n = 5) Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555571.xml
554109	Rural Indian tribal communities: an emerging high-risk group for HIV/AIDS	Background Rural Indian tribes are anthropologically distinct with unique cultures, traditions and practices. Over the years, displacement and rapid acculturation of this population has led to dramatic changes in their socio-cultural and value systems. Due to a poor health infrastructure, high levels of poverty and ignorance, these communities are highly vulnerable to various health problems, especially, communicable diseases including HIV/AIDS. Our study sought to assess knowledge, attitudes and practices regarding sexuality, and the risk factors associated with the spread of HIV/AIDS and STDs among these communities. Methods A nested cross sectional study was undertaken as part of the on going Reproductive and Child Health Survey. A total of 5,690 participants age 18–44 were recruited for this study. Data were obtained through home interviews, and focused on socio-demographics, knowledge, attitudes and behaviors regarding sexuality, HIV/AIDS and other STDs. Results The study revealed that only 22% of adults had even heard of AIDS, and 18 % knew how it is transmitted. In addition, only 5% knew that STDs and AIDS were related to each other. AIDS awareness among women was lower compared to men (14% vs.30 %). Regarding sexual practices, 35% of the respondents reported having had extramarital sexual encounters, with more males than females reporting extramarital affairs. Conclusion Lack of awareness, permissiveness of tribal societies for premarital or extra-marital sexual relationships, and sexual mixing patterns predispose these communities to HIV/AIDS and STD infections. There is a dire need for targeted interventions in order to curtail the increasing threat of HIV and other STDs among these vulnerable populations.	Background India is the second most populous nation in the world and has changing sociopolitical and demographic characteristics as well as varied morbidity and mortality patterns [ 1 ]. These changes, in conjunction with the country's high population growth rate, have exacerbated the prevailing and emerging public health challenges the country is facing. Since 1986 when the first case of human immunodeficiency virus (HIV) was reported in India [ 2 ], it has become imperative to include acquired immunodeficiency syndrome (AIDS) on its long list of public health issues that need to be addressed. As a direct result of these challenges, India has begun to assess and monitor the impact of HIV/AIDS throughout the country's various states and regions with the assistance of several international health organizations. According to 2003 estimates from UNAIDS, approximately 5.1 million individuals in India are infected with the HIV virus [ 3 ]. Furthermore, recent studies indicate that transmission of HIV is no longer confined to high-risk urban populations, but is spreading across rural settings as well [ 4 ]. This trend is a cause for concern as AIDS is increasingly hampering social and economic development throughout the country. For effective control of the spread of HIV/AIDS, it is crucial to have data on knowledge, attitudes and behavioral practices for specific population as research has shown that socio-cultural influences, traditional lifestyles, societal norms, and traditions influence HIV/AIDS transmission rates [ 5 , 6 ]. Because India's HIV/AIDS transmission pattern is predominantly heterosexual (85% of all newly reported cases) [ 7 ], subcultures that have relaxed marital structures or are tolerant of high-risk sexual practices (e.g., sex with a commercial sex worker) are particularly vulnerable to the spread of HIV/AIDS and STDs within their communities [ 7 , 8 ]. With more Indian men reporting premarital and extramarital sexual activity, women who marry as teenagers are vulnerable to HIV/AIDS infection and STDs [ 9 , 10 ]. The rapid spread of HIV/AIDS in rural Indian communities has been attributed to the country's poor health infrastructure, poverty and lack of awareness [ 4 , 10 ]. Despite these indicators, little is known about the risk factors, transmission rates, or the impact AIDS will have in these areas in the future. Traditionally, there has been little research and only a paucity of health-related research conducted among this potentially high-risk vulnerable population. Throughout India, approximately 8% of the population lives within rural tribal communities, which are collectively referred to as 'Tribes'. These communities are geographically distinct; with each tribe having its own unique customs, traditions, beliefs and practices. Even within a particular tribal entity, differences in dialect, health practices, unique customs, values, and traditions are apparent. In rural Indian communities indices of reproductive health are typically very poor: maternal mortality rate is about 230 per 100,000 live births and 61.2% of the women suffer from at least one gynecologic pathology [ 11 ]. Because tribal groups have existed on the fringe of Indian society, they may still be unaware or indifferent to the potential health threats from HIV/AIDS. Ascertaining whether or not tribal communities are potentially a high risk group warranting intervention is a necessary step in India's war on AIDS. Accordingly, we undertook this study to explore the risks for this special group of people. Methods We conducted a cross-sectional study nested within an existing enumerative study referred to as the Reproductive and Child Health Survey (RCHS). The RCHS was initially designed to enumerate and ascertain basic demographic and health profiles for all tribal members. Data collection for this particular study was done in two phases. Phase one involved adding additional questions to the original RCHS study to assess risk factors (knowledge, attitudes and behavioral practices) associated with the transmission of HIV/AIDS and other communicable diseases. Study population The study population comprised of tribal communities living in the southern region of Karnataka (Figures 1 , 2 ). Members of these tribes have traditionally derived much of their livelihood from the country's vast reserve of natural forest resources. However over the years, these tribes have been forced to migrate from their ancestral land and are currently living within poor rural communities throughout the state. The initial displacement was as result of the submergence of their traditional homelands through the construction of the Kabini dam, and the second displacement was as a result of 'Project Tiger,' a wildlife conservation project that displaced them to their current location in the southern region of Karnataka, where this study was conducted. Figure 1 Map of India showing the region of Karnataka where the study was conducted. Used with permission from India-tourism.com Figure 2 The reproductive and child health program area in the southern part of Karnataka. Used with permission from the Reproductive Child Health Survey (RCHS) project Sampling Because of the enumerative nature of the RCHS, all persons within the age group 18–44 years who participated in the RCHS study were included for phase one of this study. This age group was selected as the focus of the initial study was on reproductive health issues. Survey instruments A semi-structured questionnaire with both open- and close-ended questions was developed to collect information on knowledge, attitudes and behaviors regarding HIV/AIDS, as well as other relevant demographic information not included in the RCHS. The instrument was developed in English, translated to the native language, and subsequently back translated to English for content and language verification. The survey instrument was field-tested for validity purposes and modified accordingly. Data collection A team of ten interviewers from the local tribal communities with a minimum of high school education were selected and trained for two weeks to ensure uniform and high-quality data collection. All adults were interviewed separately to ensure confidentiality. Each interviewer read out each of the questions and response choices (where appropriate) to the interviewees and recorded all answers directly on the questionnaire. Verbal informed consent was obtained from each respondent prior to starting the interview. Data analysis Data were entered into an electronic database using Sybase Central Software (Sybase, Inc. Dublin, CA). To ensure confidentiality, all respondent identifiers were expunged to create a secondary data set that was used for the final analysis. Frequency tables were generated for selected demographics and health related categorical variables. In addition, univariate analysis was performed on relevant continuous variables. The findings are presented below. Ethical consideration Ethical clearance was obtained from ethical boards within each of the tribal communities, and appropriate government agencies were informed about the study objectives. Permission was also obtained from the Institutional Review Board (IRB) at the University of South Florida in Tampa, Florida. Verbal informed consent was obtained from the local leaders, and the individual participants. Because of the high levels of illiteracy, it was not feasible for us to obtain written consent. Results Demographic profile A total of 11,379 individuals from the 5 tribal communities had been enumerated as part of the RCHS. Of these, 5,690 were within the study age range (18–44 years) and formed the basis for this analysis. Table 1 shows the demographic profile of the study participants. The mean age of the study group was 31 years. There were more males than females; 53% vs.47%. Eighty four percent were married (91% females & 78% males). The average age at marriage was 13 years for females, and 22 years for males. Only 28% (27% female, & 30% male) of the population was literate i.e. able to read and write in any of the Indian languages. The majority of respondents (67 %) reported living in tiled roof houses with mud flooring, while only 40 % indicated easy access to potable water. Agriculture was the major source of income in these communities. The reported average daily income ranged from US $1.50 to $2.00. Approximately 35% of the respondents migrated on average three to four months each year to nearby areas for work. Table 1 Demographic profile of the study participants Characteristics Number (%) (N = 5,690) Mean age 31 years Sex Men 3,016 (53) Women 2,674 (47) Currently living in tiled-roof housing 3,812 (67) Access to potable water 2,276 (40) Migrated to find work 1,992 (35) Currently married Men 2,353 (78)* Women 2,433 (91) All 4,786 (84) Literacy* Men 814 (27)* Women 802 (30)** All 1,616 (28) Mean age of first marriage Mean years (95% CI) Men 22.0 (19.5–24.5) Women 13.0 (11.2–14.7) All 15.0 (11.2–18.8) Percentages are based on the total number of men in the study. Percentages are based on the total number of women in the study. *Literacy as defined by census operations of India is the ability to read and write in any of the Indian languages. CI = Confidence interval Unique sexual practices among tribal members The findings revealed that these tribal communities did not have a structured marital system; instead members practiced a form of serial monogamy in which they change partners and remarry every four to five years. Regarding sexual practices, 35% of the respondents reported either premarital affairs or extramarital affairs (Table 2 ). However such practices were more common in men compared to women. Furthermore, 20% of the male participants reported having had sex with a commercial sex worker (CSW) during the period the wife had had a child. Table 2 Self reported sexual practices of respondents Characteristics N = 5,690 Age at first sexual activity Mean years (95% CI) Men 17.0 (13.4–20.6) Women 13.0 (11.5–14.5) Premarital or extramarital sexual encounters Number (n) (%) Males 1,434 72.0 Females 558 28.0 Total 1,992 35 Sex with commercial sex worker within a year after spouse giving birth (men only n = 3,016) 470 20 * Percent based on the total sample of 5,690 respondents Knowledge and beliefs about HIV/AIDS and STDs Among these communities, there was a low level of knowledge on HIV/AIDS; only 22 % of all study participants (n = 1,252) had heard of AIDS (Table 3 ). Among those who have heard of AIDS, less than 20 % (n = 250) knew how HIV/AIDS was transmitted (16.8 % male vs. 8% females). About 98 % were not aware of the methods to prevent HIV/AIDS transmission. As many as 30 % (n = 376) of those who had heard of AIDS believed that "sinners" will get AIDS, while 10 % (n = 125) believed that AIDS and STDs could be prevented by the sterilization of women. Fifteen percent (n = 188) thought "AIDS is acquired by looking at a person who has AIDS," and 18 % (n = 225) believed that "AIDS is acquired by talking to a person who has AIDS." Only 5 % knew that a relationship exists between HIV/AIDS and STDs. Interestingly, 4 percent (n = 51) believed that there was a cure for AIDS. Most had not heard of STDs, and of those who had heard of them only 1 percent (n = 16) were aware of associated symptoms. Table 3 Knowledge and beliefs about HIV/AIDS and STDs Knowledge and beliefs about HIV/AIDS No. of study subjects N = 5,690 No. of study subjects who have ever heard of HIV/AIDS N = 1,252 N Percent of 5,690 Percent of 1,252 Have ever heard of HIV/AIDS 1,252 22.0 100.0 Know how HIV/AIDS is transmitted 250 4.4 20.0 Know methods to prevent the transmission of HIV/AIDS 114 2.0 9.1 Believe "sinners" will get AIDS 376 6.6 30.0 Believe AIDS and STDs can be prevented by sterilization of women 125 2.2 10.0 Believe AIDS is acquired by looking at an infected person with AIDS 188 3.3 15.0 Believe AIDS is acquired by talking to a person who has AIDS 225 4.0 18.0 Believe there is a cure for HIV/AIDS 51 0.9 4.1 Discussion In today's modern world, it is difficult to imagine societies that are still socially and culturally isolated from the rest of civilization; however, they do exist. The tribal societies throughout India have remained socially and culturally alienated from mainstream Indian society until developmental and conservation activities in tribal areas forced interactions between them. Displacement of the tribal people of southern Karnataka has led to a complex process of rapid acculturation and loss of cultural identity; as they struggle to maintain their traditional social structure, they must adopt new skills, beliefs, and practices necessary for success in their new environment. During this acculturation process, they have been faced with a myriad of public health challenges complicated by poverty, ignorance, and reluctance to abandon traditional beliefs and practices that would allow them to assimilate successfully. To date this is the first health related study among the displaced tribal communities of southern Karnataka that has attempted to assess risk threshold for the transmission and spread of HIV/AIDS and other STDs. It is not surprising that knowledge and awareness about HIV/AIDS and STDs was very low among tribal communities compared to the national figures given the degree of isolation, low literacy rates, and minimal access to information. The high level of poverty, inadequate health resources, ignorance and high-risk beliefs and practices among the tribal communities has contributed to the vulnerability of this population. As such it has created a highly susceptible population for the rapid spread of HIV/AIDS and other STDs' as well. Because tribal members are forced to migrate outside of their communities in search for work and increased wages, this may contribute to the spread of HIV/AIDS as many engage in extramarital affairs, seek commercial sex partners, or are under the threat of sexual harassment (females). While there has been limited scientific research exploring the cultural context of extramarital sexual behaviors, it is generally noted that in these communities, extramarital affairs are condoned and widely practiced especially during periods when women are pregnant or nursing or during period of travel for work [ 8 ]. This kind of behavior creates a fertile ground for HIV transmission and spread. Our data indicate that tribal women are particularly vulnerable for HIV/AIDS in this population since many of them commence sexual activity at an early age, and get married early as well. Also, they are in a culture that condones extramarital sex, and this exposes the women to a particularly precarious situation, increasing their risk for acquiring HIV. Conclusion It is evident from this study that the Indian tribal community is experiencing a latent phase that is potentially a precursor for an HIV/AIDS epidemic. There is a high prevalence of behavioral risk factors, coupled with ignorance, and inadequate health infrastructure thus creating a potential risk for rapid spread of HIV/AIDS, as well as other related diseases. In a country that is struggling to contain the spread of HIV, it is particularly important for concerned parties to pay attention to this population. Currently, virtually no resources are allocated toward the treatment of those infected with HIV/AIDS; the main stay of management is through education and preventive measures to control the spread of the scourge as they represent the most practical and cost effective strategies in this developing nation. It therefore becomes imperative and urgent to address the health concerns revealed in our study in order to formulate effective, culture-sensitive and appropriate intervention programs so that an imminent disaster (i.e. HIV/AIDS epidemic) in this remote and isolated communities can be averted. Competing interests The author(s) declare that they have no competing interests. Authors' contributions All the authors were involved in the design of the study, analysis, interpretation and development of the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC554109.xml
544865	Psychological health of family caregivers of children admitted at birth to a NICU and healthy children: a population-based cross-sectional survey	Background There is little information in the research literature on how parents of children who spend time in a neonatal intensive care unit (NICU) adapt psychologically to the demands of caregiving beyond the initial hospitalization period. Our aim was to compare parents of NICU children with parents of healthy full-term children, looking specifically at the relationship between parental psychosocial health and child characteristics, as well as the relationship between important predictor variables and psychosocial health. Methods A cross-sectional survey was sent to parents as their child turned 3 1/2 years of age. The setting was the province of British Columbia, Canada. The sample included all babies admitted to tertiary level neonatal intensive care units (NICU) at birth over a 16-month period, and a consecutive sample of healthy babies. The main outcome was the SF-36 mental component summary (MCS) score. Predictor variables included caregiver gender; caregiver age; marital status; parental education; annual household income; child health status; child behavior; birth-related risk factors; caregiver strain; and family function. Results Psychosocial health of NICU parents did not differ from parents of healthy children. Child health status and behavior for NICU and healthy children were strongly related to MCS score in bivariate analysis. In the pooled multivariate model, parental age, low family function, high caregiver strain, and child's internalizing and externalizing behavioral symptoms were independently associated with lower psychosocial health. In addition, female gender was associated with lower psychosocial health in the NICU group, whereas lower education and child's problem with quality of life indicated lower psychosocial health in the healthy baby group. Conclusions Overall, parental gender, family functioning and caregiver strain played influential roles in parental psychosocial health.	Background Neonatal intensive care is associated with a range of long-term health problems such as cerebral palsy, mental retardation, deafness, blindness and milder but more common problems such as learning disabilities and behavioral problems [ 1 - 13 ]. Although these problems create challenges for the parent responsible for the day-to-day provision of care to their child at home, the impact of caregiving on the health of parents of children discharged from neonatal intensive care units (NICUs) remains an under-explored research topic. There is a literature that focuses on the early hospitalization period. These studies show that mothers of preterm infants experience more severe levels of psychological distress in the neonatal period than do mothers of healthy full-term infants [ 14 - 17 ]. In the few studies that compare the impact of caregiving on parents of children discharged from NICUs with parents of healthy full-term children, the addition of a preterm infant into the family has been shown to have negative repercussions for the family in some studies [ 18 - 21 ], but not in others [ 22 - 24 ]. In one of the few NICU studies where parental mental health was the primary outcome measure, mothers of high and low-risk very low birth weight infants were compared with parents of healthy full-term infants [ 17 ]. The authors report that early differences between the groups at one month and two years were no longer apparent by the age of three, although parenting stress remained high throughout. In the present study, we sent a questionnaire booklet to mothers of all children admitted to a level III neonatal intensive care unit in the province of British Columbia (Canada) over a 16 month period to collect data on a range of factors in order to examine both neonatal and caregiver outcomes. Our study differs from other NICU follow-up studies in that it is population-based, focuses on preschool aged children and examines the full spectrum of NICU graduates. The aims of this paper are two-fold: (1) to compare psychosocial health of parents of NICU children with parents of healthy full-term children, looking specifically at the relationship between parental psychosocial health and child characteristics (i.e., health status, behavior problems, and birth-related risk factors); and (2) to identify predictors of parental psychosocial health (i.e., socioeconomic and demographic variables, child characteristics, caregiver strain, and family function). Methods Sample Ethical approval was gained from the University of British Columbia and participating hospitals. Our NICU sample included 2221 surviving babies admitted for more than 24 hours to one of three level III NICUs in British Columbia (BC), Canada over a 16 month period (March 1996 to June 1997). These 3 hospitals (Children's and Women's Health Centre of BC, Royal Columbian Hospital, Victoria General Hospital) provided 100% of the tertiary care NICU beds in the province. The birth mothers' name and contact details were obtained from the health records department at two hospitals and manually extracted from ledgers of the third hospital. Our list of babies was matched with provincial mortality records to exclude any babies that had died after discharge from the NICU and thereby prevent questionnaires being sent to bereaved parents. A comparison group of 718 healthy singleton full-term babies was recruited from the two hospitals with a hospital-based primary care unit (i.e., Children's and Women's Health Centre of BC and the Royal Columbian Hospital). This sample included all babies delivered over an 11 month period (March 1996 and January 1997) by primary care physicians at these two clinics. Babies with a sibling in the NICU sample and babies subsequently admitted to an NICU for more than 24 hours were excluded. Contact details for the mother were obtained from the health records department at one hospital, and directly from the primary care unit at the other. We excluded from the sample 150 babies (123 NICU; 27 healthy children) who did not meet our inclusion criteria for the following reasons: parent did not speak English (n = 95); baby died (n = 34); mother died (n = 6); and not applicable (n = 1). In addition, we excluded cases where the questionnaire was completed on the wrong child (n = 7) and where a comparison baby was subsequently admitted to a NICU (n = 7). The overall response rate (after exclusions), was 55% (54.3% NICU, 56.9% healthy baby group). The response rate for located families (82.8% of the sample was located) was 67.4% (n = 1140) for the NICU group, and 66.4% (n = 393) for the comparison group. Five NICU respondents returned a signed consent form without a completed questionnaire and were dropped from the analysis. Seventy-five percent of parents provided permission for data linkage between the questionnaire data and CNN database. The NICU sample included 181 children that were part of a multiple birth group: 171 twins; and 10 triplets. Table 1 contains sample characteristics. Most questionnaires (98%) were completed by a biological parent, most often the mother (96%). The NICU sample was composed of 1.8% fewer biological parents; 2.6% more male respondents, and 11.9% more families who earned less than $50,000 per year. Table 1 Characteristics of study sample Group; no. (%) of subjects NICU N = 1135 Comparison N = 393 Biological parent 1 1091 (97.7) 389 (99.5) Female 1 1070 (95.4) 383 (98.0) Married/common-law 962 (85.9) 344 (87.8) Age of parent, years 19–29 195 (17.8) 61 (15.7) 30–39 704 (64.1) 265 (68.3) ≥ 40 199 (18.1) 62 (16.0) Education level University 373 (33.4) 146 (37.4) Trade/technical school or community college 494 (44.3) 176 (45.1) High school graduation 185 (16.6) 50 (12.8) No high school diploma 64 (5.7) 18 (4.6) Household income, $ 2 511 (48.1) 136 (3) <30,000 247 (23.3) 58 (15.5) 30 – 49,999 264 (24.9) 78 (20.8) 50 – 79,999 333 (31.4) 145 (38.7) 80 > 218 (20.5) 94 (25.1) Male children in the sample 633 (55.8) 198 (50.6) Age of child, years 3 years 784 (69.3) 253 (64.4) 4 years 328 (29.0) 134 (34.1) 5 years 19 (1.7) 6 (1.5) 1 p < .05 (chi-square, Fischer's exact test); 2 p = .0018 (chi-square) Materials Our main measure of outcome was the SF-36 mental component summary (MCS) score [ 25 , 26 ]. The SF-36 is a well validated generic measure of adult physical and psychosocial health related quality of life (HRQL), which is composed of 36 items that measure 8 health domains. The MCS is computed from the following four domains: mental health (5 items); vitality (4 items); social functioning (2 items); and role limitations due to emotional problems (3 items). It has a mean of 50 and standard deviation of 10 and represents the mean and standard deviation of the general population (USA). Child health status was measured using the Health Status Classification Preschool Version (HSCS-PS) [ 27 ]. This measure asks about twelve health status (HS) problems that we have grouped into the following 4 categories: neurosensory (i.e., seeing and hearing); motor development (i.e., getting around, using hands and fingers, taking care of self); learning (i.e., speaking, learning/remembering and thinking/solving problems); and quality of life (i.e., pain/discomfort, feelings, behavior and general health). Each attribute has 3 to 5 levels of severity ranging from normal function to severe functional limitations. For each category of health problems, we recoded the data into the following: no problem; a mild problem; or a moderate or severe problem. Child behavior was measured with the Child Behavior Checklist 1.5–5 (CBCL/1.5–5) [ 28 ]. This questionnaire measures internalizing, externalizing and total problems, and scales can be scored categorically to indicate normal, borderline or clinical range scores. Data for birth-related risk data were obtained from the Canadian Neonatal Network Study [ 29 ] for the NICU children whose parents provided written consent for data linkage. The following variables were examined: birthweight; gestational age; small for gestational age; multiple birth, apgar score less than 7 at 5 minutes; congenital anomalies; the presence of a major morbidity (i.e., a composite score for the presence of at least one of the following: chronic lung disease (at 36 weeks); severe intraventricular hemorrhage (≥ grade 3); nosocomial infection; necrotizing enterocolitis; retinopathy of prematurity (≥stage 3)); and neonatal illness severity score [ 30 ]. Caregiver strain was measured using the Parental Impact-Time (PTT) scale from the Infant Toddler Quality of Life Questionnaire [ 31 ]. This 7-item scale asks parents to indicate limitations in the amount of time in the past 4 weeks they had for their own personal needs due to problems with their child's health (e.g., physical, emotional, cognitive, behavior, temperament). Scores on these scales can range from 0 to 100, with lower scores indicating greater caregiver strain. Family function was measured using the Family Assessment Device (FAD) [ 32 ]. Scores for this 12-item questionnaire can range from 0 to 36, with higher scores indicative of greater family dysfunction. Procedure A questionnaire booklet, which included the questionnaires described above, was sent to the address of the birth mother as her child turned 3 1/2 years of age. A consent letter was included to obtain permission to link the questionnaire data with hospital birth records. The primary caregiver in our study was defined as the person who, to that point in the child's life, had spent the most amount of time with the child. This could include the mother or father or another parent (e.g., grandparent, foster parent, guardian). We asked the primary caregiver (referred to in this paper as parent) to complete the questionnaire booklet and consent form. Non-respondents were sent a reminder letter, additional copies of the questionnaire booklet and a phone call as necessary. If the telephone number was not in service or reassigned, or a questionnaire booklet was returned to us from the post office, we implemented a comprehensive search strategy that involved searching the Internet and contacting the mothers' primary care physician. Data analysis To address the first objective, we compared the psychosocial summary score for the SF-36 questionnaire for parents of NICU children and parents of healthy children using student's T -test. T -test, ANOVA and the equivalent nonparametric tests, and Spearman correlation were used to explore relationships between MCS score and various child characteristics, including health status, behavior and birth-related risk factors. For health status and child behavior, we computed an effect size (mean difference divided by standard deviation of the group with no problems (health status) or with scores in the normal range (behavior)), to look at the magnitude of the difference in MCS score between subgroups for the NICU and healthy baby samples, and used the Cohen's guidelines for interpretation (0.2 is small, 0.5 is medium, 0.8 is large) [ 33 ]. To address the second objective, multiple regression analysis was used to examine the independent effects of, and proportion of variance in MCS scores explained by our predictor variables. For the analysis we examined a pooled model and a model where we stratified by group membership (i.e., NICU vs. healthy baby sample) to separately examine the contribution of each predictor variable for the two samples. Variables with significant (p < .05) or borderline p-values in bivariate analysis were included in the model. Certain birth-related risk factors (i.e., birthweight, congenital anomalies, illness severity score, and gestational age) were entered into the model on the basis of clinical rather than statistical importance, however, no effects were found. Potential predictor variables include the following: caregiver's gender; caregiver's age (continuous); marital status (married or common-law versus other), caregiver's education (less than high school graduation vs. other); annual household income (< or > $30,000); child health status (i.e., neurosensory; motor development; learning; and quality of life problems); child behavior; caregiver strain (continuous); and family function (continuous). For child health status and behavior variables, no problem (health status) and scores in the normal range (behavior) were the reference categories, with mild and moderate/severe (health status) or borderline and clinical range scores (behavior) entered separately, or combined and entered as dichotomous variables. We computed effect sizes to interpret the significance of beta coefficients. Results Psychosocial health comparing NICU and healthy children The unadjusted mean MCS score for parents of NICU children did not differ from parents of healthy children (48.2 versus 48.8; p = .305). We also compared MCS scores after adjusting for the three sample characteristics that differed between the two groups (i.e., proportion of biological parents; gender of subject; and those with lower household income), and no differences were found in the outcome variable. Psychosocial health by child health status problem On the HSCS-PS, 55.2% of healthy children had no health problems in any area, compared with 39.8% of NICU children (p < .001 on Chi-square). Table 2 shows the joint distribution of health status problems across the four categories for the NICU and healthy sample. These results show that the NICU sample had a higher proportion of children with more health status problems, as well as a higher proportion with moderate/severe versus mild problems. Table 2 Distribution of children with health status problems across the 4 health status categories for NICU and healthy children HSCS problems by domains NICU (N = 1104) Comparison (N = 386) no problem N 438 215 % 39.67 55.7 1 mild problem N 309 111 % 27.99 28.76 2+ mild problems N 183 37 % 16.58 9.59 1 moderate/severe problem only N 40 7 % 3.62 1.81 1 moderate/severe problem + any mild N 69 15 % 6.25 3.89 2–3 moderate/severe problems N 60 0 % 5.43 0 4 moderate/severe problems in all domains N 5 1 % 0.45 0.26 p < 0.0001, chi-square For parents of NICU children, for all 4 health status categories, parental MCS scores decreased as severity of the child health problem increased (see Table 3 ). Effect sizes comparing parents of children with no health status problems with parents of children with a moderate or severe health status problem were all moderate to large indicating important differences in parental mental health according to Cohen's benchmarks. The results for parents of healthy children show similar trends, with mainly moderate to large effect sizes. Table 3 Parental mental health summary score, 95% confidence intervals, number of subjects, p-value and effect size for child health status category Sample Type of HS problem None Mild Moderate or Severe p-value* Effect size NICU children Neurosensory 48.4 (47.7, 49.0) n = 975 46.3 (41.7, 51.0) n = 33 41.9 (36.0, 47.8) n = 17 .023 .040 .63 Motor development 49.1 (48.5, 49.8) n = 789 45.8 (44.0, 47.6) n = 174 42.0 (38.8, 45.2) n = 63 <.001 <.001 .74 Learning/remembering 49.3 (48.6, 50.0) n = 623 47.5 (46.3, 48.8) n = 298 43.4 (40.9, 45.9) n = 109 <.001 <.001 .63 Quality of life 49.8 (49.0, 50.5) n = 659 46.3 (45.1, 47.4) n = 313 39.4 (35.7, 43.2) n = 59 <.001 <.001 1.11 Healthy children Neurosensory 48.9 (47.9, 49.8) n = 361 57.0 n = 1 31.1 n = 1 .120 .154 1.89 Motor development 49.2 (48.2, 50.3) n = 333 45.6 (42.6, 48.7) n = 31 37.9 (22.6, 53.2) n = 3 .018 .002 1.18 Learning/remembering 49.6 (48.5, 50.7) n = 266 46.9 (44.7, 49.1) n = 89 45.8 (39.1, 52.5) n = 14 .037 .025 .41 Quality of life 50.1 (49.0, 51.2) n = 271 45.6 (43.5, 47.8) n = 86 36.2 (23.8, 48.6) n = 8 <.001 <.001 1.58 * first based on Anova, second based on Kruskal-Wallis non-parametric test (in italics) Psychosocial health by child behavior problem Child behavior was strongly related to parental psychosocial health in both groups of parents (see Table 4 ). Parents whose child scored in the clinical range for internalizing and externalizing symptoms and the total problem score on the CBCL/1.5–5 had the lowest mean (i.e., poorest) MCS scores. The differences between this group and the group with children scoring in the normal range resulted in large effect sizes, indicative of clinically important differences in parental psychosocial health. Table 4 Mean score, p-value and effect size for SF-36 psychosocial summary score comparing CBCL/1.5–5 normal with borderline and clinical groups CBCL scale Normal Borderline Clinical p-value Effect size NICU sample Internalizing 49.5 (48.9,50.2) n = 841 42.5 (39.6,45.5) n = 67 40.5 (37.6,43.4) n = 78 <.001 .95 Externalizing 48.9 (48.3,49.6) n = 925 41.6 (38.0,45.2) n = 45 35.0 (29.6,40.2) n = 32 <.001 1.43 Total 49.3 (48.6,49.9) n = 831 43.3 (39.7,47.0) n = 34 35.3 (31.0, 39.5) n = 39 <.001 1.45 Healthy children Internalizing 49.6 (48.6,50.6) n = 324 40.7 (36.8,44.6) n = 20 40.1 (34.0,46.2) n = 16 <.001 1.03 Externalizing 49.3 (48.3,50.3) n = 342 43.4 (37.0,49.8) n = 14 34.1 (22.5,45.8) n = 8 <.001 1.67 Total 49.4 (48.4,50.4) n = 330 41.5 (34.0,49.0) n = 12 36 (27.0, 44.0) n = 11 <.001 1.46 p-value based on Anova, (non-parametric tests: all p-values < .001) Parental psychosocial health by birth-related risk factors Within the NICU sample, MCS score did not vary by any birth-related risk factor (i.e., gestational age; small for gestational age; apgar score; multiple birth; the presence of a major morbidity; and neonatal illness severity score), with the exception of the presence of a congenital anomaly. For this variable, MCS scores were significantly lower in parents of children with versus without a congenital anomaly (mean difference = -3.8; p = .017; effect size = -.37). Children with a congenital anomaly (n = 87) had proportionally more mild and moderate/severe health status problems in all 4 categories (see Table 5 ). Table 5 Number (%) of NICU children with and without a congenital anomaly to report a problem for each health status category and p-value for Chi-square test of significance Type of HS problem Congenital anomaly None Mild Moderate or Severe p-value Neurosensory No 715 (95.6) 25 (3.3) 8 (1.1) <.001 Yes 70 (83.3) 8 (9.5) 6 (7.1) Motor development No 584 (78.3) 126 (16.9) 36 (4.8) <.001 Yes 48 (57.1) 17 (20.2) 19 (22.6) Learning/remembering No 457 (60.9) 222 (29.6) 71 (9.5) <.001 Yes 36 (42.9) 30 (35.7) 18 (21.4) Quality of life No 481 (64.2) 233 (31.1) 35 (4.7) <.001 Yes 38 (44.2) 34 (39.5) 14 (16.3) Correlates of psychosocial health in general In general, variables significantly associated with the MSC score in bivariate analysis were as follows: any health status problems (mean difference = -3.8; p < .001); neurosensory problems (mean difference = -3.7; p = 0.04); motor development problems (mean difference = -4.4; p < .001); learning/remembering problems (mean difference = -2.9; p < .001); poorer quality of life (mean difference = -4.8; p < .001); more internalizing behaviour symptoms (mean difference = -8.3; p < .001); more externalizing behavior symptoms (mean difference = -9.9; p < .001); household income below $30,000 per year (mean difference = -2.6; p < .001); female gender (mean difference = -2.6; p < .001);not living as common-law or married (mean difference = -3; p = .03); more caregiver strain (r = .41; p < .001); and lower family function (r = -.44; p < .001). Borderline significance was also found for less than high school education (mean difference = -2; p = .08). We examined a pooled model (both groups together) for a direct comparison of the NICU and healthy groups after adjustment for other variables. Due to the low number of male respondents in the healthy group, we restricted the pooled multivariable analysis to only female respondents. Predictors significantly associated with the outcome were the following: parental age (Beta = 0.15; p = 0.001); internalizing behavior (Beta = -2.06; p = 0.017); externalizing behavior (Beta = -3.24; p = 0.004); parental strain (Beta = 0.15; p < 0.001); and family function (Beta = -0.53; p < 0.001). The pooled model also showed an interaction effect between NICU admission and education (less than high school) (Beta-education = -5.94 with p = 0.009; Beta-interaction = 7.28 with p = 0.005)(see Table 6 .) For the NICU group, education did not show any effect in terms of difference in outcome, but for the healthy group, lower education was associated with a significantly lower mean MCS score. More specifically, for respondents with less than high school education, the healthy group reported lower MCS scores than did the NICU group. The results were not affected by exclusion of multiple births and cases of congenital anomalies from the analysis. Table 6 Beta coefficients, 95% confidence intervals, standardized beta coefficients and p-values for predictor variables in the multiple regression models for pooled model Pooled model Variable Beta CI-low CI-high St. beta p-value Intercept 34.21 30.26 38.17 <.0001 NICU -0.38 -1.32 0.55 -0.02 0.500 Parental age 0.15 0.08 0.23 0.08 0.001 Education -5.94 -9.67 -2.21 -0.13 0.009 Internalizing behavior -2.06 -3.47 -0.65 -0.07 0.017 Externalizing behavior -3.24 -5.08 -1.40 -0.08 0.004 Caregiver strain 0.15 0.13 0.18 0.26 <.0001 Family function -0.53 -0.60 -0.46 -0.32 <.0001 NICU-education interaction 7.28 3.01 11.56 0.14 0.005 Although other interaction terms with NICU status did not add any more significant results in the pooled model (non-significant partial F-test), we examined separate models for the NICU and the healthy baby group to further explore the association between gender and MCS score, and to evaluate the potential influence of congenital anomalies in NICU group. Correlates of psychosocial health for NICU sample Variables that were significantly associated with lower MCS scores at the bivariate level include the following: female caregivers (mean difference = -3.2; p = .037); household income below $30,000 per year (mean difference = -3.3 and p < .001); not living as common-law or married (mean difference = -5.1; p < .001); neurosensory problems (mean difference = -6.44; p = .011); motor development problems (mean difference = -7.1; p < .001); learning/remembering problems (mean difference = -5.9; p < .001); poorer quality of life (mean difference = -10.4; p < .001); more internalizing behaviour symptoms (mean difference = -9.03; p < .001); more externalizing behavior symptoms (mean difference = -13.9; p < .001); the presence of a congenital anomaly (mean difference = -3.8; p = .017); more caregiver strain (r = .411; p < .001); and lower family function (r = -.441; p < .001). Predictors that were significant in the final regression model appear in Table 7 . Female gender was an independent risk factor for lower MCS score: females scored on average 5.3 points (CI interval 2.5 to 8.0) lower, which represents a moderate effect size of 0.51 (when overall NICU parents group standard deviation (SD) 10.4 for MCS was used as the denominator). Scoring outside the normal range for internalizing and externalizing child behavior symptoms independently contributed to lower MCS scores (-1.9 and -2.8, both with wide confidence intervals), with the change representing small effect sizes of 0.18 and 0.27. More caregiver strain (i.e., lower PTT) was related with poorer MCS scores. A one point change in PTT corresponded to a 0.15 (CI: 0.11–0.19) change in MCS score. In NICU parents, the mean PTT was 86.9 and SD was 18.5. Therefore, 2 SD on the PTT would represent 5.5 points on the MCS, or an effect size of 0.53. The mean score for family function (FAD) was 8.1 and the SD was 6.4. A one point change in FAD corresponded to a 0.5 (CI: 0.62; 0.42) change in MCS. Therefore a 2 SD increase in family function score (i.e., poorer family functioning) would result in a 6.4 decrease (worsening) in MCS, representing a moderate effect size of .62. Overall, the adjusted R2 was .2884 (F = 73.96; df = 5; p = < .0001), with 5 out of 15 predictors included in the full model. Table 7 Beta coefficients, 95% confidence intervals, standardized beta coefficients and p-values for predictor variables in the multiple regression models for both samples NICU sample Healthy baby sample Variable Beta CI-low CI-high St. beta p-value Beta CI-low CI-high St. beta p-value Intercept 44.9 40.3 49.5 <.001 35.2 26.3 44.1 <.001 Parental age 0.3 0.1 0.5 0.1 .005 Female gender -5.3 -2.6 -8.0 -0.11 <.001 Education -5.00 -0.8 -9.1 -0.1 .019 Internalizing behavior -1.9 -0.1 -3.8 -0.06 .043 -4.0 -0.8 -7.2 -0.1 .014 Externalizing behavior -2.8 -0.4 -5.3 -0.07 .025 Caregiver strain 0.2 0.1 0.2 0.26 <.001 0.1 0.04 0.2 0.2 .003 family function -0.5 -0.4 -0.6 -0.32 <.001 -0.6 -0.4 -0.8 -0.4 <.001 Quality of life -6.9 -0.4 -13.4 -0.1 .039 Correlates of psychosocial health for healthy baby sample Variables that were significantly associated with poorer SF-36 MCS scores at the bivariate level include the following: younger parental age (r = .19; p < .001); household income below $30,000 per year (mean difference = -4.6; p = .005); less than high school education (mean difference = -6.22; p = 0.065); not living as common-law or married (mean difference = -6.1; p = .005); motor development problems (mean difference = -11.3; p = .043); learning/remembering problems (mean difference for any problems versus none = -2.68, p = 0.021); poorer quality of life (mean difference = -13.9; p < .032); more internalizing behavior symptoms (mean difference = -9.5; p < .001); more externalizing behavior symptoms (mean difference = -15.2; p < .018); more caregiver strain (r = .385; p < .001); and lower family function (r = -.438; p < .001). Predictors that were significant in the final regression model appear in Table 7 . The model for parents of healthy children did not include female gender (because of low numbers) and externalizing behavior symptoms, and included several variables not predictive in the NICU model (i.e., parental age; education; quality of life). Both models included internalizing child behaviors, caregiver strain and family function. In the healthy baby sample, younger parental age was related to poorer MCS score, with a one year change in age resulting in a 0.26 (CI: 0.08; 0.45) change in MCS. A ten year difference in age would correspond to a 2.6 difference in MCS, which would represent a small effect size of 0.27 (when the overall healthy baby parent group SD for MCS (9.6) was used as a denominator). Education was also associated with MCS. Compared with high school graduates, the MCS score for parents with less than a high school education were on average 5.0 lower (CI: 0.84; 9.1), which represents a moderate effect size of 0.52, although the effect could range from minimal to large due to lower precision of the beta estimate. Child internalizing symptoms, family function and caregiver strain were associated with parental MCS in a similar way as for NICU parents. However, due to lower numbers and resulting low precision in beta estimates, the effects ranged from minimal to large. Lower parent-reported child quality of life was also associated with a lower parental MCS. Parents who reported a problem with their child's quality of life had MCS scores that were 6.9 (CI: 0.37; 13.4) lower than parents who reported at least one quality of life problem compared with those who reported at least one problem. Again, due to the small numbers, the effect could range from minimal to large. In the final regression model, the adjusted R2 was .3046 (F = 25.97; df = 6; p < .0001), with 6 out of 16 predictors included in the full model. Discussion There is little information in the research literature on how parents of NICU children adapt psychologically to the demands of caregiving beyond the initial hospitalization period. We compared the psychosocial health of parents of NICU children with parents of a group of healthy full-term children using the SF-36, a popular generic measure of psychosocial HRQL. Although children admitted to a NICU at birth are at increased risk of a variety of long-term health problems, we did not find any difference in parental psychosocial health when the two groups were compared. This finding is in agreement with one of the few studies that measured mental health in parents of NICU children at preschool age. Singer et al. [ 17 ] reported that after the neonatal period, the mental health of mothers of low-risk infants did not differ from mothers of term infants, and by 3 years, they had lower levels of distress, which they suggest may be due to maternal relief after an initial period of fear and anxiety. Mothers of high-risk infants, in contrast, had more symptoms of distress at 2 years, more negative family impact at 2 and 3 years and more parental strains and illness stressors at 3 years. But by 3 years, their reported psychological distress did not differ from that of term mothers. The authors suggest that by 2 years, infant developmental scores are predictive of later outcomes, and many mothers of high-risk infants must relinquish their hopes for their children to "catch up" to healthy born children and that some psychological adaptation has taken place despite parental acknowledgment of greater family and parenting stressors. With our cross-sectional design, we are not able to confirm the trend noted by Singer, but given the lack of relationship between most birth-related risk factors and parental mental health, it is possible that the parents of high- and low-risk infants in our sample have adjusted over time. Current health status, in bivariate analysis, was strongly related with parental psychosocial health. In both groups of parents, those whose child had a neurosensory, motor development, learning/remembering or quality of life problem had poorer psychosocial health than those with children with no problems in these areas. Child behavior was also strongly related to parental psychosocial health. More specifically, parents of children who scored in the borderline or clinical range for internalizing, externalizing and total behavior problems on the CBCL/1.5–5 reported poorer psychosocial health than parents of children who scored in the normal range. These findings were consistent across both samples of parents. The only birth-related risk factor associated with parental psychosocial health was the presence of a congenital anomaly. Here the effect size was small, but points to the possibility that a congenital anomaly may affect parents mental health adversely. Researchers have reached a consensus that a minimally important difference in HRQL is close to one half of a standard deviation [ 34 ]. The differences that we found for health status and behavior were substantially larger and therefore represent clinically important differences in parental psychosocial health. However, not all of these variables showed a significant effect in the multivariate analysis, and it is possible that these variables influence other, more proximal, variables that showed stronger effects on parental psychosocial health. The factors associated with poorer psychosocial health in the multivariate models provide important information about correlates of adjustment for NICU and healthy baby families. In a more general pooled model, parental age, higher caregiver strain, lower family function, and child's internalizing and externalizing behavior were independently associated with poorer caregiver's mental health score. The effect of lower parental education was modified by NICU status of the child. In the healthy baby group, less than high school education indicated lower MCS score. Child externalizing behavior symptoms and female gender (parental) were associated with lower MCS scores in the NICU group, whereas lower parental age, less education and poorer child quality of life were associated with lower MCS in the healthy baby group. For both samples, as it is also seen in a pooled model results, low family function, high caregiver strain, and child's internalizing behavioral symptoms were independently associated with lower parental psychosocial health. For family function and caregiver strain, only a substantial departure from mean values (at least 2 SDs) would result in a clinically important moderate effect size for the NICU group. Our interpretation for the healthy baby sample is hampered by wide confidence intervals around the beta estimates, resulting in effect sizes that ranged from minimal to large. Internalizing behavior symptoms were associated with only a small effect on caregiver's MCS score, again with wide confidence intervals around the beta coefficients for both samples. A recent publication outlines the integration of a number of theoretical models into one multidimensional model that can be used to describe the caregiving process [ 35 ]. This model includes the following constructs: background and context; child characteristics; caregiver strain; intrapsychic factors; coping/supportive factors; and health outcomes. Fitting our findings within this framework, we found that poorer psychosocial health in parents was associated with background/context variables (i.e., female gender, younger age, less education); child characteristics (i.e., poorer quality of life, more child behavior problems); caregiver strain; and coping/supportive factors (i.e., family function). We suggest that future research with NICU parents be conceptually based and measure constructs found in other research to be important to caregiver health. Our study has several limitations. Because it is not possible to verify cause-effect using a cross-sectional design, we were only able to estimate the direct effect of a limited number of predictor variables on parental psychosocial health. While our study has helped to identify some possibly important caregiving variables, there are other variables important to caregiver health that we did not measure. For example, while it is possible that some parents of children with severe health problems may have received specialized or targeted services (health and/or social services) to help them cope with their child's health problems, we did not include measures to determine this. Another limitation concerns our response rate. Although it is within the range often obtained in a postal survey [ 36 ], non-response can introduce bias. Some non-respondents indicated (verbally or in writing) they were "too busy" to participate. It is also likely that some questionnaires returned to us blank were from non-English speakers. Where we had data and were able to explore response bias (NICU sample only), only a few differences in birth-related sample characteristics and outcome were found that suggests respondents had sicker babies [ 37 ]. However, our study findings about health outcomes of NICU graduates are in agreement with the larger NICU literature, so it is unlikely that the differences we found are entirely due to response bias. Conclusion Our findings would suggest that overall, parental gender, family functioning and caregiver strain played influential roles in parental psychosocial health. For child characteristics, current behavior was more influential than initial birth-related risk factors. List of abbreviations MCS – Mental Component Score NICU – Neonatal intensive care unit HS – Health status FAD – Family Assessment Device PTT – Parental Impact Time Competing interests The author(s) declare that they have no competing interests. Contributions of each author Anne Klassen contributed substantially to the study's conception and design, acquisition of data, analysis and interpretation of data; and she drafted and revised and gave final approval of the version to be published. Shoo Lee contributed substantially to the study's conception and design, acquisition of data, analysis and interpretation of data; and revised the article critically for important intellectual content and gave final approval of the version to be published. Parminder Raina contributed substantially to the analysis and interpretation of data; and revised the article critically for important intellectual content and gave final approval of the version to be published. Sarka Lisonkova contributed substantially to the analysis and interpretation of data; and revised the article critically for important intellectual content and gave final approval of the version to be published. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544865.xml
555940	Heterologous expression of the filarial nematode alt gene products reveals their potential to inhibit immune function	Background Parasites exploit sophisticated strategies to evade host immunity that require both adaptation of existing genes and evolution of new gene families. We have addressed this question by testing the immunological function of novel genes from helminth parasites, in which conventional transgenesis is not yet possible. We investigated two such novel genes from Brugia malayi termed abundant larval transcript (alt) , expression of which reaches ~5% of total transcript at the time parasites enter the human host. Results To test the hypothesis that ALT proteins modulate host immunity, we adopted an alternative transfection strategy to express these products in the protozoan parasite Leishmania mexicana . We then followed the course of infection in vitro in macrophages and in vivo in mice. Expression of ALT proteins, but not a truncated mutant, conferred greater infectivity of macrophages in vitro , reaching 3-fold higher parasite densities. alt-transfected parasites also caused accelerated disease in vivo , and fewer mice were able to clear infection of organisms expressing ALT. alt -transfected parasites were more resistant to IFN-γ-induced killing by macrophages. Expression profiling of macrophages infected with transgenic L. mexicana revealed consistently higher levels of GATA-3 and SOCS-1 transcripts, both associated with the Th2-type response observed in in vivo filarial infection. Conclusion Leishmania transfection is a tractable and informative approach to determining immunological functions of single genes from heterologous organisms. In the case of the filarial ALT proteins, our data suggest that they may participate in the Th2 bias observed in the response to parasite infection by modulating cytokine-induced signalling within immune system cells.	Background Pathogens have evolved many ingenious mechanisms to manipulate innate and adaptive host immune responses [ 1 - 6 ]. The nematode parasite Brugia malayi is a causative agent of the disease lymphatic filariasis, which afflicts over 100 million people in tropical countries. Mosquito-borne infective stage larvae gain entry to the human body during a blood meal, and establish long-lived infections characterised by down-regulation of host T cell and macrophage reactivity [ 7 , 8 ]. We have studied the profile of genes expressed in infective larvae and reported that ~5% of the mRNA transcripts from this stage correspond to two closely related genes, which we have named abundant larval transcript (alt) -1 and -2 [ 9 , 10 ]. The two genes encode proteins with 79% amino acid identity, but no similarity to any gene of known function. alt -like genes are present in other filarial nematode species [ 11 , 12 ] and are characterised by a signal peptide, a variable acidic domain, and a conserved, cysteine-rich domain. A distantly-related gene is also present in the genomes of the free-living nematodes Caenorhabditis elegans and C. briggsae but in both cases the acidic domain is absent (Gregory, Maizels and Blaxter, unpublished observation). ALT proteins are stockpiled in the oesophageal glands of infective larvae [ 12 ] and are secreted by the parasites when they encounter mammalian culture conditions. Thus, their function may be to promote parasite survival within the host physiological or immunological environment. For example, ALTs may interfere with the critical first interaction between the innate immune system and the nematode invaders. It has been established that larval stages rapidly elicit a strong Th2 response in mice [ 13 ] and induce host macrophages to adopt a counter-inflammatory phenotype [ 14 ]. Although ALT antigens are not expressed on the parasite surface, they can induce protective immunity in animal models [ 9 , 15 , 16 ], indicating that neutralization of ALT function may be sufficient to protect the host from infection. Transgenesis and targeted gene deletion have yet to be established for parasitic helminths, so it is not possible to investigate the biological role of ALT proteins by conventional reverse genetics. We reasoned, however, that if ALT function is fulfilled within the host rather than within the parasite, we can validly study these proteins by transgenic expression in a more tractable carrier species. We chose to test this approach with the protozoal parasite Leishmania , several species of which are infective to laboratory mice. Leishmania can readily be modified genetically [ 17 - 20 ] to yield lines expressing high levels of exogenous transgenes. We selected L. mexicana as it establishes infections in murine macrophages in vitro , providing experimental access to a key cell type known to be modified in filarial infection [ 14 , 21 - 23 ]. L. mexicana will also infect mice, and although the immunological factors determining resistance and susceptibility are not as well-defined as in L. major [ 24 - 26 ], it offers the advantage of slower in vivo kinetics and consequently is anticipated to be more sensitive to altering factors. Using this model, we show here that transgenic L. mexicana expressing the ALT proteins are more virulent in macrophages in vitro , and that this property is abolished by deletion of the filarial-specific acidic domain. We also show that mice infected with alt -transgenic L. mexicana harbour higher parasite burdens than controls. By studying the responses of macrophages infected with transgenic parasites, we suggest that the ALT products modulate cytokine-induced signalling and render parasites more resistant to IFN-γ-induced killing. Results Expression of B. malayi ALTs in L. mexicana Bm-alt-1 and Bm - alt-2 genes were subcloned in their entirety, including endogenous signal peptide sequences, into the recombination vector pSSU, which contains flanking sequences homologous to the 18S small subunit (SSU) rRNA locus [ 19 , 27 ] (Figure 1 ). Electroporation of L. mexicana was undertaken, permitting homologous recombination of the alt-1 and -2 sequences downstream of the strong polymerase I promoter into the sequence for the small sub-unit rRNA, which is known to be transcribed in both stages of the life cycle of the parasite. Following puromycin selection, multiple transgenic lines were isolated for each alt gene, from which representative clones were selected containing the correct insert at an appropriate integration site. Transgenic ALT expression in the free-living culture promastigote stage was confirmed in both lines by Western blot (data not shown) and immunofluorescent staining (Figure 2A–D ) of permeabilized parasites with murine anti-ALT antibodies. Not only were ALT proteins found widely distributed in the transgenic protozoa, but staining of the membrane-rich flagella indicated surface expression in the promastigote stage. This was confirmed by flow cytometric analysis of anti-ALT-stained non-permeabilized transgenic promastigotes (Figure 2E, F ; negative controls panels G, H). In vitro infection of macrophages with transfected amastigotes Bone-marrow-derived macrophages were infected with axenic transformed amastigotes of each line. Both wild-type (Figure 2I ) and alt -transfected parasites (Figure 2J ) were fully infective to macrophages, and continued to express transgene-encoded protein reactive by immunofluorescence (data not shown). Within 24 hours, ALT-1 and ALT-2 expressing lines were able to infect significantly more host cells (Figure 3A , p < 0.001), a contrast that was sharply accentuated by day 7 of culture (Figure 3B ). Moreover, >90% of macrophages infected with alt -transgenic L. mexicana harboured at least 3 parasites, compared to <50% of cells infected with the wild-type, and overall there was a 3-fold difference in mean parasite load per macrophage. This enhancement was manifest in both alt-1 and alt-2 transgenic lines and did not affect the total number of macrophages surviving through the culture period. In contrast, transfectants expressing an unrelated filarial gene, the cystatin Bm -CPI-2 [ 28 ], have no effect on survival of L. mexicana in murine macrophages (Figure 3C, D ). Thus, the observed effect is gene-dependent and is not an attribute of the transfection system. Enhanced virulence of ALT-expressing L. mexicana promastigotes Analysis of transgene function in the more complex setting of in vivo infection was also performed, in order to test more stringently whether ALT products interfere with immunity. In two experiments, the alt -transfectants elicited larger lesions more quickly than either the parental wild-type strain of L. mexicana (Figure 3E ), or a transfectant encoding GFP (not shown). In both experiments, the effect of alt transfection was to accelerate lesion development to a plateau after 8–10 weeks, while control parasites reached similar levels only after 12–15 weeks. Recovery of parasites from the footpads of infected mice also showed marked exacerbation: parasites were detected in our assay in 94% (15/16) of animals given transfected L. mexicana compared to 50% (4/8) of those receiving wild-type parasites (p = 0.03, χ 2 test) (Figure 3F ). Nitric oxide production and susceptibility to NO-mediated killing Because nitric oxide generation is known to be a primary factor in the control of parasite survival in macrophages [ 29 , 30 ], we compared levels of the NO-metabolite nitrite from J774 macrophages infected with the different parasite lines. NO production was assayed 24- and 48-hours post-infection, as the differences in parasite numbers are apparent from an early stage. Irrespective of the transgenic status of the parasite, macrophages produced near-identical amounts of NO in culture medium, measured by nitrite, at both time points (Figure 4A ); thus the ALT products do not abolish the reactive nitrogen burst. To test whether there was a quantitative reduction in either NO responses or parasite sensitivity to destruction, we infected cells that had been previously stimulated with LPS and a range of doses of IFN-γ. Survival was measured by enumeration of infected macrophages 3 days later. Figure 4B shows that alt- transfected parasites not only achieve higher infection levels in unstimulated macrophages (no IFN-γ), but survive better in cells stimulated with intermediate IFN-γ doses (0.01–0.1 U/ml) than do wild-type organisms. Interestingly, the production of nitrite was comparable at each IFN-γ dose in all parasite types (Figure 4C ), indicating that the ALT products are likely to be interfering in an NO-independent pathway of immunity. Gene expression in infected macrophages To gain insight into what other mechanisms may be at play, we tested mRNA from macrophages infected with wild-type or transfected L. mexicana against an array of 514 genes associated with immune responsiveness formatted on the Mouse Cytokine Expression Array 1.0 (R & D Systems Inc.). Two independent experiments were performed, and hybridization of [ 33 P]-labelled cDNA was measured by phosphorimaging of spots of interest. Little difference was seen in hybridization for iNOS, IL-10, IL-12 (Table 1 ) or many other known players in the pro- and counter-inflammatory cascades. The constancy of iNOS expression following transfection is consistent with the measurements of nitric oxide production discussed above. However, other pro-inflammatory players (TLR6, LPS-BP, LTBP3 and TNFSF11 and 12) were down-regulated relative to wild type in both alt -1 and alt -2 transfection, in replicate experiments. Moreover, substantial shifts in mRNA expression were consistently observed with certain products known to modulate the type-1/type-2 balance. Bone-marrow-derived macrophages, infected 7 days earlier with wild-type L. mexicana infection, showed significant ablation of GATA-3 and SOCS-1, but expression was maintained or enhanced in cells infected with alt -transgenic parasites (Figure 5A and 5B ). These effects were reproduced with both alt genes in both experiments (Table 1 ). Because array hybridization may under certain conditions be non-linear with respect to mRNA concentrations, we performed quantitative RT-PCR with GATA-3- and SOCS-1-specific primers on replicate samples of infected macrophages taken at 24 h (Figure 5C, D ) and 7 days (E, F). For GATA-3, these data reinforce the conclusion that while wild-type infection results in a 2–3-fold loss of GATA-3 expression, alt -transgenic infected macrophages maintain the level of this transcription factor throughout the course of in vitro infection (Figure 5C and 5E ). Interestingly, SOCS-1 is sharply upregulated by 24 h in all infected macrophages, but elevated expression remains evident only where alt transgenic parasites are present (Figure 5D and 5F ). Although it is possible that larger parasite numbers in the alt- transfection system may in themselves alter macrophage gene expression, parasite densities were only slightly shifted at 24 h, when GATA-3 expression was markedly different in wildtype and transfected cell infections. Up-regulation of GATA-3 and SOCS-1 in filarial infection We then tested whether GATA-3 and SOCS-1 are up-regulated by live infection with B. malayi larvae in vivo . Mice were injected i.p with 200 larvae, and 7 days later peritoneal cell populations were recovered by lavage with medium. We then performed real time PCR on an adherent macrophage enriched cell population and a non-adherent cell population, predominantly lymphocytes. As shown in Figure 6A and 6C , the macrophage-rich adherent population showed a modest increase in both GATA-3 and SOCS-1 expression, whereas a very substantial rise in both was seen in the non-adherent cells (Figure 6B and 6D ). It seems that B. malayi infection results in up-regulation of the same genes as observed with ALT in macrophages, in both macrophage and non-macrophage subsets. The acidic domain of ALT is essential for biological function The enhanced virulence phenotype conferred by alt transfection, measured by in vitro infectivity to macrophages, provides a ready and tractable system for submolecular analysis of functional domains of the ALT protein. We investigated whether the N -terminal acidic domain, which is unique to the filarial genes, was important for the immunological activity of the protein. We constructed a truncated mutant of ALT-2, named acidic domain deleted (add) , which was cloned into the pSSU vector and electroporated into L. mexicana . These transfectants expressed immuno-reactive protein (Figure 7A–C ) and were able to infect bone marrow-derived macrophages. However, add -transfected parasites did not display the greater infectivity seen with alt -transfectants, but showed a phenotype indistinguishable from wild-type parasites (Figure 7D ). Thus, the acidic domain is required for functional expression of the immunological effects of the ALT proteins. Similar analyses will permit the future definition of the critical residues required for immunomodulatory activity. Discussion Long-lived helminth parasites have evolved highly effective strategies to evade host immunity, requiring both adaptation of existing genes and evolution of new gene families [ 6 ]. With genomes that encode many thousands of proteins, these parasites are likely to be repositories of numerous novel 'immune evasion genes' with no or only weak sequence similarity to known products [ 31 ]. The imminent completion of genome sequence information for major helminth parasites [ 32 - 34 ] accentuates the problem of how to identify functional immune modulators among numerous novel gene sequences. We hypothesized that transcripts for secreted immunomodulatory proteins would be among the most abundant mRNAs at key points in the parasite life cycle. Two such genes are those that encode the abundant larval transcripts (ALT) proteins, which are released by larval parasites ready to infect the mammalian host and represent 5% of the total mRNA at this stage [ 9 ]. To test whether the ALT proteins are functional immune evasion products, we transfected each alt gene into L. mexicana and showed that infection of macrophages in vitro is exacerbated by expression of either ALT protein. Moreover, mice infected with alt -transgenic parasites display more rapid lesion development and higher parasite burdens than controls. Our results also demonstrate that alt -transfected parasites are more resistant to INF-γ-induced killing by macrophages, supporting our hypothesis that ALT proteins act to modify host immune responses in filarial infection. These data validate the transfection strategy in general and, in highlighting changes in key intracellular factors resulting from ALT expression, justify the selection of a protozoal carrier to test the function of a helminth gene. A further advantage of the system we describe here is the facility with which selected mutants can be analysed with an in vitro read-out of infectivity to macrophages. We constructed a deletion mutant lacking the N-terminal acidic domain, which shows most variation between filarial species and is not present in distantly related genes from free-living nematode organisms. This deletion showed a clear-cut abolition of the alt phenotype, indicating the essential functional nature of this sequence and paving the way for a finer analysis of structure-function relationships in a tractable experimental system. We have also extended the use of this transfection system to the analysis of other parasite genes that are hypothesised to be immunomodulatory. For example, filarial parasites produce homologues of mammalian macrophage migration inhibitory factor, MIF [ 39 , 40 ], a cytokine generally considered to be an acute pro-inflammatory agent [ 41 , 42 ]. It is, however, paradoxical that long-lived tissue pathogens produce a potentially inflammatory mediator, and we therefore used the Leishmania transfection system to test the hypothesis that long-term MIF production may promote parasite survival. L. mexicana organisms transfected with B. malayi MIF homologues were tested first in vitro , in which setting they were less infective to macrophages, rarely exceeding one parasite per host cell. This result was consistent with an immediate pro-inflammatory action of MIF on macrophages. However, when tested in vivo over a 4- or 8-week-period, MIF-transfected parasites were able to survive better than wild-type, indicating that over the longer term, filarial MIF homologues are able to exert a down-modulatory effect on host immunity (Prieto-Lafuente, manuscript in preparation). We are now using this system to analyse the gene expression profiles of macrophages infected with MIF-transfected parasites in both in vitro and in vivo settings. These data illustrate the facility with which the Leishmania transfection system can be used in parallel for in vitro and in vivo experiments, and the importance of an in vivo read-out to assay gene effects within the immune system as a whole. In addition, our work has demonstrated that Leishmania transfection for helminth gene analysis is equally applicable for two completely unrelated, but immunologically important, gene families. The application of this new strategy to transfection of L. mexicana is ideally suited to the study of macrophage modulation by genes predicted to function in this environment. Parallel investigations can also be envisaged by transfection of L. major , which will permit a more thorough analysis of T cell modulation to be undertaken. For example, resistance to L. major is clearly associated with development of a parasite-specific Th1 response, and it is possible that parasite genes that inhibit Th1 differentiation will alter the course of L. major infection in vivo . These studies are now under way. Macrophages are known to be a critical cell population in the immune response to filarial parasites at successive points in time. First, they participate in innate defences against invading larvae [ 35 , 36 ]; second, if infection becomes established, they evolve an IL-4-dependent "alternatively activated" phenotype, which is broadly immunosuppressive [ 8 , 37 ]; and third, in late stage infection, they clear the bloodstream of microfilarial forms through a nitric oxide-dependent pathway [ 21 , 38 ]. Thus, the reduction in IFN-γ-responsiveness in macrophages harbouring alt -transfected parasites has resonance for initial host susceptibility, and longer-term propensity for chronic infection, as well as the ability to eliminate the blood-borne Mf stage. A key outcome of the present study is that ALT expression is associated with up-regulation of GATA-3 and SOCS-1. GATA-3 is a pivotal transcription factor for the development and function of the Th2 pathway [ 43 , 44 ] and has not previously been reported in mouse macrophages; hence, at the present time, downstream genes activated by GATA-3 in macrophages have not been defined. Significantly, GATA-3 is required for embryonic development and has recently been shown to be essential for the differentiation [ 45 ] and effector function [ 46 ] of murine eosinophils. Thus, GATA-3 expression in macrophages may fulfil an important role, possibly in conjunction with the altered phenotype of these cells in chronic parasite infections [ 8 , 47 ]. SOCS-1 is a member of the Suppressor Of Cytokine Signalling family of proteins, which regulate signal transduction by IFN-γ and structurally related cytokines [ 48 , 49 ]. SOCS-1 is particularly important in macrophage responsiveness to inflammatory stimuli [ 50 ], countermanding IFN-γ by directly inhibiting the JAK kinase associated with the IFN-γ receptor [ 51 ]. SOCS-1 is known to inhibit the IFN-γ-dependent killing of Leishmania parasites, because macrophages from SOCS-1 null mice require 100-fold less IFN-γ to clear infection [ 52 ]. Indeed, in the absence of SOCS-1, there is generalised activation of the immune system, causing autoimmune pathology [ 53 , 54 ]. Thus, by up-regulating SOCS-1, cells expressing ALT may have down-shifted responsiveness to inflammatory cytokines, requiring exogenous stimulation to induce Leishmania killing (Figure 5B ). In the context of Brugia infection, SOCS-1 induction by ALT proteins could explain why macrophages fail to develop the IFN-γ-activated phenotype, and instead express a counter-inflammatory profile [ 8 ]. Overall, these data suggest that the ALT proteins play a role in the evasion strategy of the filarial nematode, by directly amplifying Th2 responses and/or interfering with signals necessary for the development of pro-inflammatory Th1 populations. It is interesting to note that in vivo exposure to live infective larvae of B. malayi induces a prompt Th2 response measurable by 24 h and increasing to 10 days post-infection [ 13 ]. We have repeated these experiments by intraperitoneal inoculation of live L3 and find up-regulation of GATA-3 and SOCS-1 in both adherent and non-adherent peritoneal cells (Figure 6 ). Conclusion The novel approach we have described permits, for the first time, the elucidation of gene function for a major group of biologically and medically important parasites, which in the example presented here provides non-intuitive results linking parasite secretions to host cell signalling. The transfection strategy will also accommodate a mutagenesis analysis of structure-function relationships in unique gene families. In conclusion, our system provides a solution to one of the major obstacles facing helminth parasite immunology in the post-genomic era and offers a fascinating insight into the molecular and cellular intricacies of pathogen manipulation of host immune responsiveness. Methods Mice and parasites Six- to eight-week old female C57BL/6 and CBA mice were used. L. mexicana (strain MNYC/BZ/62/M379) promastigotes were cultured in vitro in semi-defined medium/10% heat inactivated foetal calf serum (hiFCS)/1% penicillin-streptomycin (complete SDM) at 26°C. Amastigotes were cultured axenically at 34°C in Schneider's Drosophila medium (Gibco BLR) supplemented with 20% hiFCS and 3.9 g/l 2-(N-morpholino)ethanesulfonic acid (Sigma, U.K.). Transgenic parasites were cultured under the same conditions with the addition of 20 μg/ml puromycin (Sigma, U.K). Cell culture The murine macrophage cell line J774 was passaged in DMEM containing 10% hiFCS/1% penicillin-streptomycin/1% L-glutamine and cultured at 37°C in 5% CO 2 . Leishmania expression construct for B. malayi ALTs Primers were designed to amplify the entire coding region of alt-1 and alt-2 from a B. malayi L3 cDNA library. The oligonucleotides used for alt-1 were 5'-CCGCTCGAG ATG AACAAATTGCTAATAGCA-3' (sense, initiating codon in bold) and 5'-TGCTCTAGA TTA CGAGCATTGCCAACTTTC-3' (antisense, terminating codon in bold); and for alt-2 , 5'-CCGCTCGAG ATG AATAAACTTTTAATAGCA-3' (sense) and 5'-TGCTCTAGAC TAT GCGCATT GCCAACCTGC-3' (antisense). After an initial denaturation step at 95°C for 5 min the PCR was cycled between 94, 55 and 72°C (1 min each) for 35 rounds, followed by 1 round at 72°C for 10 min. The fragments were digested with XhoI and XbaI and cloned into the pSSU vector (13), yielding pSSU- alt-1 and pSSU- alt-2 . Clones were fully sequenced on both strands. DNA was extracted from pSSU- alt-1 and pSSU- alt-2 using the Qiagen Miniprep kit following the manufacturer's instructions Site directed mutagenesis An ALT- 2 mutant, in which the acidic domain of the protein (amino acids 24–49) were deleted, was generated using the Exite PCR-based site-directed mutagenesis kit (Stratagene, USA) following the manufacturer's instructions. The pSSU- alt-2 construct (see above) was used as the template in a PCR reaction containing two oligonucleotides: 5'-TGATTCTGATACACACGGGAGTGT-3' (antisense, primer phosphorylated) and 5'-TATGTAACCAAAGGGAATTTGTT-3' (sense). The cycling parameters were as follows: 1 cycle of 1 min at 94°C, 4 min at 53°C and 2 min at 72°C; followed by 10 cycles of 1 min at 94°C, 2 min at 55°C (adding 10 s after each cycle) and 1 min at 72°C; and a final cycle of 5 min at 72°C. After the PCR the nonmutated parental plasmids were digested with Dpn restriction enzyme. The undigested linear DNA was then polished with Pfu DNA polymerase and ligated at 37°C for 1 h with T4 DNA ligase. The ligated DNA was then transformed into E. coli , yielding a pSSU-ADD (acidic domain deleted) construct. The insert was sequenced to verify that the intended mutation was correctly constructed. Transfection of Leishmania Logarithmic phase promastigotes (4 × 10 7 ) were electroporated with 10 μg of PmeI linearized fragments of either pSSU- alt-1 , pSSU- alt-2 or pSSU- add . Clones were selected on 24-well plates in complete SDM supplemented with 20 μg/ml of puromycin and further propagated in a culture volume of 10 ml. Immunofluorescence Promastigotes were washed in PBS, fixed in 2% paraformaldehyde for 30 min at room temperature, and quenched in NH 4 Cl for 10 min. Wild-type parasites were incubated with dilution buffer (0.1% saponin, 2% goat serum) in the presence of anti- L . mexicana rabbit serum or anti-ALT mouse serum (from C57BL/6 mice) for 45 min. Following 3 washes with wash buffer (0.1% saponin, PBS) the parasites were incubated with either an anti-rabbit-FITC or an anti-mouse-FITC secondary antibody (DAKO, Denmark) for 45 min. They were then washed 3 times before mounting with Cityfluor (Cityfluor Ltd, UK) for microscopy. Flow cytometry Promastigotes were washed in PBS, resuspended at 1 × 10 6 /ml and fixed without permeabilization in 2% paraformaldehyde for 15 min at room temperature. After two washes in PBS, 10% FCS (FACS wash), the parasites were stained with anti-ALT-1 antibody or normal mouse serum diluted in FACS wash. This was followed by incubation with a FITC anti-mouse secondary antibody (DAKO, Denmark). Flow cytometric analysis was performed using a FACScan (Becton Dickinson) and analysed using CellQuest 3.1 software. Preparation of bone marrow-derived macrophages (BMM) BMM were obtained from femurs and tibias of 6- to 8-week old CBA mice by flushing the bones with DMEM (Gibco, BLR) containing 10% hiFCS/1% penicillin-streptomycin/1% L-glutamine. Cells were centrifuged and plated out in non-tissue culture Petri dishes at a density of 5 × 10 5 /ml in complete DMEM, supplemented with 20% (v/v) L929 cell-conditioned medium as a source of M-CSF and 20% hiFCS. After 6 days at 37°C, the cells were detached by incubation in PBS containing 3 mM EDTA and 10 mM glucose, plated and cultured in small non-tissue culture Petri dishes at 2.5 × 10 5 /ml in complete DMEM for 24 h at 37°C. Infection of BMM with L. mexicana Day 7 BMM were infected for 24 h or 7 days at 34°C with wild-type or alt -transfected amastigotes at a ratio of 10 parasites per macrophage. After the period of infection macrophages were harvested as described above, washed with PBS and fixed and permeabilized using a "Fix & Perm kit" (Pharmigen); 1 × 10 5 cells were centrifuged on to glass slides with a Cytospin. Intracellular Parasites were detected and counted by immunofluorescence as described above. Between 100 and 200 macrophages were counted for each time-point. Infections in vivo For L. mexicana in vivo infections, groups of 8 female C57BL/6 mice were injected subcutaneously in the footpad with 3 × 10 6 stationary-phase wild-type L. mexicana , alt-1 transfectants and alt-2 transfectants. Lesion size was measured weekly during the course of infection with a dial micrometer and expressed as the difference in size between the infected footpad and the contralateral uninfected footpad. For B. malayi in vivo infections, groups of 5 female C57BL/6 mice were injected intraperitoneally with 200 infective larvae recovered from crushed Aedes aegypti mosquitoes. After the experimental period the mice were euthanized by terminal anaesthetic, and peritoneal cells (PEC) were harvested by thorough washing of the peritoneal cavity with 15 ml of ice-cold RPMI supplemented with 10% FCS. The harvested PEC were plated in 24-well culture plates at 2 × 10 6 cells/well. Following 3 h at 37°C to allow cells to adhere, both the non-adherent and the adherent macrophage-enriched cell populations were harvested. Parasite quantification The number of parasites in the footpad was estimated by limiting dilution assay. Infected footpads were harvested in cold PBS after removal of the skin. Footpad tissue was dispersed through a cell strainer and resuspended in PBS-1% penicillin/streptomycin. After centrifugation the pellet was resuspended in complete SDM. The cell suspension was then serially diluted in 10-fold steps, in quadruplicate, in 96-well plates. The plates were incubated for 4 days at 27°C; the wells were then observed for parasite growth. Measurement of nitrite production J774 macrophages were plated out on 96-well plates (1 × 10 5 /well) and incubated at 37°C for 24 h. The medium was then removed, the cells were washed twice, and L. mexicana promastigotes were added for 4 h at a ratio of 10 parasites per well. IFN-γ (40 U/ml) and LPS (10 ng/ml) were then added, with or without the arginine analogue N -monomethyl-D-arginine (D-NMMA), 1 mM. Nitrite accumulation in medium over the subsequent 24–48 h was used as an indicator of NO production and was assayed by the Griess reaction in which 100 μl of Griess reagent [ 55 ] was added to 100 μl of each supernatant in triplicate wells in a 96-well plate. Plates were read at 490 nm against reference wavelength 620 nm using an ELISA plate reader. NaNO 2 was used to make a standard curve for each plate reading. Leishmanicidal assay BMM (5 × 10 4 ) were plated out on glass coverslips in 24-well plates, allowed to adhere for 24 h, and stimulated with 100 ng/ml LPS and the indicated concentrations of IFN-γ. Cells were incubated for 6 h at 37°C before adding stationary phase promastigotes of wild-type L. mexicana or transfected alt-1 and alt-2 parasites at a ratio of 10 parasites per cell. After 72 h at 37°C the cells were stained with Giemsa. The percentage of macrophages infected with parasites was determined by counting 4 samples of 100 cells. RNA isolation Total RNA was isolated from non-infected and infected BMM as well as adherent and non-adherent cell populations using TRIzol Reagent (Invitrogen, Life Technologies) according to the manufacturer's instructions. The RNA was subjected to DNase treatment (Ambion, INC) to eliminate genomic contamination, according to the manufacturer's instructions Gene array analysis Gene expression was analysed using the Mouse Cytokine Expression Array (R&D systems). The mouse cytokine-specific primers were first annealed to the total RNA, which was then reverse transcribed in the presence of SuperScript II (Invitrogen, Life Technologies) and [α- 33 P]dCTP. The radiolabelled cDNA probes generated from non-infected and infected cells were hybridized to identical membranes containing the mouse cDNA arrays. Following hybridization, high stringency washes were performed and the membranes were subjected to autoradiography. Quantification was carried out using a PhosphorImager and data analyzed with ImageQuant v1.2. Real Time RT-PCR analysis Total RNA was extracted in Trizol, as described above, and single-stranded cDNA was synthesized using MMLV reverse transcriptase (Stratagene). Relative quantification of the expression of the genes of interest was measured by real-time PCR using the LightCycler (Roche Molecular Biochemicals). PCR amplifications were performed in 10 μl volumes containing 1 μl cDNA, 2.5 mM MgCl2, 3 μmM primers and the LightCycler-DNA SYBR Green I mix (Qiagen). The reaction was performed in the following conditions: 15 min activation step at 95°C for one cycle, 15 s denaturation at 95°C, 20 s annealing of primers at 50°C and 15 s elongation at 72°C, for 50 cycles. The fluorescent DNA binding dye SYBR Green was monitored after each cycle at 80°C. Five serial 1:2 dilutions of alt-2 infected macrophages cDNA were used to produce a standard curve in each reaction. The abundances of GATA-3 and SOCS-1 were expressed as ratios of amplified product to the control, mouse S29 ribosomal protein. Primers for RT-PCR analysis were as follows: GATA-3: 5'-CTA CGG TGC AGA GGT ATC C-3' and 5'-GAT GGA CGT CTT GGA GAA GG-3'; SOCS-1: 5'-ACC TTC TTG GTG CGC GAC AGT CGC CAA-3' and 5'-GGA ACT CAG GTA GTC ACG GAG TAC-3'; and S29 ribosomal protein: 5'-ATG GGT CAC CAG CAG CTC TAC-3' and 5'-GTC CAA CTT AAT GAA GCC TAT-3'. Abbreviations ALT, abundant larval transcript protein; alt , abundant larval transcript gene or mRNA; BMM, bone marrow-derived macrophages; FACS, fluorescence-activated cell sorter; L3, third-stage infective larva; NO, nitric oxide. Authors' contributions NG-E designed and led the experimental work, including molecular constructs, cell transfection, immunological assays, array and RT-PCR analyses. She also drafted the manuscript. CB and LP-L made constructs in pSSU, maintained Leishmania cultures, participated in electroporation procedures, macrophage infection, in vivo infection and dilution assays. TA designed the transfection system and devised the Leishmania transfection methodology. TA, CCB and RMM jointly conceived the transfection strategy for Brugia genes, critically assessed progress, and made revisions to the draft manuscript. The overall study was co-ordinated by RMM who also completed the draft of the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555940.xml
519021	Recombinant antigen-based antibody assays for the diagnosis and surveillance of lymphatic filariasis – a multicenter trial	The development of antifilarial antibody responses is a characteristic feature of infection with filarial parasites. It should be possible to exploit this fact to develop tools to monitor the progress of the global program to eliminate lymphatic filariasis (LF); however, assays based on parasite extracts suffer from a number of limitations, including the paucity of parasite material, the difficulty of assay standardization and problems with assay specificity. In principle, assays based on recombinant filarial antigens should address these limitations and provide useful tools for diagnosis and surveillance of LF. The present multicenter study was designed to compare the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. Coded serum specimens were distributed to five participating laboratories where assays for each antigen were conducted in parallel. Assays based on Bm14, WbSXP, or BmR1 demonstrated good sensitivity (>90%) for field use and none of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections. Limitations of the assays are discussed. Well-designed field studies are now needed to assess sampling methodology and the application of antibody testing to the monitoring and surveillance of LF elimination programs.	Background The exponential growth of the lymphatic filariasis elimination program has highlighted the need for tools that can be used to monitor progress toward programmatic endpoints (e.g. when to stop mass treatment) as well as to conduct surveillance to detect any potential resumption of transmission. Measurement of microfilaremia, the recognized gold standard for demonstrating the impact of community-wide interventions, is not an optimal monitoring or surveillance tool because of the requirement for nocturnal blood collection in much of the world and because it is a relatively insensitive test for infection [ 1 ]. For Wuchereria bancrofti , assessment of antigenemia offers the convenience of daytime testing and greater sensitivity than testing for microfilaremia; however, both microfilaremia and antigenemia develop from months to years after exposure, reducing their utility for detection of low levels of infection or recrudescence of transmission [ 2 - 6 ]. Entomologic methods can be used to monitor filarial infection in mosquitoes and to provide point estimates of transmission intensity; however, as infection levels decline, it may become more difficult to collect sufficient numbers of mosquitoes to demonstrate with confidence that infection is absent [ 7 ]. By providing a cumulative measure of exposure to filarial infection, antibody assays may circumvent many of the limitations of methods based on direct detection of the parasite, its antigens or its DNA. Antibody detection has served as the basis for diagnostic assays for filariasis for many decades. The best of these assays are sensitive for infection but are not specific, both because they cannot distinguish current infection from past infection or exposure to the parasite and because there is some degree of cross-reactivity with other helminth infections. On the positive side, prior studies have shown that virtually all residents of filariasis-endemic areas mount antifilarial antibody responses within the first few years of life. Thus, prevalence rates of antifilarial antibodies in children may be a useful index for assessing changes in transmission of the infection [ 8 , 9 ]. Antibody assays based on crude filarial extracts are limited by cross-reactions with other nematode antigens [ 10 ]. Recombinant filarial antigens should, in principle, be more useful as the basis of diagnostic or exposure assays because of their greater specificity. As a first step in the development and validation of such assays, we conducted a multicenter evaluation of antibody-based diagnostic assays using 3 recombinant antigens, Bm14, WbSXP and BmR1. Bm14 and WbSXP belong to a family of related genes that encode proteins that are strong immunogens in many parasitic nematode infections [ 11 ]. SXP and Bm14 were originally isolated from cDNA libraries based on their strong recognition by antibodies from microfilaremic persons, and both have been developed as candidates for diagnostic assays [ 12 , 13 ]. Assays based on detection of IgG4 antibodies to Bm14 have sensitivities of 85–90% when serum specimens from microfilaremic persons are tested [ 14 , 15 ]. This antigen is reported to be equally sensitive for sera from patients infected with Brugia malayi or Wuchereria bancrofti . Comparable results have been reported with assays for SXP [ 11 , 16 , 17 ]. BmR1 encodes a secreted antigen selected from a B. malayi cDNA library by antibody screening [ 18 , 19 ]. ELISA and dipstick formats of BmR1 assays have been reported to have a sensitivity of >90% when serum specimens from persons with B. malayi or B. timori microfilaremia were tested [ 18 - 22 ]. The present study was designed to compare objectively the performance of antibody assays for filariasis based on recombinant antigens Bm14, WbSXP, and BmR1. We report here the results of this multicenter evaluation. Materials and Methods Serum Specimens Serum specimens from patients of known infection status (see Table 1 ) were sent to the U.S. Centers for Disease Control and Prevention (CDC). For persons with filariasis, infection was diagnosed by detection of microfilariae. Each specimen was assigned a code number and aliquotted into 5 tubes (100 – 200 μl per tube). A panel of coded serum samples was sent to each of the five participating laboratories along with antigens and assay kits for testing. Results were sent back to CDC for data analysis. Table 1 Sensitivity and Specificity of Antibody Assays 1 Infection Bm14 ELISA Positive/Tested (%) SXP Cassette Positive/Tested (%) BmR1 ELISA Positive/Tested (%) BmR1 Dipstick Positive/Tested (%) W. bancrofti (n = 35) 2 32/35 (91%) 30/33 (2 NC) (91%) 14/31 (4 NC) (45%) 17/30 (5NC) (56.7%) B. malayi (n = 28) 3 27/28 (96%) 7/18 (10 NC) (39%) 28/28 (100%) 27/27 (1NC) (100%) O. volvulus (n = 20) 4 11/16 (4 NC) (69%) 9/15 (5 NC) (60%) 0/20 (0%) 1/20 (5%) Loa (n = 10) 5 7/9 (1 NC) (78%) 3/7 (3 NC) (43%) 0/10 (0%) 0/9 (1 NC) (0%) Other (incl. S trongyloides, Echinococcus ; n = 20) 6 0/19 (1 NC) (0%) 0/19 (1 NC) (0%) 0/20 (0%) 0/20 (0%) 1 Specimens were collected from persons with documented infections with the listed parasites; for patients with filarial infections, microfilariae were detected. Abbreviations: NC, no consensus; specimens with a no consensus result were not included in the denominators for calculations. 2 Geographic source of specimens provided in Table 2. 3 Geographic source of specimens provided in Table 2. 4 Ten specimens were from Guatemala and 10 were from Ecuador. 5 Specimens were collected from patients in Benin. 6 Echinococcus specimens were collected in Kenya; Strongyloides specimens were from several settings where lymphatic filariasis was not endemic. Assay formats The Bm14 and BmR1 tests included in the evaluation are based on the detection of antifilarial IgG4 antibodies. The Bm14 assays were performed according to procedures described by Weil et al. [ 15 ]. Three different assay formats were used to test for BmR1: an ELISA [ 18 ], a dipstick [ 20 ] and cassette. All were produced by Malaysian Bio-Diagnostics, Research, Sdn, Bhd. A rapid cassette test for IgG antibody to WbSXP was produced by Span Diagnostics, Ltd (Sachin, India). All participating labs followed assay instructions provided by the assay or kit supplier. Analysis of Results Results of ELISA assays were determined using cut offs defined by the assay developer. For qualitative tests, each laboratory determined whether the appropriate band or spot was visible. To collate results for a given assay with a specific serum sample, a consensus result (either positive or negative) was defined on the basis of agreement among at least 4 of 5 labs. If only 3 of 5 labs obtained the same result or if 3 did and one of the two remaining laboratories did not obtain an interpretable result, this was considered to represent a lack of consensus (recorded as 'NC' or no consensus in Tables 1 and 2 ). Only two labs used the BmR1 cassette to test the specimens. Achieving consensus required two identical results for this test. Inter-laboratory agreement was assessed with Kappa coefficients, a measure of pair-wise agreement among observers making categorical judgments. For Bm14 and BmR1 ELISA, categorical assignments of positive or negative results were based on criteria established by the test developers. Table 2 Regional Differences in Antigen Recognition Infection Location (serum source) Bm14 ELISA SXP Cassette BmR1 ELISA W. bancrofti Cook Is. 10/10 9/9 (1 NC) 4/8 (2 NC) PNG 9/10 9/9 (1 NC) 6/10 India 9/10 9/10 4/8 (2 NC) Kenya 2/3 1/3 0/3 Haiti 2/2 2/2 0/2 B. malayi Indonesia 10/10 0/5 (5 NC) 10/10 India 7/7 4/6 (1NC) 7/7 Malaysia 10/11 3/7 (4 NC) 11/11 Results The Bm14 ELISA displayed comparable sensitivity for both W. bancrofti (91%) and Brugia (96%) infections, while the other two tests performed better with specimens from the homologous infections (Table 1 ). For example, the BmR1 ELISA was positive for 100% of the samples from Brugia patients, but displayed only modest sensitivity (45%) in terms of its performance with W. bancrofti samples. Results were comparable for both the BmR1 dipstick and cassette (data not shown). Similarly, the WbSXP assay was positive for 30 of 33 (91%) serum specimens from W. bancrofti patients, but only 39% of Brugia cases. BmR1 assays were remarkably specific for Wuchereria and Brugia infections, and there was little reactivity with specimens from persons with O. volvulus , Loa loa or other helminths (e.g. Strongyloides ) . The Bm14 assay, and to a lesser extent, the WbSXP assay, appeared to function as a 'pan-filaria' assay, showing reactivity with the specimens from persons with W. bancrofti , B. malayi , L. loa and O. volvulus . None of the assays demonstrated reactivity with specimens from persons with non-filarial helminth infections (Table 1 ) or with hyper-IgE syndrome (data not shown). When the geographic source of the serum specimens was considered, additional heterogeneity in responsiveness was noted. For example, although only a limited number of specimens were available for testing, 4 of 6 serum specimens from persons from India infected with B. malayi were positive using the WbSXP cassette; however, none of those from Indonesia were positive with this assay (Table 2 ). Inter-laboratory categorical agreement for the ELISA assays was quite good (Table 3 ). Rapid format tests, though convenient, often presented problems of interpretation, independent of the test. Some labs reported the presence of weak bands or dots with control sera. This resulted in a significant number of 'no consensus' results (Table 1 ) as well as the lower kappa scores associated with the rapid tests (Table 3 ). Table 3 Inter-lab Agreement for the Different Diagnostic Tests Assay Range of Kappa statistics Mean Bm14 ELISA 0.690 – 1.00 0.88 SXP Cassette 0.612 – 0.912 0.73 BmR1 ELISA 0.878 – 0.982 0.93 BmR1 Dipstick 0.817 – 0.965 0.87 BmR1 Cassette 0.546 – 1.00 0.80 Kappa statistics were derived from 10 pair-wise inter-lab comparisons. Discussion Assays based on Bm14, WbSXP, or BmR1 demonstrate adequate sensitivity for field use. The Bm14 assay appeared to function as a 'pan-filaria' assay, demonstrating antibody reactivity in the sera from patients with W. bancrofti , B. malayi , L. loa and O. volvulus . Although this cross reactivity makes the Bm14 assays useful for monitoring either bancroftian or brugian filariasis, cross reactivity with O. volvulus and L. loa may limit its utility in some areas of sub-Saharan Africa. The BmR1 assays were sensitive for B. malayi infection but relatively insensitive for W. bancrofti infection. They showed excellent specificity for Brugia and Wuchereria , with little reactivity with sera from persons infected with other parasites, including L. loa and O. volvulus . These results suggest that it may be useful to study the W. bancrofti homologue of BmR1 to determine if it is as specific for W. bancrofti as BmR1 is for Brugia . Unfortunately, recent work suggests that this is not the case [ 23 ]. For mapping the distribution of lymphatic filariasis, rapid antibody tests may provide acceptable sensitivity, depending on the geographic area where the mapping is to be done, but the potential for problems with specificity (both in distinguishing past exposure from present infection as well as differentiating filarial from non-filarial infection) still remains. For mapping W. bancrofti , there is minimal value in using antibody tests instead of the antigen tests that are currently used, but because there is no antigen test for Brugia , antibody tests might be an alternative for mapping the distribution of these infections. In Brugia -endemic areas, it will be important to demonstrate, however, the relationship between prevalence rates for microfilaremia and antibodies to validate the assay as a useful tool for programmatic decisions. At this point, it is not clear what antibody prevalence should be considered an indication to initiate mass treatment; no attempt was made in this study to distinguish between antibody responses associated with active infection and those triggered by exposure [ 24 , 25 ]. Antibody assays almost certainly will find other uses in the context of lymphatic filariasis (LF) elimination programs. For example, antifilarial antibody tests may be sensitive markers of transmission intensity or provide evidence of ongoing exposure to filarial infection long before the development of antigenemia or microfilaremia. Primates develop antibody responses to Bm14 within 4–8 weeks following B. malayi infection [ 26 ]. In a longitudinal study in Egypt, microfilaria-negative persons who were positive for Bm14 antibody at baseline were more likely to be microfilaria-positive after one year than were Bm14-negative persons [ 15 ]. Less is known about the kinetics of antibody responses to BmR1. Since antibody responses provide an early indicator of infection, assays for antifilarial antibodies should be useful for surveillance following initiation of LF elimination programs. As LF programs reach their planned end point (5 or more years of > 80% drug coverage in targeted populations), it will be necessary to determine whether or not transmission has been interrupted and whether mass drug administration can be stopped. Parasitologic testing, whether for microfilaremia or antigenemia, will require testing of thousands of persons to demonstrate that infection levels are below 0.1%, the level established by the Global Program as the end point for defining the elimination of LF. Since antibody responses develop in the absence of demonstrable infection, detecting incident antibody responses should provide a more sensitive measure of transmission than microfilaria or antigen detection. Children born following the cessation of transmission should be antibody-negative, while older children and adults may have evidence of residual antibody reactivity [ 8 , 9 , 27 ]. A testing strategy based on screening of children could be exploited for ongoing surveillance in the aftermath of LF programs and may not require screening of as many children as called for by current testing guidelines. The absence of antibody in appropriately chosen populations would strongly suggest that transmission has been interrupted. Additional studies are needed to test the value of antibody testing as a tool for certifying the elimination of filariasis transmission. Operational use of antibody assays requires far more practical experience with the assays than we now have. Of greatest concern is the specificity of the tests employed. For example, ELISA tests often use a statistical approach to establish cutoff values for positive results. A test that is 99% specific will predictably have some false positive results if large numbers of samples are tested. Further work will be needed to establish rates of antibody positivity that exceed the number that are likely to occur by chance. In addition, it will be necessary to develop and validate algorithms for confirming the presence of infection or ongoing transmission in situations where low antibody prevalence rates are detected. Despite these caveats, we believe that antibody tests based on antigens like Bm14, BmR1, and Wb-SXP will prove to be useful tools that can be used to facilitate decision making by program managers in the context of filariasis elimination programs. Competing Interests GW, RN, and PK have relationships with companies interested in developing commercial applications of the Bm14, BmR1 and WbSXP assays, respectively. Authors' Contributions GW, RN, PK, and VBL were responsible for the initial development of the assays. All of the authors participated in the planning of this multicenter study. DG was responsible for coordinating specimen shipment and database management. Participating labs included PL and DG from CDC, RN from the Universiti Sains Malaysia, PK and VBL from Anna Center for Biotechnology, CS from NIH and GW from Washington University School of Medicine. PL and EO were responsible for coordinating the study. PL wrote the first draft of the manuscript, but all of the authors participated in the editing of subsequent versions.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC519021.xml
543449	Coccidioides posadasii infection alters the expression of pulmonary surfactant proteins (SP)-A and SP-D	Background Coccidioidomycosis or Valley Fever is caused by Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known. Methods In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays. Results We found that in lavage fluid samples of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % (SEM 3.5, p < 0.001), 38 % (SEM 5.8, p < 0.001) and 4 % (SEM 1.3, p < 0.001) of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens. Conclusion Our results suggest that the C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.	Background Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus Coccidioides immitis or posadasii [ 1 ]. It is endemic in the southwest regions of US, Northern Mexico and parts of Central America [ 2 ]. C. posadasii or C. immitis , are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [ 3 ]. The primary infection is acquired by inhalation of air-borne, mycelial phase arthroconidium that converts into endosporulating spherule in the lung. Clinical manifestations of the disease range from pulmonary infection to a more severe fatal mycosis involving extra-pulmonary tissues in 1–10% of the infected people [ 1 - 4 ]. Previous studies suggest that Th1 cell mediated immunity protects individuals against Coccidioides [ 5 , 6 ]. However, information is lacking regarding the pulmonary innate immune components that may play a critical role in regulation of immune responses against Coccidioides . At alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids (88–90%) and proteins (10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function [ 7 ]. Among four different surfactant proteins, surfactant proteins-A (SP-A) and D (SP-D) are members of the "Collectin" family [ 8 ]. In the past, several studies have suggested that both SP-A and SP-D play an important role in innate host defense against various viral, fungal and bacterial pathogens [ 9 , 10 ]. More evidence for the pulmonary collectins' role in host defense comes from studies on SP-A- deficient mice that are susceptible to intra-tracheal Group B Streptococci [ 11 ], Pseudomonas aeruginosa [ 12 ], and Respiratory Syncytial Virus [ 13 ]. Also, intranasally administered SP-D has been found to reduce replication of Respiratory Syncytial Virus in the lungs of infected mice [ 14 ]. Both SP-A and SP-D, have been classified as secretory pattern-recognition receptors that can bind to a variety of pathogens and help in clearance [ 9 , 15 ]. Recent evidences indicate that in addition to their pathogen recognition property, SP-A and SP-D also play an important role in stimulating immuno-regulatory pathways [ 15 ]. However, the collectins' role in coccidioidomycosis is not known. This study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of C. posadasii and C. posadasii protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens. Methods Mice BALB/c and C57BL6 mice (6 weeks old female) from Jackson Laboratory (Bar Harbour, ME) were used in this study. Both mouse strains are susceptible to C. posadasii infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with C. posadasii . And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee. Coccidioides posadasii C. immitis (now posadasii ) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [ 1 ]. The arthroconidia were harvested in endotoxin-free 0.15 M saline (Baxter Health Care Products, Deerfield, IL) from 6–8 weeks old mycelial phase cultures grown on GYE plates. The arthroconidia suspension was passed over a sterile cotton column to remove hyphal elements and arthroconidia were enumerated by hemacytometer counts. The viable cfu counts were confirmed pre- and post-infection by plate cultures on GYE agar. All the experiments with C. posadasii were carried out in Biosafety Level-3 facility at UTHSCSA. Intranasal Challenge with C. posadasii Arthroconidia Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia (n = 30, fresh harvest of C. posadasii arthroconidia) suspended in endotoxin-free 0.15 M NaCl (Baxter Health Care Corp, Deerfield, IL) using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl. Preparation of Coccidioide -FKS vaccine and Immunization of Mice C. posadasii (strain Silveira )arthroconidia were used to prepare FKS as described earlier [ 16 ]. Briefly, arthroconidia were inoculated in modified Converse medium containing Tamol and cultured while shaking at 180 rpm at 40°C in 20 % CO 2 incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30 C. posadasii arthroconidia, 15 days after last immunization, as described above. Fungal Burden Assay Mice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load. The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates (BD Biosciences, Franklin Lakes, NJ) and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight. Collection and Processing of BALF At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution (Baxter Health Care Corp, Deerfield, IL) three times, via an angiocatheter (BD Biosciences, San Diego, CA) placed in the trachea. The volume of the input solution was kept constant (3 ml total) and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters (Nalge Nunc International, Rochester, NY) and stored at -80°C for further analysis. Total Protein and Lipid Analysis The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit (Pierce, Rockford, IL) against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine (DPPC, Avanti Polar Lipids, Alabaster, AL) standard solutions [ 17 , 18 ]. Briefly, the lipid extract of BALF specimens and DPPC standard solutions was completely dried under compressed nitrogen gas. The dried lipids were dissolved in chloroform and mixed with 1 ml of 2.7% ferric chloride and 3% ammonium thiocyanate in glass tubes. The mixture was vortex mixed for 1 min and centrifuged at 200 rpm for 5 min. The bottom red lower layer of phospholipids and ammonium ferro-thiocyanate complex was collected and absorbance was read at 488 nm. Western Blotting The BALF and lung tissue homogenate samples (total protein 10–50 μg) were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes (Schleicher & Schuell, Keene, NH) overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit (obtained from Dr. Richard J. King, UTHSCSA, San Antonio, TX) or anti-mouse SP-D antibody (kindly provided by Dr. Jo Rae Wright, Duke University Medical Center, Durham, NC). After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat (Sigma Chemical Co, St. Louis, MO). The immunoreactive bands were observed by alkaline phosphatase conjugate system (Biorad, Hercules, CA). Purified human SP-A (kindly provided by Dr. Richard J. King, UTHSCSA, San Antonio, TX) and recombinant human SP-D (kindly provided by Dr. Erika C. Crouch, Washington University in St. Louis, St. Louis, MO) were run with the samples. The Coccidioidal antigens: lysates and filtrates of Coccidioidin (CDN), prepared as a toluene-induced lysate of young C. posadasii mycelia (obtained from Dr. Rebecca A. Cox, UTHSCSA, San Antonio, TX, [ 19 ]) were also run to check the cross-reactivities of anti-SP-A and SP-D antibodies to fungal antigens. Enzyme-Linked Immunosorbent Assay (ELISA) for SP-A and SP-D The concentrations of SP-A and SP-D were measured in BALF samples as described earlier [ 20 ]. The antibodies against SP-A and SP-D reacted with 34 kDa (SP-A) and 43 kDa (SP-D) immunroreactive bands in BALF and lung tissue homogenates (Fig 1 ). For measuring the lavage concentrations of SP-A and SP-D, the indirect ELISA procedure was used [ 20 ]. Briefly, the wells of Immulon 4 strips (Dynatech, Chantilly, VA) were coated overnight with purified human SP-A or recombinant human SP-D antigens (standards) and diluted BALF (three different dilutions) in 0.1 M NaHCO 3 , pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 50 μl of 2 N H 2 SO 4 and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml. Figure 1 Western blot for (A) SP-A and (B) SP-D proteins in mouse lung. Lanes (a, b): 2.5 μg total lavage fluid protein (c, d): 100 μg of total lung tissue homogenate protein from two healthy, non-infected BALB/c mice, and (e): 10 ng purified human SP-A or recombinant SP-D protein. Binding of SP-A and SP-D to Coccidioidal Antigens A microtiter well based method [ 21 ] was used to study the SP-A and SP-D interactions with Coccidioidal antigens (CDN-lysate and CDN-filtrate). Briefly, microtiter wells (Immulon 4; Dynatech, Chantilly, VA) were coated with 50 μl of CDN-lysate (10 μg/ml diluted in 0.1 M NaHCO 3 buffer, pH 9.6) or CDN-filtrate (10 μg/ml diluted in 0.1 M NaHCO 3 buffer, pH 9.6) or BSA (10 μg/ml diluted in 0.1 M NaHCO 3 buffer, pH 9.6) at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl 2 and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody (Sigma, St. Louis, MO) was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent (Sigma Chemical Co. St. Louis, MO). The reaction was stopped by adding 2 N H 2 SO 4 and read at 405 nm spectrophotometrically. The coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier [ 22 ]. The alkaline phosphatase-conjugated rat anti-mouse IgG antibody (Zymed, San Francisco, CA) served as secondary detection antibody. Statistics Statistical analyses of the data (t-test or ANOVA) were done using Prism Software (Graphpad Software, San Diego, CA). The p value <0.05 was considered significant. Results Pathological status All of the C. posadasii infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table 1 ). Abscess like lesions were quite evident on gross examination of the lung. The total wet lung weights were increased in C. posadasii infected mice. Table 1 Body weights (g) of C. posadasii infected and non-infected BALB/c mice (n = 10 of each type). Values are shown as Mean (SEM) of one representative experiment of two independent experiments. Days post challenge → Mice ↓ Day 0 Day 10 Non-infected 18.36 (0.29) 19.46 (0.23) ** C. posadasii infected 17.77 (0.39) 16.35 (0.53) , # * p < 0.01 as compared to non-infected control mice at day 0. p < 0.05, # p < 0.0001 as compared to C. posadasii infected mice at day 0 and non-infected mice at day 10, respectively (t-test). The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection (p < 0.05, Table 2 ). In contrast, the phospholipid concentration was reduced in BALF samples from C. posadasii infected mice (58 μg) when compared to non-infected saline injected controls (165 μg, p < 0.05). Table 2 Total protein and phospholipid contents in BALF samples from non-infected and C. posadasii infected BALB/c mice (n = 5 of each type). Values are shown as Mean (SEM) from one represenative experiment of two independent experiments. Mice Total protein (μg) Total phospholipid (μg) Non-infected 326.0 (26.9) 165.5 (10.8) C. posadasii infected 788.7 (248.6) 58.7 (15.7)* * p < 0.05 as compared to non-infected control mice (t-test). The amounts of SP-A, SP-D and phospholipid are reduced in BALF samples from C. posadasii infected mice The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples (Fig 1 ). The upper bands of approximately 68 kDa and 85 kDa size (dimer of SP-A and SP-D protein) were also visible in lanes e of Figure 1A and 1B due to incomplete reduction, respectively. There were no differences in the detectable isoforms of SP-A or SP-D in the BALF samples from infected mice as compared to non-infected control mice (data not shown). The antibodies did not cross-react with Coccidioidal antigens in CDN lysate or CDN filtrate (Fig 2 ). The amounts of SP-A and SP-D were significantly reduced in BALF samples from C. posadasii infected BALB/c mice when compared to saline injected, non-infected control mice after 10 days of intranasal challenge (p < 0.001) (Fig 3A ). No significant changes were observed in the amounts of SP-A and SP-D in BALF samples collected from BALB/c mice, 5 days after intranasal challenge with C. posadasii (data not shown). Figure 2 Western blot of CDN-lysate and CDN-filtrate for crossreactivity with (A) anti-human SP-A and (b) anti-mouse SP-D antibodies. Lanes (a): 20 μg, (b): 10 μg and (c): 1 μg CDN-filtrate protein. Lanes (d): 20 μg, (e): 10 μg and (f): 1 μg CDN-lysate protein and last lane: 10 ng purified human SP-A protein or recombinant SP-D protein. Figure 3 SP-A and SP-D levels in BALF samples from (A) C. posadasii infected BALB/c mice (ng/μg protein, % of non-infected control mice, n = 5 of each type) (B) FKS immunized, C. posadasii infected C57BL6 mice (protected mice) (ng/μg protein, % of FKS immunized non-infected mice, n = 5 of each type). The data are shown from one representative experiment of two independent experiments. * p < 0.001 (ANOVA) The fungal colonies of C. posadasii were recovered from both lung and spleens of infected animals indicating the presence of active infection (Fig 4 ). Recovery of fungus in the spleen provides evidence of dissemination of C. posadasii to extra-pulmonary organs. Figure 4 Fungal load in the lung and spleen tissues of C. posadasii infected BALB/c mice (n = 5). The numbers of fungal colonies (CFU) were normalized with organ weight (g). Lavage SP-A and SP-D levels are unaltered in C. posadasii protected mice No significant changes were seen in SP-A or SP-D levels in BALF samples of protected (FKS immunized, C. posadasii arthroconidia infected) C57BL6 mice when compared to FKS immunized, non-infected mice (Fig 3B ). Also, there was no significant change in total protein content in BALF samples of FKS immunized, C. posadasii arthroconidia infected mice (890.9 μg) versus FKS immunized, non-infected mice (538.4 μg). SP-A and SP-D bind to Coccidioidal antigens We further examined the binding of SP-A and SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate) coated onto microtiter wells (Fig 5 ). Both SP-A and SP-D bound to coccidioidal antigens, but not to BSA in a concentration-dependent manner (Fig 5 ). Binding of SP-A to CDN-lysate and CDN-filtrate antigens was saturable, and maximum SP-A binding was reached between 2.5–5 μg/ml and 5–10 μg/ml, respectively (Fig 5A ). Similarly binding of SP-D to CDN-lysate and CDN-filtrate antigens was also saturable, and maximum SP-D binding was reached between 5–10 μg/ml (Fig 5B ). Figure 5 Binding of (A) SP-A and (B) SP-D to Coccidioidal antigens (CDN-lysate and CDN-filtrate). The binding of 1–10 μg/ml purified human SP-A or recombinant SP-D proteins was detected in CDN-lysate or CDN-filtrate or BSA coated wells (0.5 μg/well). Results are from one representative experiment of two independent experiments performed in duplicate. Values are shown as mean+SEM. In some cases, the error bars are smaller than the symbols. Discussion In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of C. posadasii infected mice, but were intact in lungs of C. posadasii protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of C. posadasii and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne C. posadasii arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against C. posadasii . Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including Pneumocystis carinii [ 23 ] Cryptococcus neoformans [ 24 ], Aspergillus fumigatus [ 25 ] and Candida albicans [ 26 ]. SP-D has been shown to bind to C. albicans and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [ 27 ]. On the other hand, surfactant proteins also induce phagocytosis, activation and killing of A. fumigatus conidia and C. neoformans by alveolar macrophages and neutrophils [ 28 , 29 ]. In support of these findings, further evidence comes from a study by Madan et al., that suggests that the introduction of recombinant SP-D improves the lung function and increases the survival rate of mice infected with A. fumigatus [ 25 ]. The decrease in amounts of SP-A and SP-D during C. posadasii infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of C. posadasii by antigen presenting cells and downstream immune regulation. Besides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in C. posadasii infected mice (Table 2 ). Earlier, Sheehan et al., [ 29 ] and Hoffman et al., [ 30 ] have reported similar findings of reduced surfactant phospholipid level in BALF samples of rats and humans infected with Pneumocystis carinii [ 29 , 30 ]. To date, however, the information is lacking concerning how surfactant phospholipids may be involved in host defense [ 31 ]. Likewise, the mechanisms underlying the decrease of BALF surfactant in murine model of Coccidioidomycosis remain to be defined. We speculate that the reduction in the collectins and phospholipids could be either due to metabolic dysfunction of pulmonary type II epithelial cells during C. posadasii infection or due to their utilization in the binding and uptake of C. posadasii by local antigen presenting cells. The metabolic pathways of pulmonary type II epithelial cells may be affected by secondary inflammatory mediators, such as TNF-α or IL-1β, secreted by inflammatory cells during C. posadasii infection. A variety of host inflammatory mediators and substances such as, cytokines (TNF-α) and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [ 32 , 33 ]. In the present study we found slightly increased levels of SP-D in C. posadasii protected mice, but not of SP-A (Fig 2 ). We speculate that the difference could be due to diverse mechanisms for regulation of SP-A and SP-D expression. Probably the cytokines and chemokines that are released as a result of FKS vaccination (protective immune response) increase the SP-D expression, but do not affect the SP-A expression. As reported earlier, the expression of SP-A and SP-D is differentially regulated during lung infection [ 34 ]. In future, more studies are warranted to understand the mechanism of the alterations in levels of surfactant phospholipids, SP-A and SP-D. At present, the treatment of C. posadasii infected patients with disseminated disease is not very effective [ 35 ]. Treatment with anti-fungal agents is most often related to relapse of the infection and side effects on the body. Earlier, the FKS based immunization has been found protective against Coccidioides infection in murine model of coccidioidomycosis, but failed in humans [ 35 ]. More pre-clinical studies on developing different vaccine strategies are underway. Since C. posadasii and C. immitis are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [ 3 , 35 ]. Conclusions In future, clearance experiments after in vivo administration of artificial or natural surfactant in C. posadasii infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by C. posadasii or other virulent respiratory pathogens. Authors' Contributions SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC543449.xml
374243	Accelerated Evolution of the ASPM Gene Controlling Brain Size Begins Prior to Human Brain Expansion	Primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by global reduction in cerebral cortical volume. The microcephalic brain has a volume comparable to that of early hominids, raising the possibility that some MCPH genes may have been evolutionary targets in the expansion of the cerebral cortex in mammals and especially primates. Mutations in ASPM , which encodes the human homologue of a fly protein essential for spindle function, are the most common known cause of MCPH. Here we have isolated large genomic clones containing the complete ASPM gene, including promoter regions and introns, from chimpanzee, gorilla, orangutan, and rhesus macaque by transformation-associated recombination cloning in yeast. We have sequenced these clones and show that whereas much of the sequence of ASPM is substantially conserved among primates, specific segments are subject to high Ka/Ks ratios (nonsynonymous/synonymous DNA changes) consistent with strong positive selection for evolutionary change. The ASPM gene sequence shows accelerated evolution in the African hominoid clade, and this precedes hominid brain expansion by several million years. Gorilla and human lineages show particularly accelerated evolution in the IQ domain of ASPM . Moreover, ASPM regions under positive selection in primates are also the most highly diverged regions between primates and nonprimate mammals. We report the first direct application of TAR cloning technology to the study of human evolution. Our data suggest that evolutionary selection of specific segments of the ASPM sequence strongly relates to differences in cerebral cortical size.	Introduction The human brain, particularly the cerebral cortex, has undergone a dramatic increase in its volume during the course of primate evolution, but the underlying molecular mechanisms that caused this expansion are not known. One approach shedding light on the molecular mechanisms of brain evolution is the analysis of the gene mutations that lead to defects in brain development. Among the best examples of such defects is the human primary microcephaly syndrome. Primary microcephaly (MCPH) is an autosomal recessive neurodevelopmental disorder in which the brain fails to achieve normal growth. The affected individuals have severe reduction in brain size; however, the gyral pattern is relatively well preserved, with no major abnormality in cortical architecture ( McCreary et al. 1996 ; Mochida and Walsh 2001 ). Moreover, there are no recognizable abnormalities in the organs other than the central nervous system. The most common cause of MCPH appears to be mutations in the ASPM gene ( Roberts et al. 2002 ). The ASPM gene encodes a 10,434-bp-long coding sequence (CDS) with 28 exons, and spans 65 kb of genomic DNA at 1q31. ASPM contains four distinguishable regions: a putative N-terminal microtubule-binding domain, a calponin-homology domain, an IQ repeat domain containing multiple IQ repeats (calmodulin-binding motifs), and a C-terminal region ( Bond et al. 2002 ). Though the exact function of the human ASPM in the brain needs to be clarified, the homologue in the fruit fly, Drosophila melanogaster, abnormal spindle (asp), is localized in the mitotic centrosome and is known to be essential for both the organization of the microtubules at the spindle poles and the formation of the central mitotic spindle during mitosis and meiosis. Mutations in asp cause dividing neuroblasts to arrest in metaphase, resulting in reduced central nervous system development ( Ripoll et al. 1985 ; do Carmo Avides et al. 2001 ; Riparbelli et al. 2001 ). In the mouse (Mus musculus) brain, the Aspm gene is expressed specifically in the sites of active neurogenesis. Expression in the embryonic brain was found to be greatest in the ventricular zone, which is the site of cerebral cortical neurogenesis ( Bond et al. 2002 ). This expression profile suggests a potential role for Aspm in regulating neurogenesis. Interspecies comparisons of ASPM orthologs have shown their overall conservation, but also a consistent correlation of greater protein size with larger brain size ( Bond et al. 2002 ). The increase in protein size across species is due mainly to the increased number of IQ repeats, suggesting that specific changes in ASPM may be critical for evolution of the central nervous system. In an attempt to reconstruct the evolutionary history of the ASPM gene, we isolated large genomic clones containing the entire ASPM gene in several nonhuman primate species. Sequence analysis of these clones revealed a high conservation in both coding and noncoding regions, and showed that evolution of the ASPM gene might have been under positive selection in hominoids. These clones could also provide important reagents for the future study of ASPM gene regulation in its native sequence context. Results Comparison of Genomic Organization of the ASPM Genes in Primates Homologues from chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) were isolated by transformation-associated recombination (TAR) cloning in yeast (Saccharomyces cerevisiae), the technique allowing direct isolation of a desirable chromosomal region or gene from a complex genome without constructing its genomic library ( Kouprina and Larionov 2003 ). The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast. Since up to 15% divergence in DNA sequences does not prevent selective gene isolation by in vivo recombination in yeast ( Noskov et al. 2003 ), for cloning purposes, a TAR vector was designed containing short human ASPM -gene-specific targeting hooks specific to the exon 1 and 3′ noncoding regions (see “Materials and Methods” ). The TAR cloning scheme for isolating the ASPM gene homologues from nonhuman primates is shown in Figure 1 . The yield of ASPM -positive clones from chimpanzee, gorilla, orangutan, and rhesus macaque was the same as that from the human DNA, suggesting that most homologous regions from nonhuman primates can be efficiently cloned by in vivo recombination in yeast using targeting hooks developed from human sequences. Figure 1 Isolation of the Syntenic Genomic Regions Containing the ASPM Gene from Human, Chimpanzee, Gorilla, Orangutan, and Rhesus Macaque by TAR Cloning The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae . For isolation, genomic DNA is transformed into yeast spheroplasts along with a TAR vector that contains targeting hooks homologous to the genomic DNA sequence. CEN corresponds to the yeast Chromosome VI centromere; HIS3 is a yeast selectable marker. Recombination between the vector and the genomic DNA fragment results in cloning of the gene/region of interest as YAC. Chromosomal regions with sizes up to 250 kb can be isolated by TAR cloning. For cloning purposes, TAR vector was designed containing a 5′ hook specific to exon 1 and a 3′ hook specific to the 3′ end of the human ASPM . Transformation experiments were carried out with freshly prepared spheroplasts for each species. To identify ASPM -containing clones, the transformants were combined into pools and examined by PCR for the presence of the unique ASPM sequences not present in the vector. The yield of ASPM -positive clones from primate species was the same as that from the human DNA (3%). Because the TAR procedure produces multiple gene isolates, six independent TAR isolates for each species were checked. The detectable size of the cloned material corresponded to that predicted if the entire ASPM gene had been cloned, i.e., all gene-positive clones contained circular YACs with approximately 65-kb DNA inserts. Alu profiles for each species were determined and found to be identical for each species, suggesting that the isolated YACs contained nonrearranged genomic segments. Finally the YACs were retrofitted into BACs, and their restriction patterns were examined by three restriction endonuclease digestions. No differences between ASPM clones for each species were found. We have compared complete gene sequences from primate species with a 65-kb, full-size human ASPM gene. All the analyzed genes are organized into 28 exons encoding a 3,470–3,479-amino-acid-long protein. ASPM genes start with an approximately 800-bp-long CpG island, that harbors promoter sequences, 5′ untranslated regions, and the first exon ( Figure 2 ). ASPM sequences share a high degree of conservation ( Figure 2 H), and pairwise DNA identity ranges from 94.5% for macaque and gorilla to 99.3% for the human–chimpanzee comparison ( Table 1 ). Multiple alignment of the genes revealed a low proportion of indels. Only ten insertions/deletions equal to or longer than 50 bp have been found, all of them located within introns ( Figure 2 B). Seven detected insertions were mainly associated with repetitive DNA: two (AT) n microsatellite expansions, three Alu insertions, including retroposition of AluYi9 in the orangutan–gorilla–chimpanzee–human clade, and retroposition of a new macaque-specific AluY subfamily similar to human AluYd2 . Analysis of eight different macaque individuals showed that this particular insertion is polymorphic in the macaque population (data not shown), and thus the insertion appears to be very recent. One macaque-specific 245-bp-long insertion is linked to expansion of a 49-bp-long, minisatellitelike array. The remaining macaque-specific insertion (50 bp) is nonrepetitive. A closer analysis suggests that the insert is not a processed pseudogene of known genes (data not shown). Figure 2 Structure and Evolution of the ASPM Gene in Primates The scale of all plots corresponds to the consensus sequence obtained based on a multiple alignment of five ASPM genes. (A) Schematic representation of the alignment. Promoter regions, exons, and introns are marked in gray, red, and blue, respectively. White segments correspond to gaps. (B) Positions of long (50 bp or longer) insertions/deletions. “O” denotes orangutan, “M” macaque, “OGCH” the orangutan–gorilla–chimpanzee–human clade, and “GCH” the gorilla–chimpanzee–human clade. (C) Positions of polymorphic bases derived from the GenBank single nucleotide polymorphism (SNP) database. (D) Positions of the CpG island. The approximately 800-bp-long CpG island includes promoter, 5′ UTR, first exon, and a small portion of the first intron. (E) Location of an approximately 3-kb-long segmental duplication. (F) Positions of selected motifs associated with genomic rearrangements in the human sequence. Numbers in parentheses reflect number of allowed differences from the consensus motif (zero for short or two ambiguous motifs, two for longer sites). (G) Distribution of repetitive elements. The individual ASPM genes share the same repeats except of indels marked in (B). (H) DNA identity and GC content. Both plots were made using a 1-kb-long sliding window with 100-bp overlaps. The GC profile corresponds to the consensus sequence; the individual sequences have nearly identical profiles. Table 1 Pairwise Identity of Aligned Primate ASPM Genes The pairwise identities were calculated for five complete ASPM genes and therefore include all promoter regions, introns, and exons. The values above the diagonal show DNA identities (in percent) calculated after removing indels. Under the diagonal are values for comparisons with gaps Of the two detected deletions, the macaque-specific 72-bp-long deletion appears to be associated with nonrepetitve DNA. The second one, an 818-bp-long deletion in orangutan, was probably caused by homologous Alu–Alu recombination (see below and Figure S1 ). The remaining indels are related to expansion/contraction of a short minisatellite array. It was caused either by a 53-bp expansion in the gorilla–chimpanzee–human clade or by two independent deletions/contractions in the macaque and orangutan lineages. An approximately 3-kb-long intronic segment between exons 4 and 5 is present in several copies in the genome ( Figure 2 E; Figure S2 ). Closer analysis of the human genome confirmed that copies of this region are homologous to 24 segmental duplications located mainly in telomeric regions of Chromosomes 1–8, 10, 11, 16, 19, 20, and Y. Based on the sequence similarity and the presence of an L1P4 LINE insertion at the 5′ end, the most closely related are three duplications at 7q11–13. The most similar copy is located on Chromosome 7 and shares 93% identity with the ASPM intronic segment. Five duplications are located on Chromosome 1; the closest copy is found 27 Mb away from the ASPM gene. We looked for several common motifs associated with genomic breakpoints in cancers ( Abeysinghe et al. 2003 ). Figure 2 F shows the positions of such potentially unstable oligonucleotides. Interestingly, the orangutan-specific deletion ( Figure 2 B) has its 5′ breakpoint located just 1 bp upstream of a sequence 100% identical to the chi-like consensus motif GCWGGWGG (see Figure S1 ). The chi motif is recognized by the RecBCD-mediated recombination pathway in prokaryotes and seems to be associated with rearrangements in the human genome ( Dewyse and Bradley 1991 ; Chuzhanova et al. 2003 ). Both deletion breakpoints in the orangutan deletion are located within 5′ parts of two Alu sequences, suggesting that the deletion was created by homologous Alu–Alu recombination. Similar homologous recombinations with breakpoints located near chi-like motifs in 5′ regions of Alu sequences were described previously ( Chen et al. 1989 ; Rudiger et al. 1995 ). In summary, despite the presence of a few indels, coding and noncoding regions of ASPM homologues show a marked degree of conservation. Evolution of the ASPM Protein We have analyzed ASPM CDSs from six primate species: human, chimpanzee, gorilla, orangutan, rhesus macaque, and African green monkey (Cercopithecus aethiops) . Except for orangutan and rhesus macaque, two or more ASPM CDSs were used for analysis. ASPM proteins showed the same overall length and domain structure ( Figure 3 A). The IQ repeat domain contains the same number of repeats, suggesting that their expansion occurred in early primate evolution. The CDSs are, as expected, more conserved than the complete gene sequences with promoter and intronic regions ( Table 2 ; Table 3 ). Only six short indels were identified ( Figure 3 B). Figure 3 Structure of ASPM CDSs and Evolution in Primates The scale of all plots corresponds to the 3,480-amino-acid-long protein alignment; positions in the CDS were scaled accordingly. (A) Structure of the human ASPM CDS and protein. The first scheme shows positions of major domains in the ASPM protein ( Bond et al. 2002 ). The putative microtubule-binding domain is in gray, the calponin-homology domain in orange, IQ repeats in blue, and the terminal domain in black. Positions of exons in the CDS are drawn in the second block. To separate individual exons, odd numbered exons are colored in black and even numbered ones in white. (B) Positions of insertions/deletions in the protein sequences. Coordinates correspond to the human protein sequence. “O” denotes orangutan, “G” gorilla, “M” macaque, “Gm” African green monkey, and “OGCH” the orangutan–gorilla–chimpanzee–human clade. (C) Substitutions in hominoid CDSs relative to the common ancestor. The expected ancestor CDS was derived using ML codon reconstruction implemented in PAML. African green monkey and rhesus macaque were outgroups. Nonsynonymous/synonymous (ω = Ka/Ks) ratios were free to vary in all branches. Positions marked in green correspond to synonymous changes relative to the ancestral sequence; the red bars indicate nonsynonymous changes. (D) Synonymous (red) and nonsynonymous (green) changes in ancestral lineages leading to human. aOGCH–aGCH is the ancestral lineage from the orangutan divergence to the gorilla divergence; aGCH–aCH represents the lineage from the gorilla divergence to the chimpanzee common ancestor. aCH–human corresponds to the human lineage after the chimpanzee divergence. There are seven synonymous and 19 nonsynonymous human-specific substitutions. Methods and description are the same as in (C). (E) Positions of polymorphic bases for different CDSs of African green monkey, gorilla, chimpanzee, and human. Positions marked in green correspond to synonymous polymorphisms, and the red bars indicate nonsynonymous sites. Numbers of compared sequences are in parentheses; in the case of human we show nine polymorphic positions (four synonymous and five nonsynomous) from the GenBank SNP database. ASPM mutations detected in MCPH patients are shown separately in (F). (F) Positions of 19 mutations reported for MCPH patients ( Bond et al. 2002 ; Bond et al. 2003 ). All the reported mutations introduce premature stop codons. Mutation sites located within CpG dinucleotides are highlighted in red. (G) Positions of CpG dinucleotides in the human CDS. (H) Comparison of Ka and Ks rates with codon adaptation index (CAI). Ka and Ks values are for all branches (fixed ω ratio); CAI is an average for all five primates (note that CAI differences are very small between the five species). The window was set to 300 bp (100 amino acids) with a 30-bp (10-amino-acid) step. (I) Conservation at the nucleotide and protein level in primates. Y-axis corresponds to proportions of conserved (identical) positions in the CDS and the protein alignment. The plot was obtained using 100-amino-acid-long, overlapping windows, and the step was set to 10 amino acids. In the case of CDS conservation, the window was 300 bp and step 30 bp. Table 2 Pairwise Identity of ASPM CDSs The pairwise identities were calculated for six CDSs. The values above the diagonal show DNA identities (in percent) calculated after removing indels. Under the diagonal are values for comparisons with gaps Table 3 Pairwise Identity of ASPM Proteins The pairwise identities were calculated for six protein sequences. The values above the diagonal show DNA identities (in percent) calculated after removing indels. Under the diagonal are values for comparisons with gaps From the DNA and protein conservation profiles ( Figure 3 I), ASPM segments evolve differently along the length of the CDS. N- and C-terminal regions and the region corresponding to exons 5–15 are conserved. In contrast, exons 3 and 4 and the complete IQ repeat domain (positions 1,267–3,225) are more variable. Indeed, nonsynonymous substitutions in hominoid primates ( Figure 3 C) and in ancestral lineages ( Figure 3 D) and nonsynonymous polymorphism ( Figure 3 E) are nearly absent in the conserved central (exons 5–15) and C-terminal regions. This pattern indicates different rates of evolution along the ASPM protein, visualized by plots of synonymous Ks and nonsynonymous Ka rates ( Figure 3 H) and supported by phylogenetic analysis (see below and Figure 4 ). It is notable that the comparison of the primate and mouse proteins also revealed the same pattern of conservative and nonconservative regions along ASPM protein ( Figure S3 ). Figure 4 Phylogenetic Trees and ω ratio for Complete ASPM and Three Selected Segments Trees and ω (Ka/Ka) ratios were computed using the ML method for codons implemented in PAML. Branch lengths represent ML distances for codons, i.e., using both synonymous and nonsynonymous nucleotide sites, and in all branches the ω ratio was set free to vary. All trees are drawn to the same scale. Branch labels mark the ω ratios for corresponding branches. Values in square brackets show ω for additional cDNA sequences whenever available. Default values and branch lengths were calculated from genomic copies. Selected tested hypotheses are listed. ω H stands for the ω rate in the human lineage, ω C in the chimpanzee lineage, ω CH in the common human–chimpanzee ancestral lineage after the gorilla divergence, ω G in the gorilla lineage, and ω 0 in all other branches. Single asterisks indicate p < 0.05, χ 2 1 = 3.84; double asterisks indicate p < 0.01, χ 2 1 = 6.63. (A) Phylogeny for the complete ASPM CDS. In addition to testing different ω values in the human lineage, we also tested the hypothesis that the complete gorilla–chimpanzee–human clade evolved at a constant rate, different from the rest of the tree (compared to the one-ratio model, boxed). (B) The ASPM phylogeny derived from a conserved segment from exon 5 to the beginning of the IQ domain (amino acids 676–1,266). The branch connecting the human and chimpanzee common ancestor with the gorilla–chimpanzee–human common ancestor had no substitutions, therefore the ω ratio could not be calculated. (C) IQ domain (amino acids 1,267–3,225). We also tested the hypothesis that the gorilla and human lineages evolved at the same ω rate, different from the rest of the tree (compared to the one-ratio model, boxed). (D) Phylogeny of eight primate sequences from a 1,215-amino-acid-long segment of exon 18 (amino acids 1,640–2,855). We also tested the hypothesis that the gorilla and human lineages evolved at the same ω rate, different from the rest of the tree (compared to the one-ratio model, boxed). Analysis of the nonsynonymous/synonymous substitution ratio (ω = Ka/Ks) revealed an elevated value in the human branch ( Figure 4 A). According to the likelihood ratio test, the human ω rate is significantly different from the rate in the rest of the tree ( p < 0.05). Also the model that the complete gorilla–chimpanzee–human clade is evolving at one ω rate different from that in the rest of the tree is well supported ( p < 0.01). Because ASPM consists of regions with different degrees of sequence conservation (see Figure 3 ), we separately analyzed a conserved region (exons 5–15 plus a small part of exon 16) and a variable IQ repeat domain. As can be seen ( Figure 4 B) the conserved region has all branches shorter, indicating overall a slower rate of evolution. In the human lineage, the ω ratio equals zero; however, the test for whether the human branch has a different (lower) ω rate than the rest did not yield significant values. In contrast, the tree based on the variable IQ repeat domain exhibits ω values greater than one for the human and gorilla branches ( Figure 4 C). The likelihood ratio test supports the model in which human and gorilla lineages evolved under a significantly higher ω ratio than the rest of the tree. Similar results were obtained for exon 18 with additional sequences from two New World monkeys ( Figure 4 D). As seen from Figure 4 A– 4 D, different sequences from African green monkey, gorilla, and chimpanzee individuals result in different ω values for their corresponding terminal branches. One chimpanzee sequence also produced an ω ratio greater than one for exon 18 ( Figure 4 D). It is worth noting that neither codon bias nor selection on third codon positions seemed to influence the synonymous rate Ks strongly ( Table S1 ). Therefore, the high Ka/Ks ratios in human and gorilla are likely to be products of adaptive evolution. Sequencing of two CDSs in African green monkey, three in gorilla, and three in chimpanzee allowed us to look for ASPM polymorphism in those species (see Figure 3 E). Human polymorphism data from ASPM mutant haplotypes are not representative of wild-type variation so were not used in these comparisons. For African green monkey, five synonymous and five nonsynonymous changes were found between two sequences. The gorilla and chimpanzee CDSs in particular showed an apparently high degree of replacement polymorphism. Gorilla polymorphism included 35 point mutations (15 silent mutations and 21 replacements). Chimpanzee sequences differed in five synonymous and 11 nonsynonymous sites. In order to interpret this seemingly high level of observed polymorphism, intraspecific diversity was compared to interspecific diversity using the McDonald and Kreitman test ( McDonald and Kreitman 1991 ). In the case of chimpanzee polymorphism compared to divergence with human, we could not reject the null hypothesis that polymorphism and divergence between species were significantly different (William's adjusted G statistic = 0.083, chi-square with 1 d.f., not significant; values based on PAML-generated Ka and Ks values using the free ratio model). Gorilla polymorphism was compared to divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. In this case we can reject the null hypothesis (William's adjusted G statistic = 122.45, chi-square with 1 d.f., p < 0.001) to conclude that the pattern of gorilla polymorphism is therefore different from the divergence pattern. Indeed gorilla polymorphism is less than variation resulting from divergence: within species, the ω ratio is 1.43 for gorillas compared to 2.2 for the divergence between the gorilla common ancestor and the human–chimpanzee common ancestor. Intraspecific variation, although seemingly unusual in showing so many replacement substitutions in both chimpanzee and gorilla, is less than or in line with what we have observed for ASPM divergence between species. Therefore, relaxation of selection cannot explain the high nonsynonymous/synonymous substitution ratios among African hominoids, further supporting the idea that adaptation has occurred in ASPM. Discussion In this study, we applied TAR cloning technology to investigate molecular evolution of the ASPM gene, which is involved in determining the size of the human brain and in which mutations lead to MCPH. The ASPM homologue in the fruit fly is essential for spindle function, suggesting a role for this gene in normal mitotic divisions of embryonic neuroblasts. Complete gene homologues from five primate species were isolated and sequenced. In agreement with the predicted critical role of ASPM in brain development, both coding and noncoding regions of ASPM homologues showed a marked degree of conservation in humans, other hominoids, and Old World monkeys. The differences found in noncoding regions were small insertions/deletions and lineage-specific insertions of evolutionarily young Alu elements into introns. Analysis of nonsynonymous/synonymous substitution ratios indicates different rates of evolution along the ASPM protein: part of ASPM evolved under positive selection while other parts were under negative (purifying) selection in human and African ape lineages. Such “mosaic” selection has been previously described for other proteins ( Endo et al. 1996 ; Crandall et al. 1999 ; Hughes 1999 ; Kreitman and Comeron 1999 ). When our work was completed, the paper by Zhang supporting accelerated evolution of the human ASPM was issued ( Zhang 2003 ). However, because the author did not analyze the gorilla gene homologue, he concluded that accelerated sequence evolution is specific to the hominid lineage. Our finding that selection on ASPM begins well before brain expansion suggests that the molecular evolution of ASPM in hominoids may indeed be an example of a molecular “exaptation” ( Gould and Vrba 1982 ), in that the originally selected function of ASPM was for something other than large brain size. In the case of ASPM, rapidly evolving residues are mainly concentrated in the IQ repeat domain containing multiple IQ motifs, which are calmodulin-binding consensus sequences. While there is no direct evidence yet, it is likely that the function of human ASPM is modulated through calmodulin or calmodulinlike protein(s). Previous interspecies comparisons of ASPM proteins have shown a consistent correlation of greater protein size with larger brain size mainly because of the number of IQ repeats ( Bond et al. 2002 ). For example, the asp homologue of the nematode Caenorhabditis elegans contains two IQ repeats, the fruit fly — 24 IQ repeats, and the mouse—61 IQ repeats, and there are 74 IQ repeats in humans ( Bond et al. 2002 ). ASPM homologues in the nonhuman primates examined here contain the same number of IQ repeats as human, supporting the idea that repeat expansion occurred prior to the anthropoid divergence (which gave rise to New World monkeys, Old World monkeys, and hominoids) and possibly even earlier in primate evolution. IQ motifs are seen in a wide variety of proteins, but the ASPM proteins in primates are unique, because they have the largest known number of IQ repeats. Given the proposed role of ASPM in regulating divisions of neuronal progenitors, both the number of repeats and the particular amino acid substitutions in the IQ repeats may be strongly related to brain evolution. Human ASPM gene mutations which lead to MCPH provide a direct link between genotype and phenotype. ASPM is yet another example on the growing list of positively selected genes that show both accelerated evolution along the human lineage and involvement in simple Mendelian disorders ( Clark et al 2003 ). However, ASPM is unique because its distinctive pattern of accelerated protein evolution begins several million years prior to brain expansion in the hominid lineage. Absolute brain size in orangutans (430 g in males; 370 g in females) is barely different from that in gorillas (530 g in males; 460 g in females) and common chimpanzees (400 g in males; 370 g in females) ( Tobias 1971 ), yet accelerated ASPM evolution began in the common ancestor of gorillas, chimpanzees, and humans, approximately 7–8 million years ago. Only much later did brain expansion begin in hominids, starting at 400–450 g roughly 2–2.5 million years ago and growing to its final current size of 1350–1450 g approximately 200,000–400,000 years ago ( Wood and Collard 1999 ). Therefore genotypic changes in ASPM preceded marked phenotypic changes in hominoid brain evolution, at least at the level at which they have currently been studied. The molecular changes in ASPM may predict the existence of differences in early neurogenesis between orangutans, on the one hand, and gorillas, chimpanzees, and humans, on the other, which may manifest as more subtle differences in brain anatomy than gross changes in brain volume. How might evolutionary changes in the ASPM protein affect cerebral cortical size? One potential mechanism might be that changes in ASPM induce changes in the orientation of the mitotic spindle of neuroblasts. Normally, neural precursor cells can have mitotic spindles oriented parallel to the ventricle or perpendicular to the ventricle. Mitoses in which daughter cells are oriented next to one another at the ventricular zone are typically “symmetric” in that a single progenitor cell generates two progenitor cells, causing exponential expansion of the progenitor pool. In contrast, mitoses that generate daughter cells that are vertically arranged are typically “asymmetric” so that one daughter cell becomes a postmitotic neuron, whereas the other daughter cell remains as a progenitor, causing only a linear increase in cell number. Control of this proliferative symmetry can cause dramatic alterations in cerebral cortical size ( Chenn and Walsh 2002 ), and so changes in ASPM could regulate cortical size by making subtle changes in spindle orientation. Alternatively, evolutionary changes in ASPM may not themselves have led to increase in the size of the brain, but instead perhaps ASPM might be essential to insure faithful DNA replication and proper chromosome segregation. In rodents, a surprising number of cerebral cortical neurons are aneuploid ( Rehen et al. 2001 ). Perhaps directed selection of specific domains of ASPM helps insure faithful chromosome segregation to allow a larger number of cerebral cortical neurons to be formed without an unduly high incidence of chromosome aneuploidy. Functional genomics studies are clearly needed to elucidate the exact nature of the molecular mechanisms affected by ASPM gene evolution in hominoids. Here, we have demonstrated the utility of TAR cloning for evolutionary sequence comparisons among humans and other primates. In addition, the ASPM TAR clones isolated in these studies could provide valuable reagents for studying ASPM gene regulation in its natural sequence context. Overall, we anticipate this technology will be extremely useful in studying the evolution of other genes that may be responsible for uniquely human traits. Note The related paper by Evans et al. (2004 ) was published in Human Molecular Genetics shortly after this paper was submitted. Materials and Methods TAR cloning of the ASPM gene homologues by in vivo recombination in yeast To isolate the full-size ASPM gene from the human (Homo sapiens), chimpanzee (Pan troglodyte s ), gorilla (Gorilla gorilla), orangutan (Pongo pygmaeus), and rhesus macaque (Macaca mulatta) genomes, a TAR vector containing two unique hooks was constructed. Two targeting sequences were designed, 131 bp 5′ and 151 bp 3′, from the available human genomic sequence of ASPM (positions 155,758–155,888 and 92,922–93,071 in the BAC RP11–32D17 [GI:16972838]). The targeting sequences were PCR amplified from the genomic DNA using two specific primers ( Table S2 ). PCR products were cloned into a polylinker of the basic TAR vector pVC604 as Apa I– Sal I and Sal I– Xba I fragments. Before transformation experiments, the TAR cloning vector was linearized with Sal I to release targeting hooks. Genomic DNA samples were prepared from chimpanzee, gorilla, orangutan, and rhesus macaque fibroblast culture cell lines (Coriell Institute for Medical Research, Camden, New Jersey, United States) in agarose plugs. Spheroplast transformation experiments were carried out as previously described in Kouprina and Larionov (1999 ). To identify clones positive for ASPM , yeast transformants were examined by PCR using diagnostic primers specific for exon 2 and exon 27 of ASPM ( Table S2 ). Integrity of yeast artificial chromosomes (YACs) and the issue of their stability during propagation in yeast were examined. DNA was isolated from ten subclones carrying the ASPM YACs for each primate, and their size was analyzed by Not I digestion followed by CHEF. Each subclone carried a YAC of similar size, indicating that these clones were stable in yeast. Alu profiles of the clones were checked by Taq I digestion of 1 μg of total yeast DNA isolated from transformants. Samples were run by electrophoresis, transferred to a nylon membrane, and hybridized with an Alu probe. YACs were retrofitted into bacterial artificial chromosomes (BACs) by homologous recombination in yeast using a BAC/Neo R retrofitting vector, BRV1, and then transformed into a recA DH10B E. coli strain ( Kouprina and Larionov 1999 ). Before sequencing, the integrity of inserts in BACs was confirmed by Not I, Hin dIII, Eco RI, and Pst I digestions. The promoter regions of the chimpanzee, gorilla, orangutan, and rhesus macaque (approximately 3.2 kb) and exon 18 of the red-chested mustached tamarin (Saguinus labiatus) and black-handed spider monkey (Ateles geoffroyi) (approximately 4.7 kb) were PCR amplified using a pair of specific primers ( Table S2 ) from primate genomic DNAs (Coriell Institute for Medical Research) and then TA-subcloned for further sequencing. RT-PCR of ASPM coding regions RNAs were extracted from primate cell lines (African green monkey [Cercopithecus aethiops] kidney, COS-7 [American Type Culture Collection, Manassas, Virginia, United States], chimpanzee peripheral lymphoblast, EB176 [JC], and gorilla peripheral lymphoblast, EB [JC] [European Collection of Cell Cultures, Wiltshire, United Kingdom]) using TRIzol reagent (Invitrogen, Carlsbad, California, United States). Reverse transcription and 5′- and 3′-RACE reactions were performed using SMART RACE cDNA Amplification Kit (BD Biosciences, San Jose, California, United States). Sequencing Chimpanzee, gorilla, orangutan, and rhesus macaque TAR clones containing full-size ASPM genes were directly sequenced from BAC DNAs ( Polushin et al. 2001 ). Forward and reverse sequencing of the promoter and exon 18 as well as fragments of coding regions of the ASPM homologues were run on a PE-Applied Biosystem 3100 Automated Capillary DNA Sequencer (Applied Biosystems, Foster City, United States). Primer pairs for cDNA sequencing were designed based on the human ASPM mRNA sequence. Primer sequences are available upon request. All sequences were named and numbered according to the clone/accession identifier. Sequence analysis Genomic sequences were aligned using MAVID ( http://baboon.math.berkeley.edu/mavid/ ) ( Bray and Pachter 2004 ); proteins and protein-coding DNA sequences were aligned by DIALIGN2.1 ( http://bibiserv.techfak.uni-bielefeld.de/dialign/ ) ( Morgenstern 1999 ). Alignments were manually edited in the SEAVIEW editor ( http://pbil.univ-lyon1.fr/software/seaview.html ) ( Galtier et al. 1996 ). We have used a number of programs from the EMBOSS package ( http://www.hgmp.mrc.ac.uk/Software/EMBOSS/ ) for sequence analysis. Short nucleotide patterns associated with genome rearrangements were searched using FUZZNUC (EMBOSS). We searched for the following recombinogenic motifs: chi-like octamer (GCWGGWGG), immunoglobulin heptamer (GATAGTG), translin (ATGCAGN(0,4)GCCCWSW and GCNCWSCTN(0,2)GCCCWSSW), topoisomerase II (RNYNNCNNGYNGKNYNY), topoisomerase IId (GTNWAYATTNATNNR), topoisomerase IIi (YYCNTASYGGKYYTNNC), and V(D)J recombinase (CACAGTGN(12/23)ACAAAAACC). For short or highly ambiguous patterns (topo-isomerase II), no mismatches were allowed; for longer motifs (translin, V(D)J recombinase) up to two mismatches were permitted. Prediction of CpG islands was performed by CPGPLOT (EMBOSS) with default parameters (length ≥ 200; CpG/GpC ≥ 0.6; GC ≥ 0.5). CENSOR ( http://www.girinst.org/Censor_Server-Data_Entry_Forms.html ) ( Jurka et al. 1996 ) and REPEATMASKER ( http://repeatmasker.genome.washington.edu/cgi-bin/RepeatMasker ; developed by A.F.A. Smit and P. Green) were used for identification of repetitive elements. Minisatellites were detected by TANDEM REPEAT FINDER ( Benson 1999 ). ASPM segmental duplications in the human genome were detected by local BLAT searches ( http://genome.ucsc.edu/cgi-bin/hgBlat ) ( Kent 2002 ). First, we used ASPM genomic sequences with all repeats masked to detect segmental duplications. Full-size duplications were then obtained by BLAT alignment with full (i.e., repeat-containing) ASPM sequence. Primate CDSs were deduced from the ASPM gene alignment with human sequences. Synonymous and nonsynonymous substitutions were detected by SNAP ( http://www.hiv.lanl.gov/content/hiv-db/SNAP/WEBSNAP/SNAP.html ). Codon maximum likelihood (ML) in CODEML in PAML v. 3.13 ( http://abacus.gene.ucl.ac.uk/software/paml.html ) ( Yang 1997 ) has been applied for reconstruction of phylogenetic trees, reconstruction of ancestral sequences, and detection of positive selection. Branch lengths and ancestral sequences were reconstructed using a free ω ratio for individual branches. The methodology of likelihood ratio tests is described elsewhere ( Yang 1998 ). For large alignments, the initial input trees for PAML were estimated by ML implemented in PHYLO_WIN ( http://pbil.univ-lyon1.fr/software/phylowin.html ) ( Galtier et al. 1996 ). Segmental duplications were clustered by a neighbor-joining method implemented in the same program. Distance measurements for examining intraspecific/interspecific diversity were calculated in PAUP (Swofford, D. L. 2003. PAUP v. 4.0b10; Sinauer Associates, Sunderland, Massachusetts, United States; http://paup.csit.fsu.edu/index.html ) and corrected for multiple substitutions using the Tamura-Nei algorithm. Supporting Information Commentary Selection operating on codon usage may increase the ω ratio by lowering the rate of synonymous substitutions ( Sharp and Li 1987 , 1989). Therefore, we tested the correlations between the CAI ( Sharp and Li 1987 ) and the rate of synonymous substitutions (Ks). We found no significant association between the tested variables. Moreover, interspecies comparisons disclosed that CAI is nearly identical for all compared species, and no CAI increase over other species was detected for human or gorilla (data not shown). On the other hand, there was a significant negative correlation between CAI and both protein and DNA identity. A partial correlation analysis revealed that the significant positive linear correlation between Ka and CAI was merely caused by the strong negative correlation of Ka with DNA and protein identity. When we controlled for identity, the correlation between Ka and CAI disappeared (data not shown). These results may indicate that at positively selected sites, protein changes are preferred over optimization of codon usage, and thus mutations causing amino acid replacements do not immediately produce optimal codons. It should be noted that selection on codon usage seems to be generally relaxed in mammals ( Duret and Mouchiroud 2000 ). Mammalian codon usage as well as the rate of nonsynonymous substitutions can be potentially biased by selection favoring a high GC content (or even saturation by G and C) at the third codon positions (GC3) ( Bernardi and Bernardi 1985 ; Aota and Ikemura 1986 ). However, ASPM is an AT-rich gene (GC content 36.4%–37%) and, as expected ( Bernardi and Bernardi 1985 ; Aota and Ikemura 1986 ), the third codon positions are also AT-rich (GC3 content, 30.6%–31.4%) and thus far from saturation. In summary, neither the codon bias nor selection on the third codon seems to strongly influence the synonymous rate Ks. Therefore the high Ka/Ks ratio in human and gorilla is likely to be the product of adaptive evolution. Figure S1 Recombination Breakpoints in the Orangutan-Specific 818-bp-Long Deletion Both orangutan breakpoints are located within 5′ portions of two Alu elements. The sequence conservation is marked by different shades of gray. Both Alu elements are compared to their corresponding AluSp and AluSz subfamily consensus sequences. Gorilla, chimpanzee, and human sequences located 1 bp downstream of the 5′ breakpoint share a perfect match with the chi-like octamer consensus sequence GCWGGWGG (first box, positions matching the chi consensus are shown in black). On the other hand, the 3′ breakpoint sequences are diverged from the chi consensus (second box). Both Alu elements in the alignment are shown from the first position and end at the same position, and thus positions in one element correspond to positions in the other Alu copy. As can be seen, the breakpoint position in the first AluSp repeat exactly corresponds to the breakpoint position within the second AluSz element, suggesting homologous recombination between the two repeats. (163 KB PDF). Click here for additional data file. Figure S2 Segmental Duplications of the Fourth Internal Intron From left to right: phylogeny, chromosomal position, band name, identity to ASPM segment (percent same), and a schematic alignment of segmental duplications. The ASPM segment (black) shares similarity with 24 segmental duplications that contain additional sequences and are present on several human chromosomes. The ASPM copy and three duplications on Chromosome 7 share the same L1P4 terminal insertion, which is absent from all other duplications. The tree on the left shows evolutionary relationships among the duplications estimated by the neighbor-joining method. (169 KB PDF). Click here for additional data file. Figure S3 Comparison of Mouse and Human ASPM Proteins The amino acid identity in the conserved regions is 85.44%, 49.39%, and 68.74% for exon 3, exon 4, and the IQ domain, respectively. In addition, while the alignment of conserved regions is completely gap-free, the variable domains exhibit several gaps including a large deletion in the mouse IQ domain (human positions 2655–2943). (97 KB PDF). Click here for additional data file. Table S1 Primers Used in This Work Upper case letters indicate sequences homologous to ASPM and lower case letters indicate cloning sites. (118 KB PDF). Click here for additional data file. Table S2 CDS and Protein Correlations All correlations were obtained for the same 100-amino-acid-/300-nucleotide-long, nonoverlapping windows. The first value shows the correlation coefficient; p -value is in parentheses. The section over the diagonal is calculated using the Pearson (linear) correlation coefficient; under the diagonal are correlations obtained using the Spearman's rank coefficient—nonparametric). Nontrivial or interesting significant correlations are shown in bold and italics. The CAI represents the mean for all species (the CAI values are nearly identical for individual species). The ω ratio, Ka, and Ks (rows/columns 2, 3, and 4) correspond to all branches of the phylogenetic tree. They were obtained using a ML model with one fixed ω ratio for all branches. Click here for additional data file. Accession Numbers The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession number for the human ASPM mRNA sequence used in this study is NM_018136. The sequence data from chimpanzee, gorilla, orangutan, and rhesus macaque full-length ASPM have been submitted to GenBank under accession numbers AY497016, AY497015, AY497014, and AY497013. The sequence data from chimpanzee, gorilla, and African green monkey ASPM cDNA have been submitted to GenBank under accession numbers AY508452, AY508451, and AY486114. The sequence data from spider monkey and tamarin exon 18 have been submitted to GenBank under accession numbers AY497017 and AY497018.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC374243.xml
555559	Activation of p53, inhibition of telomerase activity and induction of estrogen receptor beta are associated with the anti-growth effects of combination of ovarian hormones and retinoids in immortalized human mammary epithelial cells	Background A full-term pregnancy has been associated with reduced risk for developing breast cancer. In rodent models, the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P). However, the effects of pregnancy levels of E/P in humans and their underlying mechanisms are not fully understood. In this report, we investigated the growth inhibitory effects of pregnancy levels of E/P and both natural and synthetic retinoids in an immortalized human mammary epithelial cell line, 76N TERT cell line. Results We observed that cell growth was modestly inhibited by E/P, 9-cis-retinoic acid (9-cis RA) or all-trans-retinoic acid (ATRA), and strongly inhibited by N-(4-hydroxyphenyl) retinamide (HPR). The growth inhibitory effects of retinoids were further increased in the presence of E/P, suggesting their effects are additive. In addition, our results showed that both E/P and retinoid treatments resulted in increased RARE and p53 gene activity. We further demonstrated that p53 and p21 protein expression were induced following the E/P and retinoid treatments. Furthermore, we demonstrated that while the telomerase activity was moderately inhibited by E/P, 9-cis RA and ATRA, it was almost completely abolished by HPR treatment. These inhibitions on telomerase activity by retinoids were potentiated by co-treatment with E/P, and correlated well with their observed growth inhibitory effects. Finally, this study provides the first evidence that estrogen receptor beta is up-regulated in response to E/P and retinoid treatments. Conclusion Taken together, our studies show that part of the anti-growth effects of E/P and retinoids is p53 dependent, and involve activation of p53 and subsequent induction of p21 expression. Inhibition of telomerase activity and up-regulation of estrogen receptor beta are also associated with the E/P- and retinoid-mediated growth inhibition. Our studies also demonstrate that the potency of retinoids on cell growth inhibition may be increased through combination of estrogen and progesterone treatment.	Background It is well documented that women who experience a full-term pregnancy early in their lives have a significantly reduced risk for developing breast cancer [ 1 , 2 ]. The mechanisms by which pregnancy affects maternal breast cancer incidences are not fully understood. Previous studies suggest that the protective effect of pregnancy can be mimicked with a defined regimen of estrogen and progesterone combination (E/P) in rodent models [ 3 , 4 ]. However, the effects of pregnancy levels of E/P in human and their underlying mechanisms have not been investigated. Retinoids are a promising class of chemopreventive agents against breast cancer because of their antiproliferative and proapoptotic properties [ 5 , 6 ]. Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) are nuclear transcription factors that modulate the biological effects of retinoids. Most retinoid forms, including 9-cis-retinoic acid (9-cis RA) and all-trans-retinoic acid (ATRA), activate RAR family members, whereas RXR family members are activated by 9-cis RA. N-(4-hydroxyphenyl) retinamide (HPR), a synthetic derivative of ATRA, may weakly interact with retinoid receptors [ 7 ]. The 76N TERT cells were derived from a reduction mammoplasty specimen [ 8 , 9 ]. They are normal human mammary epithelial cells immortalized by plasmids containing hTERT, the human catalytic subunit of the reverse transcriptase protein of telomerase [ 10 ]. hTERT-expressing normal cell clones have been shown to have an extended life span without any change in karyotype [ 11 ]. The 76N TERT cells in culture could continuously grow about 60 population doublings [ 8 ], and the level of p53 protein has been shown to remain consistent at early or late passages [ 9 ]. Unlike the tumor cell lines widely used in breast cancer researches such as MCF-7 and MDA-MD-231 cells, which have undergone several steps in tumorigenesis, the 76N TERT cell line represents a system that is immortal but does not yet have the capacity to form a tumor. Hence, it is potentially a better model to study the genetic changes, and to test the effects of carcinogenic or chemopreventive agents on the development of mammary tumors. In this study, we investigated whether E/P induce growth inhibition in 76N TERT cells; and the molecular mechanisms by which E/P inhibited 76N TERT cell growth. For comparison purpose, the anti-growth effect of both natural and synthetic retinoids was examined in parallel in this immortalized mammary epithelial cell line. We also investigated whether we could increase the responsiveness of retinoids by using retinoids in combination with E/P. Our studies demonstrate that 1) inhibition of cell growth by E/P and retinoids could be partially mediated through a p53-dependent mechanism; 2) induction of p21 expression, inhibition of telomerase activity, or up-regulation of estrogen receptor beta (ERβ) by E/P and retinoids may contribute to their anti-growth effects; 3) combination of retinoids with E/P lead to increased inhibitory effects on cell growth. Results Expression of RARs, RXRs, estrogen receptors and progesterone receptors in 76N TERT cells As the ability of estrogen, progesterone and retinoids to influence cell proliferation is mediated by their respective receptors in most cases, we first examined the expression of these receptors in 76N TERT cells. Using Western blot analysis, the proteins of RAR (RARα, RARβ and RARγ), RXR (RXRα, RXRβ and RXRγ), and ER (ERα and ERβ) were observed as suggested by the manufactures (Figure 1 ). The antibody for the progesterone receptor (PR) detected a protein between 85 and 125 KDa (Figure 1 ). The existences of RARβ and ERβ in 76N TERT cells were further confirmed by quantitative RT-PCR at the mRNA level (RARβ data not shown, ERβ data see Figure 6B ). Figure 1 Expression of RAR, RXR, ER and PR in 76N TERT cells. Whole cell lysates were analyzed by using anti-RAR, RXR, ER, and PR antibodies as described under "Materials and Methods". Blots shown are representative of 2–3 experiments with similar results. Figure 6 Induction of ERβ expression by E/P and retinoids in 76N TERT cells . Cells were treated with indicated retinoids with or without E/P for 72 hours. A. Western blot analysis of ERβ protein. Relative ERβ expression was normalized to actin protein and expressed as fold changes compared to vehicle treatment. Blot shown is representative of 3 experiments with similar results. B. Quantitative RT-PCR analysis of ERβ genes. Data are the means ± SE from 3 experiments. , significant differences from their own controls. Inhibition of 76N TERT cell growth by E/P and retinoids We then tested the influence of E/P or retinoids on 76N TERT cell growth, using [ 3 H]thymidine incorporation assay. The concentrations of E/P and retinoids used in the experiments were chosen based on previous studies and are clinically or physiologically relevant [ 3 , 4 , 12 ]. Treatment of cells with E/P or all three retinoids resulted in decreases in cell proliferation. As shown in Figure 2 , treatment of 76N TERT cells with 1 μM 9-cis RA or 2 μM ATRA significantly decreased the cell growth by 28.8% and 24.5% respectively. In comparison, cells were more responsive to HPR. Treatment with 2 μM HPR exhibit a significant 71.4% decrease in cell growth relative to controls. Combination of 1 ng/ml of β-estradiol and 1 μg/ml of progesterone, a regime that mimics the protective effects of pregnancy, also resulted in a significant 32.2% decrease in cell proliferation. In the presence of E/P, the inhibitory effects on cell growth of 9-cis RA, ATRA and HPR were further increased to a respective 56.6%, 53.3% and 86.8%, indicating that the anti-proliferative effect may be additive between E/P and retinoids. Figure 2 Inhibition of 76N TERT cell growth by E/P and retinoids . Cells were treated with indicated retinoids with or without E/P for 24 hours and then labelled with [ 3 H]thymidine as described under "Materials and Methods". Data are the means ± SE from 3–4 experiments. , significant differences from control. Activation of RARE and p53 gene by E/P and retinoids Many biological responses to retinoids are thought to be mediated through receptors by binding to retinoic acid response elements ( RARE s) and regulation of transcriptional activity [ 5 , 6 ]. In addition, cross-talk between ER and RAR pathways has been previously reported [ 13 , 14 ]. Given this, a comparison of RARE gene activation in response to E/P and retinoids was carried out using cells transfected with a RARE -luciferase reporter gene construct. As shown in Figure 3A , 76N TERT cells exhibited a respective 3.3-, 5.4-, and 2.5-fold activation of RARE gene in response to 9-cis RA, ATRA or E/P alone. In contrast, HPR caused no significant change in luciferase activity relative to the control. In the presence of E/P, the effects of 9-cis RA and ATRA on RARE gene activation were essentially the same as without E/P. Figure 3 Activation of RARE and p53 gene by E/P and retinoids in 76N TERT cells . Cells were transiently transfected with the RARE ( A ) or p53 ( B ) reporter plasmid, and then treated with retinoids for 24 hours with or without E/P. Luciferase activity was measured and normalized as described under "Materials and Methods". Results are the means ± SE from 3 experiments. , significant differences from control. Functional p53 provides a protective effect against tumor growth [ 3 , 15 ]. We next examined whether E/P and retinoids could enhance the transcriptional activity of p53 using 76N TERT cells transfected with a p53 -responsive luciferase reporter gene construct. As shown in Figure 3B , a 2.3-, 1.8-, 1.8- and 2.2-fold induction of luciferase activity was observed by treatment of cells with 9-cis RA, ATRA, HPR and E/P respectively. Co-treatment with E/P and retinoids showed no additional activation of p53 gene as compared to their treatments alone. Induction of p53 and p21 protein expression by E/P and retinoids We then performed Western blot analysis to test whether increased p53 gene activity is paralleled by increased p53 protein expression. Treatment with 9-cis RA, ATRA, HPR or E/P alone slightly increased (about 1.5-fold) the expression of p53 protein. Consistent with the data on p53 gene activation, no additive effects between E/P and retinoids on induction of p53 protein were observed (Figure 4 ). Figure 4 Induction of p53 and p21 protein by E/P and retinoids in 76N TERT cells . Cells were treated with indicated retinoids with or without E/P for 72 hours. Whole cell lysates were subjected to Western blot analysis using anti-p53, p21 and Bax antibodies. Relative expression of each protein was determined using the same membrane, and normalized to actin protein. Data are fold changes compared to vehicle treatment. Blot shown is representative of 4 experiments with similar results. Activated p53 could induce the transcription of either p21 to cause growth arrest, or Bax to induce apoptosis [ 15 ]. We therefore investigated whether increased p53 protein expression can modulate the expression level of p21 or Bax proteins in 76N TERT cells. In Figure 4 , exposure to retinoids or E/P alone did moderately increase the p21 protein level, with a 1.7-, 2.7- and 2.1-fold increase in p21 expression for ATRA, HPR and E/P respectively. The increases of p21 protein expression with combined E/P and retinoids were similar to that observed when E/P and retinoids were used alone. In addition, no significant effects by E/P or retinoids on Bax protein expression were observed in 76N TERT cells. Inhibition of telomerase activity by E/P and retinoids Activation of telomerase is an early event in the development of breast cancers that may lead to cellular immortality, a critical and rate-limiting step in oncogenesis [ 16 , 17 ]. Activated p53 has been associated with regulation of the telomerase activity [ 18 - 20 ]. To evaluate the effects of E/P and retinoids on telomerase activity in 76N TERT cells, cells were treated for different time periods with E/P and retinoids, and the levels of telomerase activity were determined by a quantitative real-time PCR method. As shown in Figure 5 , treatment with E/P decreased telomerase activity in a time-dependent manner with a 63.3% inhibition at 72 hours, whereas vehicle treatment had no effect at any time during the experiment. The maximum inhibition on telomerase activity was observed at 24 hours for both 9-cis RA and ATRA treatments, with a respective of 68.9% and 69.4% decrease. In comparison, the effects of HPR occurred more rapidly, with a complete inhibition at 16 hours, and persisted throughout the treatment. The inhibitory effects of 9-cis RA, ATRA or HPR and E/P seemed to be additive, as in the presence of E/P, 9-cis RA, ATRA or HPR showed increased inhibitions at various time points. These effects correlate well with their observed growth inhibitory effects in 76N TERT cells, suggesting that inhibition of telomerase activity by E/P and retinoids may contribute to their additive effects on inhibition of cell growth. Figure 5 Inhibition of the telomerase activity by E/P and retinoids in 76N TERT cells . Cells were treated with indicated retinoids with or without E/P for the indicated time periods. Telomerase activity was determined as described under "Materials and Methods". Data are the means ± SE from 2–3 measurements. , significant differences from their own controls. Induction of ERβ expression by E/P and retinoids There is growing evidence that ERβ could be an inhibitor of tumorigenesis of breast cancer [ 21 - 23 ]. We examined whether there were any changes in the expression of ERβ in response to E/P and retinoid treatment in 76N TERT cells. After treatment of cells with retinoids or E/P for 72 hours, the amount of ERβ protein was determined by Western blot analysis. As shown in Figure 6A , the normalized ERβ protein showed a respective 2.1-, 2.3-, and 1.5-fold increase in response to ATRA, HPR and E/P, as compared to the vehicle treatment. We also carried out a quantitative analysis of ERβ mRNA expression in response to E/P and retinoid treatment in 76N TERT cells using a real-time PCR assay. As shown in Figure 6B , there was a 2.0-, 2.3- or 2.8-fold induction of ERβ mRNA levels in HPR, E/P or combination of HPR and E/P treated cells, respectively. Discussion Similar to retinoids, a full-term pregnancy has been associated with beneficial effects on breast cancer risk over the long term [ 1 , 2 ]. The mechanisms by which pregnancy affects maternal breast cancer incidences are not fully understood. Studies have showed that higher concentrations of progesterone elicit a growth-inhibiting response from normal and cancerous breast cells [ 24 , 25 ], and are inversely related to breast cancer incidence [ 26 ]. In this study, we examined the ability of pregnancy levels of E/P and retinoids to affect the growth of the immortalized normal mammary epithelial cells. Our results demonstrated that three isoforms for RAR and RXR (α, β, γ), two isoforms for ER (α and β), and PR receptor proteins are expressed by 76N TERT cells. Treatment with 9-cis RA, ATRA, HPR or E/P inhibited 76N TERT cell proliferation and resulted in the activation of p53 gene, followed by increased expression of p53 protein and p21 protein, and inhibition of telomerase activity. Additionally, we first report here that the expression of ERβ is induced in response to E/P and retinoid treatment at both the transcriptional and translational levels. Importantly, we demonstrate that the inhibitory effects of retinoids on cell growth are more effective in the presence of E/P, and correlate well with their inhibitory effects on telomerase activity in 76N TERT cells. Our data suggest that the growth inhibitory effects of E/P and retinoids may involve the activation of p53 pathway in 76N TERT cells. First, our results showed that both E/P and retinoid treatments lead to the increased p53 gene activity. Secondly, we demonstrated that the protein expression of p53 and p21 were increased following the treatment. It has been shown that p21 can inhibit cyclin A/cdk2 kinase activity and subsequently result in cell cycle arrest [ 27 , 28 ]. Our data is in line with the previous findings that in normal mammary epithelial cells, retinoids induce cell cycle arrest which is associated with an increase in p21 expression [ 29 ]; and that in both rats and mice, p53 is activated in response to E/P and this activation is sustained to induce p21 [ 3 ]. Thirdly, our data showed that treatment with E/P or retinoids decreased the telomerase activity in 76N TERT cells. Although a few reports suggest that telomerase activity appears to be independent of p53 expression or mutation [ 30 , 31 ], the majority of the evidence to date support the involvement of p53 in regulation of telomerase activity in mammary epithelial cells and breast cancer [ 18 - 20 ]. The molecular mechanisms of regulation of telomerase activity by p53 may involve down-regulation of hTERT transcription or the interaction of p53 with other transcription factors [ 19 ]. However, our data also suggest the possibility that inhibition of cell growth by E/P and retinoids may be independent of p53 pathway in 76N TERT cells. Our data show that the enhancing effects of retinoids on p53 gene activation or on the p53 and p21 protein expression were not further augmented by the addition of E/P, unlike their inhibitory effects on cell growth, indicating that other mechanisms besides the p53 pathway are likely to be involved. A p53-independent cell cycle arrest by retinoids has been previously suggested in a number of breast carcinoma cells [ 27 , 32 ]. The p53-independent inhibitory effects of retinoids on cell growth can be exerted through various mechanisms including regulation of other genes that play critical roles in cell cycle progression such as c-myc [ 18 ], inhibition of activator protein mediated transcription [ 33 ], or induction of caspase-independent cell death via calcium and calpain [ 34 ]. However, the mechanisms of E/P-mediated p53-independent cell growth inhibition are still unknown, and are currently under investigation using cell lines with different functional p53 systems. Clearly, our data suggest that there are some overlaps between E/P- and retinoid-mediated growth inhibition in 76N TERT cells, considering that in response to E/P and retinoid treatments, same effectors such as RARE and p53 gene, p53 and p21 protein, and the telomerase activity were affected. Additionally, there also seems to be cross-talk between the E/P- and retinoid-mediated growth inhibition. Previous studies have suggested that there is cross-talk between ERβ and RAR pathways [ 13 , 14 , 35 ]. Here, we demonstrated that the RARE gene activity was increased in response to E/P treatment. Furthermore, for the first time, we showed that treatment of immortal cells with E/P or retinoids could induce the expression of ERβ, both at the mRNA and protein level. The expression of ERβ often is found to be decreased markedly in the early stages of mammary carcinogenesis [ 22 ]. Loss of ERβ expression has been suggested as one of the events leading to the development of breast cancer [ 36 ]. Our data may reveal another important mechanism by which E/P and retinoids produce their anticancer function, indicating ERβ may represent a possible therapeutic target in breast cancer prevention. More importantly, our data show that the growth-inhibitory effects of retinoids were potentiated by co-treatment with E/P in 76N TERT cells. These observations indicate that different mechanisms may be involved in E/P- and retinoid-mediated inhibition of cell growth. Our results of their differential inhibitory effects on telomerase activity may provide some explanation for this. Although E/P and all three retinoids inhibited telomerase activity, the time courses of their actions were different. While retinoids produced their maximal inhibitory effects around 16 to 24 hours after treatment, E/P required 72 hours to reach its maximal inhibition, suggesting different mediators may be utilized to decrease telomerase activity by E/P and retinoids. ATRA and 9-cis RA have been previously reported to inhibit cell growth and decrease telomerase activity through down-regulation of the expression of hTERT telomerase gene [ 37 ]. On the other hand, progesterone treatment has been shown to down-regulate telomerase activity by modulation of cell cycle phases [ 38 ]. Previous studies have also provided evidence that the function of p53 in suppression of telomerase activity is separable from its cell cycle checkpoint function [ 20 ]. Therefore, it is likely that even though E/P and retinoids treatments both activate p53 pathway, they may use different mechanisms to inhibit telomerase activity. The different mediators involved in the inhibitory effects of E/P and retinoids on telomerase activity may contribute to their additive effects on inhibition of 76N TERT cell growth. The detailed mediator mechanisms down-stream of p53 and up-stream of telomerase activity for both E/P and retinoid pathways remain to be defined. Several lines of evidence suggest that the mechanisms through which HPR regulates cell growth seem different than those by 9-cis RA and ATRA in 76N TERT cells. In the [ 3 H]thymidine incorporation experiments, our results showed that whereas only 25–30% inhibition was observed for 9-cis RA and ATRA, 70% inhibition was reached by HPR. In addition, in the RARE -luciferase activity assay, 9-cis RA and ATRA induce about 3- to 5-fold activation on RARE gene activity. In contrast, HPR treatment resulted in no significant change. Finally, our data showed that 9-cis RA or ATRA treatment caused a moderate inhibitory effect on telomerase activity. In comparison, the telomerase activity is almost completely abolished by HPR treatment. The time courses of their inhibition of telomerase activity were different as well. While 9-cis RA and ATRA maximally inhibited the telomerase activity around 24 hours after treatment, HPR produced its maximal effect at 16 hours post-treatment. An obvious explanation for these different responses observed between 9-cis RA, ATRA and HPR is that these retinoids most likely possess different mechanisms for their actions. As suggested by numerous investigators, 9-cis RA and ATRA may function through the classical retinoid pathways involving the RARs and RXRs. On the other hand, in addition to activation the retinoid receptors [7a], HPR may also function through alternative pathways such as down-regulation of the IGF system [ 39 ], activation of TGF-beta [ 40 ], induction of genes which have antiproliferative activity [ 41 ], inhibition of aromatase activity and expression [ 42 ], and involvement of cellular signals such as reactive oxygen species [ 43 ] and the sphingolipid ceramide [ 44 ]. Conclusion In summary, our data demonstrate that 76N TERT cells express RAR, RXR, ER and PR, and represent a potential useful model to investigate the genetic changes, and the carcinogenic or chemopreventive effects of new agents on the development of mammary tumors. In addition, our data clearly suggest that part of the anti-growth effects of E/P is mediated through a p53-dependent pathway, as well as the involvement of the inhibition of telomerase activity and induction of ERβ. Comparing the E/P- and retinoid-mediated inhibitory effects on cell growth, there are overlaps, cross-talks and distinct effectors between these pathways. Furthermore, our studies suggest that retinoids may be more effective when combined with E/P to prevent breast cancer development. This increased potency and sensitized response to retinoids with combined E/P treatment might have several important clinical implications for anti-cancer agents that mimic E/P effects. Firstly, it might allow the currently used RA regimens to show improved response in cancer prevention. Secondly, it may be sufficient to overcome some acquired or intrinsic RA resistance in cancer cells. Finally, it may lower the required does of either classes of anticancer agents used, leading to less side effects or toxicity. Overall, our studies better the understandings of the common and the unique mechanisms by which E/P and retinoids regulate cell growth, and may help us to design or to improve the clinical applications of anti-cancer agents. Methods Chemicals ATRA, 9-cis RA, HPR, β-estradiol and progesterone were all purchased from Sigma (St. Louis, MO, USA) and dissolved in ethanol. The final concentration was 2 μM for ATRA and HPR, 1 μM for 9-cis RA, 1 ng/ml for β-estradiol and 1 μg/ml for progesterone. These concentrations were chosen based on previous studies and are clinically or physiologically relevant [ 3 , 4 , 12 ]. Culture of 76N TERT cells Cell line was originally supplied and cultured as described by Band et al. [ 8 ]. The culture medium D-MEM/F-12, fetal bovine serum, penicillin, streptomycin, and gentamicin were from Gibco (Carlsbad, CA, USA). All the other cell culture reagents were from Sigma (St. Louis, MO, USA). Cells were grown in D-MEM/F-12 mixture (1:1, vol/vol) containing 15 mM HEPES buffer and 2.5 mM L-glutamine, supplemented with 1% fetal bovine serum, 12.5 ng/ml epidermal growth factor, 10 nM triiodothyronine, 50 μM freshly made ascorbic acid, 2 nM estradiol, 1 μg/ml insulin, 2.8 μM hydrocortisone, 0.1 mM ethanolamine, 0.1 mM phosphorylethanolamine, 10 μg/ml transferrin, 15 nM selenite, 1 ng/ml cholera toxin, 35 μg/ml bovine pituitary extract, 100 units/ml penicillin, 100 mg/ml streptomycin, and 20 μg/ml gentamicin. Cells were maintained in 95% humidified air plus 5% CO 2 and sub-cultured weekly. All experiments were performed on cells with passage numbers from 6 to 15. [ 3 H]Thymidine Incorporation Assay Cells were seeded into 24-well plates at 5 × 10 4 cells per well and incubated at 37°C overnight. Cells were then treated in triplicates with indicated retinoids in the presence or absence of E/P for 24 hours. After labelling cells with 1 μCi/ml of [ 3 H]thymidine (Amersham, Arlington Heights, IL, USA) for 24 hours, cells were harvested by washing with PBS and 10% TCA, solubilizing with the mixture of 0.1% SDS and 0.1N NaOH. Aliquots were taken for the quantification of radioactivity by the Tri-Carb 2900 TR Liquid Scintillation Analyzer (Perkin Elmer, Wellesley, MA, USA). Incorporation of [ 3 H]thymidine was expressed as a fold change from vehicle control under the same conditions. Luciferase Reporter Assays Cells were seeded in 24-well plates at 5 × 10 4 cells per well. Cells were transiently transfected with 0.5 μg of either a RARE -luciferase or a p53 -luciferase plasmid along with 0.05 μg of pCMV-Renilla luciferase using the SuperFect transfection reagent (QIAGEN, Valencia, CA, USA), following the manufacturer's recommended procedure. Twenty-four hours after transfection, triplicate cultures were treated with retinoids for 24 h in the presence or absence of E/P. The cells were then washed and lysed. The luciferase activities were measured using the DUAL-luciferase Assay System (Promega, Madison, WI, USA), and normalized by pCMV-Renilla luciferase activity for each sample. Real-Time PCR Telomerase Activity Assay Cells were lysed in CHAPS lysis buffer (Chemicon International, Temecula, CA, USA) and incubated at 4°C for 30 min. The lysate was then centrifuged at 12000 × g for 20 min at 4°C, and the supernatant was collected. The protein concentration was measured in each extract using the BCA protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Telomerase activity was determined in duplicates by a quantitative real-time PCR telomerase detection Kit (Allied Biotech, Ijamsville, MD, USA) according to the manufacturer's protocol, using Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA). Quantitative RT-PCR for ERβ gene analysis Cells at subconfluence were treated with retinoids in the presence or absence of E/P for 72 hours. Total RNA was extracted with TRIZOL Reagent from Gibco (Carlsbad, CA, USA) according to the instructions of the manufacture. Single-stranded cDNA was made from 1 μg of total RNA with the Cells-to-cDNA kit (Ambion, Inc., Austin, TX, USA) at 42°C for 15 min. The primers for ERβ were 5'CGA TGC TTT GGT TTG GGT GAT 3' (forward) and 5'GCC CTC TTT GCT TTT ACT GTC 3' (reverse). The primers for GAPDH were 5'CCA TGG AGA AGG CTG GGG 3' (forward) and 5'CAA AGT TGT CAT GGA TGA CC 3' (reverse). All primers were from Integrated DNA Technologies, Inc. (Coralville, IA, USA). cDNA (1 μl) was amplified in duplicates in Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, USA) by using Brilliant SYBR Green QPCR Master Mix from Stratagene (La Jolla, CA, USA). The reaction was carried out at 95°C for 10 min to denature, 40 cycles of 95°C for 30 sec, 55°C for 60 sec, 72°C for 60 sec. ERβ gene was quantified and normalized with external standard GAPDH. Western blot analysis Cells were treated with retinoids in the presence or absence of E/P for 72 hours. Cell lysates were obtained by incubating cells for 30 minutes at 4°C in a buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% NP40, 100 mM NaF, 10 mM MgCl 2 , and protease inhibitor cocktail (Sigma, St. Louis, MO, USA), followed by centrifuged at 12,000 rpm for 20 min. Protein content was determined using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Cell lysates (~30 μg protein) were separated on 10% polyacrylamide gels in the presence of 0.1% SDS with prestained low-range molecular-weight standards (Biorad, Richmond, CA, USA). After transfer, the membranes were blocked and then probed with antibodies against interested proteins as suggested by manufactures, followed by incubation with a peroxidase-conjugated secondary antibody. Immunoreactive bands were developed with an ECL reagent from Amersham (Arlington Heights, Il, USA). All blots were probed with anti-actin to normalize for loading differences. Quantification of gels was carried out using ImageJ software (NIH, Bethesda, Maryland, USA). The p53 (DO-1), p21 (F-5) and RARγ (G-1) mouse monoclonal antibodies, and RXRα (D-20) polyclonal antibody were from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Anti-ERβ (Ab-2), ER (Ab-1), PR (Ab-1), RARα and RARβ mouse monoclonal antibodies, and Bax (Ab-1) polyclonal antibody were from Calbiochem (San Diego, CA, USA). Monoclonal anti-actin, RXRβ and RXRγ were from Sigma (St Louis, MO, USA). Statistical Analysis Statistical differences were analyzed by T-test. Levels of statistical significance were set at p < 0.05. List of abbreviations 9-cis RA: 9-cis-retinoic acid; ATRA: all-trans-retinoic acid; E/P: ?β-estradiol and progesterone; ERβ: estrogen receptor beta; HPR: N-(4-hydroxyphenyl) retinamide; PR: progesterone receptors; RARs: retinoic acid receptors; RXRs: retinoid X receptors. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JZ designed and carried out most of the assays and drafted the manuscript. YT carried out the real-time PCR experiments. SS conceived of the study and participated in its coordination. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555559.xml
514539	Tests for finding complex patterns of differential expression in cancers: towards individualized medicine	Background Microarray studies in cancer compare expression levels between two or more sample groups on thousands of genes. Data analysis follows a population-level approach (e.g., comparison of sample means) to identify differentially expressed genes. This leads to the discovery of 'population-level' markers, i.e., genes with the expression patterns A > B and B > A. We introduce the PPST test that identifies genes where a significantly large subset of cases exhibit expression values beyond upper and lower thresholds observed in the control samples. Results Interestingly, the test identifies A > B and B < A pattern genes that are missed by population-level approaches, such as the t-test, and many genes that exhibit both significant overexpression and significant underexpression in statistically significantly large subsets of cancer patients (ABA pattern genes). These patterns tend to show distributions that are unique to individual genes, and are aptly visualized in a 'gene expression pattern grid'. The low degree of among-gene correlations in these genes suggests unique underlying genomic pathologies and high degree of unique tumor-specific differential expression. We compare the PPST and the ABA test to the parametric and non-parametric t-test by analyzing two independently published data sets from studies of progression in astrocytoma. Conclusions The PPST test resulted findings similar to the nonparametric t-test with higher self-consistency. These tests and the gene expression pattern grid may be useful for the identification of therapeutic targets and diagnostic or prognostic markers that are present only in subsets of cancer patients, and provide a more complete portrait of differential expression in cancer.	Background Studies of differential expression of individual genes often find genes that are up-regulated in some tumors, and down-regulated in others. Microarray studies typically seek to identify differentially expressed genes using use fold-change [ 1 ], t-tests [ 2 ], and models [ 3 - 6 ]. Studies of global gene expression patterns in cancer have focused largely on the identification of novel cancer subtypes via classification [ 7 - 13 ] or the identification of differentially expressed genes [ 14 - 18 ]. Such studies typically use fold-change [ 1 ], t-tests [ 2 ], and models [ 3 - 6 ]. The methods of analysis for identifying differentially expressed genes in data from microarray experiments vary widely [ 20 - 45 ], but all are focused on the question of whether genes are over-expressed or under-expressed in samples in group A (e.g., tumor, or treatment, or metastastic, or responder) compared to samples in group B (e.g., normal, or control, or quiescient, or nonresponder). These patterns can efficiently be referred to as AB (overexpressed in A) and BA (underexpressed in A) patterns. Typically, researchers use study designs that favor biological replication to maximize the ability to detect reproducibly genes that are differentially expressed in a patient population, at a sacrifice of the ability to detect individual-specific patterns of differential expression with technical replication. Most cancers are diseases with heterogeneous etiologies; moreover, the development of every primary tumor in different individuals is a unique biological event. Thus, the expression levels of genes in the individual patient are also important; some important gene dysregulation may be highly specific to each individual. Statistical methods that average gene expression may hide important expressotypes (expression phenotypes). Current tests that compare mean group expression intensities are not likely to find genes that are in fact significantly dysregulated in only a proportion of the individuals in the case population, unless the magnitude of differential expression is very high in the subset of individuals. Unsupervised clustering can be used to attempt to identify unknown partitions, or subgroups within patients, but clustering is not a well-defined method for finding differentially expressed genes, and, upon discovery of novel groups, researchers are restricted to comparing group means, and cannot identify genes that may be dysregulated in subsets of patients where the combined patterns of dysregulation patterns do not suggest coherent subgroups. Results A remarkable pattern emerges when the PPST test is applied to published cancer data sets, including breast cancer [ 7 ], ovarian cancer [ 16 ], colon cancer (epithelial-rich normals only [ 17 , 47 ]), lymphoma [ 18 ], and lung cancer [ 19 ] at the 99th percentile. We find an abundance of AB and BA pattern genes, with roughly the same number of genes called significant under the parametric t-test. We also find large numbers of genes with significant ABA test scores, and some with 'BAB' pattern genes (Table 1 ). There is a marked tendency in most data sets for more ABA (cancer-normal-cancer) type genes than BAB pattern genes. These patterns are also reflected in 'expression pattern grids' of gene with significant s3 (ABA) statistics (Fig. 1 ). These patterns are reproducible at more stringent levels of α (Table 1 ). The capability of the PPST test to identify genes that are in fact differentially expressed in only a subset of patients is made evident by a comparison of genes that are found to be significant under the PPST test, but missed by, for example, the t-test (even without Bonferroni-type adjustments). These are listed in Table 2 , for the lymphoma data18, and notably include B-cell growth factor 1 (IL4; ABA pattern). Furthermore, 'classic' oncogenes such as cyclin D1 are found by the PPST test in the lung cancer data set [ 19 ] are not reported to be significant by the t-test. Cyclin D1 ranks 1009th among significant genes in the colon cancer data set under the t-test but ranks 90th under the PPST test (AB/BA pattern results only). Discussion Our initial results are compelling in that they suggest that we can expect biomarkers of high clinical significance for subsets of patients to be important for distinct subsets of patients. This also suggests that clinical validation of the utility of biomarkers should examine panels of expression biomarkers, not individual biomarkers. Disruption of genomic function via these patterns cannot be studied in the population level biomarker framework for the simple reason that methods that compare, say, group means, will find no difference between the sample groups if the number of case samples found in the two tails are even approximately equal. This is a sensible approach even from within the framework of population-based hypothesis testing, because the PPST test can be expected to be more robust to one or two outliers that might mislead simple parametric tests. Note that a number of genes are 'nearly significant' under the t-test but are strongly significant under the PPST test for the AB/BA patterns (e.g., Table 2 ). Our re-analysis of two independently generated data sets on astrocytoma progression demonstrates the utility of extending analysis to include a search for genes that are differentially expressed in a subset of patients. Of the tests examined, the parametric t-test showed the least internal consistency, while the PPST exhibited the highest internal consistency in identifying progression markers. Comparison to the non-parametric t-tests demonstrates that PPST is most similar to the nonparameteric t-test, but is more self-consistent. While the ABA test showed the least internal consistency across populations, it also exhibited low overlap with any other test, so the genes reported are unique and tend not to be found by others tests, matching expectations. Our results are consistent with Knudsen's 'two-hit' hypothesis on the genomic etiologies of cancer [ 49 ] with some insight into the diversity of genomic pathologies (functional 'hits') that may be relevant in patient populations. Studies of differential gene expression – and its role in the etiology of cancer and its responses to treatment – should seek these types of genes in addition to population-wide biomarkers, because they represent a subset of the genes that are expressed differentially in a significant subset of cancer patients. We recommend a major shift in perspective on the study on gene expression dysregulation away from the study of 'tumor populations' – which do not exist – toward the study of genomic pathologies in individual patients. For example, tumor subtypes are typically characterized by morphological characters, and these classifications may conflict with important expressotype subtypes that do not follow classical morphological tumor classes. Imposition of these subtypes on a study design may interfere with identifying expressotypes that provide high diagnostic, prognostic and theranostic value to the individual – and sets of individuals with similar expressotypes. This view is also consistent with the Hanahan-Weinberg model of oncogenesis [ 50 ], which envisions multiple possible mechanistic strategies to the acquisition of characteristics and capabilities of cancers including self-sufficiency in growth signals, insensitivity to anti-growth signals, tissue invasion & metastasis, limitless replicative potential, sustained angiogenesis and evasion of apoptosis. We also expect that individual cancers in different patients will be found to have evolved unique sets of solutions to each of these problems. Current prevailing methods for finding differentially expressed genes such as fold-change and t-tests do not allow for such complexities. Our comparison of the methods (Table 3 ) highlights the uniqueness of the ABA test. It is an extension of the PPST test; it specifically focuses on genes that are differentially expressed in subsets of patients. This ability is extremely important in search of genes with expression patterns that correlate with drug response. The ABA and the two-tailed t-test are not comparable because the ABA test allows us to find genes that the t-test specifically cannot (genes that are simultaneously overexpressed in some patients while underexpressed in others). Such test will have high variance (leading to a low t-test score) and low mean difference, and will thus not be significant. The PPST and the ABA tests extend our abilities beyond the t-test. Other improvements or even superior alternatives to these tests may be possible. The performance of these tests and all tests described to date for the AB type patterns and now for ABA patterns should be compared using extensive numerical simulations and cross-validation. Developments are needed to determine how best to select a threshold to allow deliberate control of the false positive and false negative error rates. Conclusions The two major conclusions these results suggest are (1) that the most commonly applied tests for identifying differentially expressed genes will miss important genes that are dysregulated in only a fraction of patients, and (2) that important aspects of differential expression may be, to a degree, highly individualistic in most cancers. Some potentially important genes with this form of unusual differential expression (ABA; Table 2 ) would be missed by methods that compare group means, because the means of the two sample groups would be approximately identical, and the variance in tumors would be high, leading to a large error term. The high internal consistency of PPST compared to the non-parametric t-test and our observation that the PPST test exhibited high consistency with the nonparametric t-test suggests that the PPST test may be of interest to researchers interested in identifying both population-level biomarkers and biomarkers important to a subset of patients. An online implementation of this test, it source code (Java), and that for many other methods of analysis, are accessible online in the Cancer Gene Expression Data Analysis tool . It is hoped that the development and application of more approaches like this will lead to a more complete representation of differential expression, leading to more meaningful and specific hypotheses of dysregulation, and thus a better comprehension of how diverse genomic pathologies contribute to the etiologies of cancers, and thereby facilitate the identification of targets that may lead to individual-specific therapies. Methods We have developed a test we call the Permutation Percentile Separability Test (PPST), which attempts to refute a null hypothesis that is slightly different from A = B, but which is capable of detecting AB, BA, ABA and BAB patterns. Under this test, we are interested in the question "are there are statistically significant number of samples in group A (e.g., tumor) that exhibit expression intensities beyond a particular percentile of the observed expression intensities in group B (e.g., normal)?" and vice versa. By 'statistically significant' we mean that the number of samples that exhibit apparent overexpression (or underexpression) exceeds that expected under the null distribution. To test these hypotheses, we count the number of samples in both groups that are found beyond the n th percentile of the samples in the opposite group. This provides two scores, s 1 , and s 2 , for each gene (PPST scores). s 1 is the number of samples in group A that are beyond the upper percentile (say, 95 th ) of group B plus the number of samples in group B that are below the lower 95 th percentile of group A. This measure will tend to be large when all samples in both groups are significantly distinct from the alternate group in the same way (comparisons consistent with A > B). It can also be significant when a surprising number of samples in only one group varies from the expression levels in the alternate group. s 2 is the number of samples with correspondingly opposite pattern (comparisons consistent with B > A). Sample class label permutations are used to generate an arbitrarily large number of permuted data sets. These scores s 1 and s 2 are calculated in each permuted data set to produce unique null distributions for each gene. For the sake of convenience of interpretation, we use - s 2 when reporting s 2 to denote underexpression. Genes with values of s 1 beyond the specified acceptable Type 1 error risk (e.g., α = 5%) are determined to be significantly overexpressed in sample group A relative to B. Individuals in sample group A with expression intensity values over the 95 th percentile of sample group B for a given gene may be considered overexpressed. Similarly, genes with values of s 2 beyond the specified Type 1 risk for s 2 are deemed underexpressed in sample group B relative to A. Varying the percentile threshold allows direct control over the false discovery rate. Test for ABA patterns (ABA Test) Genes that exhibit both significant s 1 and s 2 scores in this comparison may be considered 'ABA pattern genes' (Fig. 1 ); however, for stronger inference, permutation tests are also used to calculate s 3 , to determine, for a given gene , the number of samples from one group (A) that can expected to be distributed both in the upper and lower n th percentile tails of the intensity distribution of that gene in the other group (B); i.e., in the ABA ( s 3 ) or BAB ( s 4 ) pattern. These scores are not redundant to but rather allow for exploration of distribution-wise (upper and lower) false discovery rates. The application of the PPST test to find ABA patterns is called the 'ABA' test. Under the ABA test, differential expression of a gene may be deemed to be significant in both directions at once, i.e., simultaneously significantly over-expressed and under-expressed in a surprising number of patients in the case population. Both the PPST test and the ABA test will perform optimally when the variation in expression intensities in the normal sample population is well characterized. A collection of published microarray data sets we have placed 'on-tap' in the caGEDA (Gene Expression Data Analysis) web application [ 51 ] were subjected to the PPST test and the ABA test. To avoid idiosyncracies that can result from the study of extreme values, we ran the tests at a fairly relaxed Type 1 error risk (α = 0.05 in both tails, or α = 0.10 overall). To compare the self-consistency of the parametric t-test, the nonparametric t-test, the PPST test and the ABA test, we re-analyzed two published data sets from independent astrocytoma progression studies [ 52 , 53 ]. Details of these studies are available in the original papers. In brief, Khatua et al. [ 52 ] studied global gene expression profiles from 6 early stage and 7 late-stage astrocytoma patients, while van den boom et al. [ 53 ] studied global gene expression profiles from 8 early stage and 8 late-stage astrocytoma patients. We calculated the overlap in the gene lists using our online Overlap tool . Abbreviations PPST: permutation percentile separability test. ABA test: a test that can detect genes with both A > B (gene is overexpressed in sample A compared to sample group B) and B < A (gene is overexpressed in sample B compared to sample group A) patterns. s1, s2, s3, s4: measures of the number of samples that exhibit expression intensities beyond a specified percentile in an alternate group; used as scores in the determination of significance under the PPST and ABA tests. Author contributions JLW conceived of the PPST and ABA tests and executed the analyses, SP encoded the methods in caGEDA, TG provided direction, input and scientific motivation for pursuing a test with the capabilities of the PPST and ABA tests, MJB provided, direction, input and coordination of the research. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514539.xml
548520	Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice	Background In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx. Results A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer. Conclusions This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA.	Background Photoreceptor cells play a primary role in vision by capturing light energy and converting it into neural stimuli. These sensory neurons are a shared element in all organisms capable of sensing light. In humans, genetic diseases of the eye are common and the primary site of disease is most frequently photoreceptors (for review see [ 1 - 3 ]). Photoreceptors have been well studied, particularly with respect to the biochemistry and physiology of phototransduction. Insight into the development of vertebrate photoreceptors, however, has lagged behind our understanding of function. Only recently have the first molecular mechanisms regulating photoreceptor development been identified (for review see, [ 2 , 4 ]). Crx (cone-rod homeobox) is an otx-family homeobox gene expressed predominantly in photoreceptors, from early in their development through to the adult ages [ 5 - 7 ]. Crx gene expression is critically dependent upon Otx2, another member of the same homeobox family which is expressed in early photoreceptor cells [ 8 ]. In rod photoreceptors, Crx appears to work in concert with Nrl, a leucine zipper protein that is also restricted in its expression in the retina to rod photoreceptors [ 9 ]. Many photoreceptor-specific genes have putative Crx-binding elements in their regulatory regions [ 10 ], including rhodopsin [ 11 ] and arrestin [ 12 ]. Mutations in Crx have been associated with several human diseases that lead to blindness, including cone-rod dystrophy 2 [ 6 , 13 , 14 ], retinitis pigmentosa [ 14 ], and LCA [ 14 - 16 ]. Based on these data, Crx was hypothesized to play a critical role in the differentiation and maintainance of photoreceptor cells [ 5 , 7 ]. LCA is the most severe genetic disease of photoreceptors (see [ 17 ], for recent review). Affected infants exhibit a complete or near complete absence of vision from birth. Mutations in retinal specific genes, such as Crx, have been associated with LCA [ 14 , 15 ], as well as GUCY2D [ 18 ], RPE65 [ 19 ], AIPL-1 [ 20 ], CRB-1 [ 21 ], and RPGRIP-1 [ 22 ]. There also may be as many as three additional genetically linked loci where genes have not been identified [ 23 ]. Crx mutations in LCA are varied, and include a putative dominant mutation that is proposed to encode a dominant-negative form of Crx [ 14 , 15 ]. Recessive mutations also have been reported and at least one allele encodes a protein with decreased DNA-binding activity [ 16 ]. Histopathological and ultrastructural studies of LCA should enable a better understanding of the disease process, and the design of suitable therapies. Few such studies exist for human LCA (reviewed in [ 17 ]) and the majority of such studies examine the globes of adults with LCA, after the tissue has undergone secondary changes. Only a single study exists where the developing eye of an infant was examined [ 24 ]. Animal models for LCA have recently been reported and have already served to broaden our understanding of the pathology of this disease [ 25 - 28 ]. Since LCA is a clinically and genetically heterogeneous disorder, additional mouse models are in order to allow a full understanding of the many ways in which photoreceptor development can go awry. In addition to their importance as a locus of disease, photoreceptor cells serve as an excellent model for studies in neuronal differentiation. Photoreceptor cells are highly polarized. At their apex, these neurons have a membranous outer segment, which contains proteins involved in the phototransduction cascade. Loss of function mutations in rhodopsin [ 29 ], or the structural protein, peripherin [ 30 ], result in an inability to form outer segments. At the other extremity, photoreceptors terminate with synaptic endings that make contact with the processes of horizontal and bipolar cells [ 31 , 32 ]. Rod spherules establish an invaginating synapse with rod bipolar dendrites and axonal endings of horizontal cells. This synapse is characterized by the presence of a ribbon in the presynaptic cytoplasm. Cone pedicles make invaginating synapses with the dendrites of on-cone bipolar cells and horizontal cells and basal junctions with the dendrites of off-cone bipolar cells. The factors regulating the formation of the photoreceptor synapses are completely unknown. At least one photoreceptor synaptic protein, HRG4, contains a potential Crx target sequence in its transcriptional regulatory sequence [ 33 ]. Few studies of LCA animal models have extended their examination of retinal pathology to the ultrastructural level. Certain features of neuronal differentiation, such as synapse formation, can be detected definitively at this level of analysis. With the hope of understanding the neuropathology of LCA in greater detail, we have analyzed the differentiation of the outer retina in Crx-/- mice at the ultrastructural level. These retinas exhibit several prominent defects. Crx-/- photoreceptors demonstrate a complete block in outer segment formation at the elongation stage. Further, these cells exhibit abnormal synaptic morphology, thereby broadening the function of Crx to photoreceptor synaptogenesis. This represents the first report strongly implicating the process of synapse formation in LCA. Results Multiple pathologies in the outer segment layer in Crx-/- mice A standard knock-out protocol was used to generate mice in which the homeodomain of Crx-/- was targeted and deleted. Generation of these Crx-/- mice has been reported elsewhere [ 34 ]. In this study, in order to characterize further the role of Crx in photoreceptor morphogenesis, the outer retinae from Crx-/- mice were examined using transmission electron microscopy. At postnatal day 21 (P21), when Crx+/+ photoreceptors exhibited robust outer segments (Figure 1A , os), Crx-/- retinas were without a recognizable outer segment layer (Figure 1B ). Crx-/- photoreceptors had inner segments, demonstrating at least limited photoreceptor polarization in the Crx mutant, but the inner segments were extremely short (Figure 2 ). Furthermore, the majority of inner segments showed some morphological differentiation, having approximately as many mitochondria as the control (Figure 1 and 2 ). Occasionally, an inner segment undergoing lysis was noted, appearing swollen or with vacuoles and swollen mitochondria (data not shown). Figure 1 Transmission electron microscopy of the outer retina at P21 in (A) Crx+/+ and (B) Crx-/- retinas. pe, pigmented epithelium. os, outer segments. is, inner segments. onl, outer nuclear layer with photoreceptor nuclei. Scale bar = 5 μm for A and B. Figure 2 Transmission electron micrograph of the outer segment layer of Crx-/- retina at P21. Inner segments of Crx-/- photoreceptors exhibit numerous mitochondria (m indicated by arrow) as in Crx+/+ (Figure 1A). pe, pigmented epithelium. is, inner segments. onl, outer nuclear layer. Scale bar = 2 μm. Photoreceptor inner segments and outer segments are joined by a non-motile connecting cilium that exhibits a characteristic 9 + 0 arrangement of microtubule doublets when viewed in cross-section. At P21, in Crx-/- retinas, numerous cross sections of connecting cilia were noted (Figure 3A and 3B ). Sporadically, connecting cilia contained other than the typical complement of microtubule doublets. For example, in Figure 3A , the connecting cilium labelled by arrowhead 1, shows 7 + 0 doublets. The majority exhibited the characteristic 9 + 0 doublets (arrowhead 2 and 3 in Figure 3A and Figure 3B ). These observations indicate that in addition to inner segment formation, ciliogenesis is also largely intact in Crx-/- photoreceptors. Further, in Crx-/- retinas the retinal pigmented epithelium (PE) appeared normal, at least up to P21 (data not shown), the oldest age examined. Figure 3 Transmission electron micrograph of Crx-/- retina at P21 (A and B), and scanning electron micrograph of Crx-/- at P10 (C) of outer segment layer. (A) Evidence of ciliogenesis in the photoreceptor layer of Crx-/- retina. Nonmotile connecting cilia were observed in cross section (arrowheads 1,2, and 3, for examples). Connecting cilium 1 (arrowhead 1) demonstrated seven microtuble doublets, while cilium 2 and cilium 3 exhibited nine. In A, a displaced cell nucleus (n) appearing pyknotic and abnormal deposition of matrix (mx) material of unknown identity were seen, along with large amounts of membranous vesicles (arrow) which filled the photoreceptor space and appeared to be released from inner segments. Scale bar = 3.7 μm. (B) Nonmotile connecting cilium in cross section, from a Crx-/- photoreceptor, demonstrating characteristic 9+0 radial array of microtubule doublets. Scale bar = 88 nm. (C) Scanning electron micrograph (SEM) of membranous vesicles (arrow shows one example) shed from inner segments of Crx-/- photoreceptors at P10. Figure shows inner segments viewed from the scleral side with the pigmented epithelium removed. Scale bar = 1 μm. In addition to the complete absence of outer segments, Crx-/- retinas exhibited three other notable pathologies in the outer segment layer. First, an abnormal deposition of matrix of unknown identity was noted (Figure 3A , mx). Second, sporadically displaced nuclei were found residing in the space abutting the PE. Occasionally, these nuclei appeared pyknotic (Figure 3A , n); but, more frequently exhibited the heterochromatin pattern typical of photoreceptors (data not shown), strongly suggesting that they belonged to ectopic photoreceptors. The third pathological entity noted in the outer segment layer were numerous small vesicles (Figure 3A arrow) 100 to 200 nm in diameter. They appeared to be emerging from the inner segments, as scanning electron microscopic images showed spherical structures budding from the inner segments (Figure 3C , arrow). In order to characterize further the morphogenesis of Crx-/- photoreceptors, the developing outer segment layer was viewed by scanning electron microscopy at P7, P14 and P21 (Figure 4 ). In Crx+/+ retinas, photoreceptor inner segments, connecting cilia, and the first rudimentary outer segment structures were noted at P7. In the Crx-/- retina, only an occassional connecting cilium was noted emerging from inner segments at this stage (Figure 4A and 4B ). This observation was confirmed by comparison with transmission electron micrographs (Figure 5 ). These differences are the earliest noted differences between Crx+/+ and Crx-/- photoreceptors. At P14, elongating outer segments were noted on Crx+/+ photoreceptors, occasionally demonstrating a paddle-like structure at their apex (Figure 4C , os). In Crx-/- retinae, the vast majority of photoreceptors at this stage demonstrated connecting cilia without outer segments (Figure 4D , cc). Sporadically, Crx-/- photoreceptors would exhibit an irregular structure extending from a connecting cilium (Figure 4D , cc) perhaps representing a malformed outer segment. Such structures were also observed at P21 (Figure 4F , cc). These putative, abnormal outer segments were only rarely noted in Crx+/+ at P14, and never at P21 (Figure 4C and 4E , cc). Further, in Crx-/- photoreceptors, unusually long connecting cilia were noted (Figure 4F , cc). Serial examination of Crx-/- photoreceptors at P7, P10, P14, and P21 by TEM, demonstrated a distinctive lack of any structure interpretable as orderly stacks of discs or forming discs. These data demonstrate a complete absence of normal outer segment formation in the Crx mutant mouse, and the arrest of development of the photoreceptor appendage at the elongation stage of outer segment formation. Figure 4 Outer segment morphogenesis in Crx-/- photoreceptors. Scanning electron microscopy of developing photoreceptors viewed from the scleral side with the pigmented epithelium removed at P7, P14, and P21 for Crx+/+ (A, C, and E) and Crx-/- (B, D, F) littermates. In Crx+/+ retina, numerous connecting cilia (A, cc) were evident at P7 with rudimentary outer segments. After P7, in Crx+/+ outer segment elongation occurs. Initially, outer segments have a paddle-like structure (C, os) which is later shed (E, os). In Crx-/- photoreceptors, few connecting cilia were observed at P7 (B, cc). After P7, connecting cilia were more numerous and occasionally a malformed outer segment was noted extending from a connecting cilium (D and F, cc). These were rarely observed in Crx+/+ and only at P14 (C, cc*). At P21, abnormally long connecting cilia are noted in Crx-/- (F, cc). Scale bars = 10 μm Figure 5 Transmission electron micrographs of Crx-/- photoreceptors in the photoreceptor layer at P7. (A) Photoreceptor layer of Crx+/+ retina demonstrating nascent outer segment structures (arrow) emerging from photoreceptor connecting cilia (cc). (B) Crx-/- photoreceptors exhibited connecting cilia (cc) at this early stage, however, nascent outer segment structures were not observed. Scale Bar = 1 μm. Finally, the morphology of the malformed Crx-/- photoreceptors was compared to rhodopsin-/- and peripherin-/- photoreceptors. Rhodopsin and peripherin are two photoreceptor-specific genes whose expression is significantly downregulated in the Crx-/- retinae [ 10 , 34 , 35 ]. Loss of function mutations in each of these genes separately have been reported to result in a failure to elaborate outer segments [ 29 , 30 ]. Photoreceptors from these two mutant mice examined by SEM from the scleral side appeared highly similar to Crx-/- photoreceptors (compare Figure 4F to Figure 6A and 6B ). Figure 6 Scanning electron microscopy of peripherin-/- (A) and rhodopsin-/- (B) photoreceptors at P21, viewed from the scleral side with the pigmented epithelium removed. cc, connecting cilium. is, inner segment. Scale bar = 10 μm. Crx is necessary for the formation of photoreceptor terminals In a previous study, we demonstrated that forced expression of a dominant-negative allele of Crx in developing rods blocked formation of both rod spherules in the outer plexiform layer (OPL) and outer segments [ 7 ]. To expand on these studies, the ultrastructure of photoreceptor synapses was examined in Crx-/- retinas. In Crx+/+ retinas at P21, newly mature rod spherules were abundant (Figure 7A ). The sperules were blunt or club-shaped structures with a single ribbon associated with a single invaginating synapse (Figure 7A , arrow indicates one example; Figure 8A and 8B ). Two processes from horizontal cells were situated on either side of the synaptic ridge (Figure 8B , labelled H) and one or more dendrites of rod bipolar cells occupied a central position (Figure 8B , bipolar labelled B). Cone terminals are large, flat pedicles that exhibit many invaginating synapses containing separate sets of horizontal and bipolar cell processes. Each pedicle contains numerous ribbons. These terminals were much less common than spherules in Crx+/+ retinas at P21 (Figure 7 , box). In the OPL of Crx-/- retinas, photoreceptor terminals were highly disorganized at P21 (Figure 7B , arrows). Processes containing synaptic vesicles and ribbon-like structures were apparent, suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. In addition, many terminals appeared to contain multiple ribbons (Figure 8C and 8D , r) not tethered to the plasma membrane and occasionally in perinuclear positions (Figure 8D ). Figure 7 Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas. (A) In Crx+/+ retina at P21, newly formed rod spherules were abundant (arrow demonstrates one example). The spherules were club-shaped and contained a single invaginating synapse with one ribbon complex. Cone terminals form large, flat pedicles that contain many invaginating synapses with separate ribbon structures. These terminals were more scarce, but apparent in Crx+/+ retinas at P21 (one example in box). (B) In the outer plexiform layer (OPL) of Crx-/- retinas, photoreceptor terminals appeared highly disorganized at P21 (arrows). Well-formed pedicles and spherules were not evident. Putative terminals containing ribbon-like structures were apparent, suggesting at least limited generation of synapse components. Many terminals appeared to contain multiple ribbon-like structures, instead of a singule ribbon. For example, terminal 1 and 2 contained two ribbons each, whereas terminal 3 appeared to contain only one. opl, outer plexiform layer. Scale bar = 2 μm. Figure 8 Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas at P21. (A) Crx+/+ rod spherules contained a single invaginating synapse with one ribbon complex. The spherule was a blunt or club-shaped structure. (B) Crx+/+ rod terminals contained a single ribbon structure (r). Two processes, from horizontal cells (h), contacted the rod laterally. An additional process, the postsynaptic bipolar dendrite (b), was situated more centrally. Terminals were filled with synaptic vesicles. One coated vesicle originatinf from the cell membrane was observed (arrow). (C) In the OPL of Crx-/- retinas, photoreceptor terminals appeared highly disorganized. Putative terminals containing synaptic vesicles and ribbon-like structures were apparent (arrows), suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. Further, many terminals appeared to contain multiple ribbon-like structures (r). The majority of these ribbons were not associated with the synaptic membrane, but instead were found free floating and, in some instances, were perinuclear (D, arrow). H, horizontal cell; B, bipolar cell; N, nucleus; r, ribbon. (A) Scale bar = 500 nm, (B) Scale bar = 250 nm, (C and D) Scale bar = 500 nm. Discussion In this study, an ultrastructural analysis of Crx-/- photoreceptors was carried out. As Crx mutations have been associated with Leber's congenital amaurosis, the findings in this study broaden our understanding of the pathology of this disease. Two prominent pathologies were characterized in the Crx-/- retina: (1) An absolute block in outer segment morphogenesis was noted, with the block occuring at the elongation stage of outer segment formation; (2) Crx-/- photoreceptors exhibited a severe perturbation in synapse formation. This represents the first report of a synaptogenesis defect in an animal model of LCA. Crx-/- photoreceptors cannot complete outer segment morphogenesis Mutations in Crx represent one of a collection of gene mutations that lead to an outer segment formation defect. Homozygous null mutations in the peripherin/RDS gene [ 36 ] or in rhodopsin [ 29 ] lead to a failure of outer segment formation. The deficits in peripherin-/- and Crx-/- photoreceptor morphogenesis were found to be very similar. Vesicular structures in Crx-/- photoreceptors were observed that were similar to those previously noted in the rds/peripherin-/- mouse. It was initially proposed that these vesicles were due to the breakdown of outer segment membranes that were not properly recruited to the outer segments in the absence of peripherin, or were from the result of the breakdown of the microvilli of Müller cells [ 30 ]. Strong support in favor of the former explanation was provided by Nir and colleagues who demonstrated the presence of rhodopsin protein in these vesicles using immunoelectron microscopy against a rhodopsin epitope [ 37 ]. Further, as shown here, the vesicles appear to bud from the inner segments themselves. In developing photoreceptors, an extraordinary growth process occurs whereby the outer segment is generated from the nascent connecting cilium (see [ 38 ] and references therein). Peripherin/RDS and ROM-1 proteins (localized in disc rims) and the opsin proteins (localized throughout the discs) have important roles in the structural integrity of mature outer segments (see [ 39 , 29 ]). ROM-1-/- mice produce disorganized outer segments with large disks [ 40 ]. Crx, by virtue of being a transcription factor, presumably controls genes that are responsible for the building and perhaps maintenance of the outer segment structure, including rhodopsin and peripherin. Using northern blots [ 34 ], microarrays [ 10 ], and serial analysis of gene expression (SAGE) [ 35 ], we have defined a large number of genes that are altered in their expression level in Crx-/- mice. We found that rhodopsin expression was severely diminished in Crx-/- animals, and peripherin mRNA was reduced by approximately 30%. Transgenic mice with variable levels of expression of wild type rhodopsin exhibit rod degeneration [ 41 ], indicating the importance of the level of rhodopsin expression. In addition, the timing of rhodopsin expression may be very important, as indicated by studies in Drosophila. In Drosophila , rhodopsin ( ninaE ) is expressed in photoreceptors R1–R6. In ninaE null mutants, the rhabdomere, a structure analogous to vertebrate outer segments, fails to develop in R1–R6 photoreceptors [ 42 ], reminiscent of the situation in rhodopsin-/- mice [ 29 ]. An intriguing experiment by Kumar et al. [ 43 ] demonstrated a temporal requirement for rhodopsin expression during rhabdomere development. In ninaE null flies, a ninaE transgene under the control of a heat shock promoter was subjected to various temperature shifts during development. Heat shock during the normal time of rhodopsin onset resulted in substantial and long-lasting rescue of photoreceptor structure and transient rescue of photoreceptor physiology. However, expression shortly before or after this critical period failed to rescue, suggesting that rhodopsin expression during a discrete window of time in development is essential for proper rhabdomere morphogenesis. This result is consistent with observations in the rat wherein rhodopsin onset occurs with strict timing in the developmental history of most rods in vivo [ 44 ]. Thus, through its regulation of rhodopsin levels, or perhaps through control of the kinetics of the up-regulation of rhodopsin beginning at about P6, Crx may be regulating outer segment morphogenesis. The similarty of the two cases may extend further. At present, the closest Crx relative in Drosophila is Otd, the founding member of the class of homeobox genes to which Crx belongs. Interestingly, in one hypomorphic allele of Drosophila otd , otd uvi , photoreceptor morphogenesis is also disrupted [ 45 ]. We found that there are many other genes that are dependent upon Crx. Those that are expressed at a lower level in the Crx-/- retina, such as rhodopsin and peripherin, comprise many that are either enriched or specific to photoreceptors in their expression [ 35 ]. They include enzymes that are important in lipid metabolism, protein folding and transport, as well as in other processes that one might envision would be important in building a structure such as the outer segment. In situ hybridization using probes from this collection of genes has revealed that some of them have their RNA localized to the inner segment, a finding typical for proteins targeted to the outer segment. Future analyses of the function of these genes might reveal their role in outer segment biogenesis. Finally, polarization of photoreceptors was found to be largely intact, as was ciliogenesis. Another LCA gene, CRB1, and a related gene CRB3, have been implicated in ciliogenesis in in vitro models [ 46 ]. The Drosophila homologue of CRB1, Crumbs, has been implicated in photoreceptor morphogenesis [ 47 ]. However, the spontaneously occurring mouse mutant in CRB1, the Rd8 mouse, develops shortened outer segments that subsequently degenerate [ 48 ], suggesting that photoreceptor polarization and synaptogenesis are intact in this mutant. While CRB1 and Crx have been both linked to LCA, further work is necessary to determine if their function is linked in retinal development. Synaptogenesis is perturbed in Crx-/- photoreceptors The Crx-/- mouse demonstrates the most severe abnormality of photoreceptor synapses reported to date. The peripherin-/- mouse develops a normal complement of photoreceptor terminals which then degenerate as the photoreceptors are lost [ 30 ]. Also, similarly in rhodopsin (Rho) and cyclic nucleotide-gated channel, alpha-3 (CNGA3) double knockout mice (Rho-/-, CNGA3-/-), synapses are reported to form normally [ 49 ]. These observations demonstrate that photoreceptor synaptogenesis can occur in the absence of outer segment formation. In keeping with this observation is the fact that some electroretinogram activity is present in peripherin-/- mice, suggesting that minimal phototransduction is present in these mice, enough to drive activity at the photoreceptor synapse. In vitro studies wherein synapse elements are formed in the absence of proper outer segment development and, therefore, possible absence of light-dependent photoreceptor activity, have indicated the independence of phototransduction and synapse formation, at least for the initial stages [ 50 , 51 ]. These data then suggest that the fact that the Crx-/- photoreceptors do not have proper synaptic endings is not due to a lack of outer segment formation. A more likely explanation is that Crx plays a role in photoreceptor synapse formation, perhaps by regulating directly, or indirectly, important genes in this process. Using immunohistochemistry, we examined the expression of common pre-synaptic terminal proteins, including KIF3a, SV2, and synaptophysin, and were unable to observe qualitative differences between Crx-/- and control tissue at P14 (data not shown). Examination of their RNA levels by SAGE showed no significant difference for all 3 genes, though very few tags were recovered from these genes and thus the analysis of RNA levels may not be significant [ 35 ]. However, since other genes expressed in photoreceptors were significantly altered in their expression level in the Crx-/- mouse, there are many candidates that could be important for photoreceptor morphogenesis. Tags from three genes from proteins expressed in photoreceptor terminals were found to be decreased in a statistically significant fashion, namely the HGF-regulated tyrosine kinase substrate, the CRIPT protein, and synaptotagmin 1 (Blackshaw and Cepko, unpublished data). An example of a gene that was increased in the Crx-/- retina is HRG4 (a homologue of the C. elegans Unc119 gene) (Blackshaw and Cepko, unpublished data) which encodes a component of the ribbon synapse [ 33 ]. The fact that it is upregulated might indicate a response to the lack of proper terminal structures. Several other genes encoding putative cytoskeletal elements also were increased (e.g. microtubule associated protein 4) or decreased (e.g. cofilin 1) in the Crx-/- retina, with P values of <.005. It is not known whether any of these genes are involved in building or regulating synaptic structures, but they are now genes that might lead to a better understanding of the construction and function of the relatively unique structure of the ribbon synapse. Abnormal photoreceptor terminal formation was noted in a study that examined retinal development in the laminin beta2 chain-deficient mouse [ 52 ]. Several pathologies were noted in these mice. First, laminin beta2 chain-deficient mice displayed abnormal outer segment elongation, but a more mild phenotype than that of the Crx-/- mice; the outer segments were reduced by 50% in length. Also photoreceptor terminals were perturbed in laminin beta2 mutants, but again the phenotype was more subtle then that of Crx-/- mice. The outer plexiform layer of the beta2-deficient retinas demonstrated only 7% normal invaginating synapses, while the remainder had various pathologies, including floating synaptic ribbons, as seen here. The mechanistic relationship of these two molecules, if any, in photoreceptor morphogenesis is unknown to date. The mRNA for laminin beta2 was not detected in the SAGE study of the relative RNA levels in Crx-/- and Crx+/+ and thus we cannot comment on whether the levels of RNA for laminin beta2 were altered. Crx-/- mice are a model for LCA Crx has been implicated in three photoreceptor diseases that result in human blindness, cone-rod dystrophy2, Leber's congenital amaurosis, and retinitis pigmentosa (for review, see [ 53 ]). The cone-rod dystrophies (CRDs) are characterized by loss of cone-mediated vision in the first decade of life or later, with concomitant or subsequent loss of rod-mediated vision [ 54 ]. Conversely, RP is notable for initial loss of rod function, followed by loss of cone-mediated vision [ 55 ]. The majority of known genes responsible for human genetic blindness, encode proteins expressed almost exclusively, or exclusively, in photoreceptors, particularly in the outer segment [ 35 ]. Many of these proteins are required for phototransduction or outer segment structure. The mechanisms whereby mutations in rod-specific genes, such as rhodopsin, lead eventually to cone degeneration in RP remain obscure. Mutations in Crx were the first, and still one of a very few examples of a transcription factor mutation leading to photoreceptor disease. LCA is a disease in which there is little or no photoreceptor function in infancy; thereby, likely developmental in etiology ([ 17 , 56 ] for review). The Crx-/- mouse may be an excellent model for studying the pathology of this disorder, particularly the subtype of the disorder where Crx mutations are involved. The vast majority of histopathological studies of LCA in human tissue have been derived from adult patients with LCA where secondary changes are likely to be present. Indeed in animal models of LCA, secondary reactive and/or degenerative changes occur early after the abnormal formation of retinal tissue [ 57 ]. The only study in human tissue derived from a human 33-week retina with proposed RPE65 mutations was reported to have abnormal retinae at this early stage [ 24 ]. These authors report cell loss, including thinning of the photoreceptor layer. In addition, they claim in the text to have seen aberrant synaptic and inner retinal organization, although their examination of photoreceptor synapses unfortunately are not presented in the data section of the paper. Given the scarcity of available human tissue, the characterization of the primary pathology of LCA will require animal models. In the current study, we present data that argue that, in addition to outer segment morphogenesis, synaptogenesis may also be critically impaired in at least a subset of LCA. Methods Mice Crx-/- mice were generated as reported elsewhere [ 34 ]. Rhodosin-null mice [ 29 ] were obtained from Paul Sieving (University of Michigan). Rds mice were acquired from Jackson Laboratory. Transmission electron microscopy Littermate Crx-/- and wildtype pups were perfused in 1% formaldehyde and 0.5% glutaraldehyde at various postnatal stages. The eyes were then enucleated, and the cornea and lens were removed. The eye cup was immersed in fixative (1% formaldehyde and 2.5% glutaraldehyde) for 3 to 4 hours at 4°C. The sclera was then partially removed and the retinas were sliced into small pieces and fixed (1% paraformaldehyde and 2.5% glutaraldehyde) overnight at 4°C. These procedures were found optimal for maintaining the structural integrity of the photoreceptor outer segments. After fixing, the tissue was washed 2X in PBS for thirty minutes per wash. The tissue was then postfixed in a 1% osmium tetroxide/1.5% potassium ferrocyanide mixture for 2 hours at 4°C. Staining was carried out for 30 minutes in 1% uranyl acetate in maleate buffer (pH = 6.0) at room temperature followed by 1% tannic acid in 0.1 M cacodylate buffer (pH = 7.4) for thirty minutes. The specimens were then dehydrated and embedded in Epon/Araldite. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Jeol JEM-1200EX electron microscope. Scanning electron microscopy Specimens used for SEM required removal of the retinal pigment epithelium (PE), enabling visualization of the outer surface of the neural retina. Retinae from Crx-/-, rhodopsin-/-, RDS, or wildtype eyes were dissected free from PE in a dispase solution and fixed in 1.25% glutaraldehyde and 1.0% formaldehyde overnight at 4°C. Tissue was then washed 5X in cacodylate buffer and dehydrated in ascending grades of ethanol. Tissues were subsequently critical point dried in carbon dioxide. All specimens were mounted and coated with sublimated gold-palladium by the sputtering technique. Micrographs were obtained with a Jeol JSM-35CF scanning electron microscope. Authors' contributions EM and ER conducted transmission electron microscopy. EM performed scanning electron microscopy. TF and EM generated, characterized and maintained the Crx-/- mouse line. CC participated in the design and coordination of the study and all data analysis. EM and CC drafted the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548520.xml
545945	Prognostic significance of metallothionein expression in renal cell carcinoma	Background Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival. Patients and methods The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data (histological cell type, tumor nuclear grade, pathologic stage and patients' survival). Results Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25–50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival. Conclusions The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter.	Background Metallothioneins (MTs) were firstly discovered by Margoses and Valle in 1957 [ 1 ] as cadmium (Cd) binding proteins. Later, Piscator [ 2 ] documented a marked increase of MT in Cd exposed rabbits, as a metal detoxification mechanism. MTs are a family of heavy metal binding proteins with a large degree of sequence homology that have been described in most vertebrate and invertebrate species. They are single-chain proteins, with molecular weight of approximately 6000 Da, characterized by a very high proportion of cysteine residues (30%), resulting in several high affinity Cd and/or zinc (Zn) binding sites [ 3 ]. There are two major isoforms, referred to as MT-I and MT-II [ 4 ], resolvable through ion exchange chromatography, that have closely related but distinct amino acid sequences and are distributed in most adult mammalian tissues. Recently, a further charge-separable MT isoform (MT-0) [ 4 ], and genes for two MT isoforms with restricted tissue distribution MT-III (brain neurons) [ 5 , 6 ] and MT-IV (stratified epithelia) [ 7 ] have been described. The potential for wider tissue distribution of MT-III was suggested by recent studies demonstrating the presence of MT-III mRNA and protein in the adult and developing human kidney [ 8 , 9 ]. MT-I and MT-II isoforms are usually expressed in low levels, but are inducible by a variety of metal ions, hormones, inflammatory cytokines and xenobiotics [ 10 - 12 ]. Induction of MTs is important in detoxification and metal ion homeostasis [ 9 ], in protection against reactive oxygen species [ 10 ] and in tissue regeneration [ 13 - 15 ]. MT expression deficiency implicated in carcinogenesis [ 16 ] and possible relation of MT over expression and resistance of tumors to anti-cancer therapy [ 17 ] has provided evidence of the importance of MT expression in cancer. MT over expression, detected immunohistochemically, has been described in a variety of human tumors, in relation to different stages of tumor development and progression [ 18 ]. The involvement of MT and Zn, in processes such as p53 gene activation and protein structure has been referred [ 16 , 19 ]. There is evidence that some human tumors contain high levels of MT, nevertheless, the importance of MT expression in carcinogenic evolution and in patients' survival is not yet fully understood. In organs such as kidney, colon and liver, normally implicated in metal ions homeostasis, MT protein is apparently expressed. It would be of great scientific importance to elucidate the pattern of MT expression in tumors developed from these organs, since it could not only delineate the role of MT in carcinogenic transformation but could also provide prognostic information for patients' outcome. The aim of the present study was to examine the expression of MT in human renal cell carcinoma (RCC) and to correlate the MT positivity, the pattern and extent of MT expression with tumor histological cell type and nuclear grade, pathologic stage and patients' survival. Patients and methods Forty three consecutive patients, 31 men and 12 women, who underwent nephrectomy for RCC comprised the group of our study. Their age ranged from 33 to 85 years (mean age 59.6 ± 11.1). Tumors were histologically classified as clear cell type in 32 cases, papillary type in 2 cases, chromophobe cell type in 4 cases and sarcomatoid type in 5 cases. The 8, 2, 16, 6 and 9 tumors were pathologically staged as T1 or T2N0M0, T2N+M0, T3N0M0, T3N+M0 and T4N+M+, respectively as per the TNM classification [ 20 ]. The grade of nuclear atypia according to Fuhrman Grading system [ 21 ] was: grade I in 11 cases, grade II in 15 cases, grade III in 6 cases, and grade IV in 11 cases. The patients were followed up from 2 up to 144 months (5 lost in follow up), mean 65.4 months, median 39 months. Immunohistochemistry Sections of 5 μm thickness were deparaffinized in xylene and rehydrated in graded alcohol series. To remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% (v/v) hydrogen peroxide in methanol in dark, for 30 min, at room temperature. Non-specific antibody binding was then blocked using normal rabbit serum (Dakopatts, Glostrup, Denmark) diluted 1:5 in phosphate buffered saline (PBS), for 20 minute. A mouse (IgG1k) monoclonal antibody that reacts with both human MT-I and -II isoforms (Zymed, San Francisco, California, USA) was used in this study. The sections were then incubated for 1 hour, at room temperature, with the primary antibody diluted 1:50 in PBS. After three washes with PBS, sections were incubated for 30 minute at room temperature with rabbit, peroxidase conjugated, anti-mouse serum (Dakopatts) diluted 1:200 in PBS and rinsed three times with PBS. Sections were then incubated with swine, peroxidase conjugated, anti-rabbit serum (Dakopatts) diluted 1:100 in PBS and rinsed three more times with PBS. The resultant immune peroxidase complexes were developed in 0.5% (v/v) 3,3'-diaminobenzidine hydrochloride (DAB; Sigma, Saint Louis, MO, USA) in PBS containing 0.03% (v/v) hydrogen peroxide. Sections were counterstained with Harris' hematoxylin and mounted in gelatin (Sigma). Control slides included in MT immunostaining procedure consisted of specific tissues previously shown to express MT (lung cancer) as positive controls, whereas the primary antibody was replaced by PBS in the case of negative controls. Scoring system The stained sections were independently assessed by the pathologists (A.K., S.T., E.M.) without prior knowledge of the clinical data as previously described [ 22 ]. Specimens were considered as "positive" for MT when more than 5% of tumor cells within the section were positively stained. The intensity of staining was graded as mild (+), moderate (++), and intense (+++). To further evaluate the importance of staining extent, cases were stratified into 3 groups according to the percentage of cells staining positive for MT: group A, 0–25%; group B, 26–50%; and group C, >50% of MT positive cells. The pattern of MT staining was also characterized as cytoplasmic only, nuclear only, and both cytoplasmic and nuclear. Statistical analysis Correlation between immunohistochemical data (MT intensity and extent of staining) and clinicopathological data (histologic cell type, nuclear grade, pathologic stage) was assessed using the Chi-square test. The association of intensity and extent of MT immunohistochemical staining with survival was determined by comparing Kaplan-Meier survival curves constructed for different patient groups, and were compared using the log-rank test. Results Tubular cells but not glomeruli and interstitial cells of normal autologous renal tissue stained positive (both in the nucleus and the cytoplasm) for MT, although the intensity and extent varied significantly. Positive MT expression was prominent in 21 out of 43 cases (49%), while 22 out of 43 ones (51%) were MT negative. As far as the intensity of staining is concerned, low/moderate intensity was observed in 8 cases, while intense staining was evident in 13 cases. The pattern of positive MT immunostaining observed was either cytoplasmic (7 out of 21 cases, 33.3%) or cytoplasmic and nuclear (14 out of 21 cases, 66.6%). In certain cases of clear cell RCC membranous staining was also observed. Nuclear pattern of staining only, was not observed in any of the RCC cases examined (Figure 1 ). The extent of MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 out of 43 cases, in a percentage 25 up to 50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. Figure 1 Detection of MT expression by immunohistochemistry. Intense membranous, cytoplasmic and nuclear staining in a case of clear cell type RCC (x330). There was no significant difference between MT intensity of staining and the histological types of RCC cases examined (χ 2 = 5.61, p = 0.46). No statistically significant differences were also observed between MT intensity of staining and stage (χ 2 = 9.24, p = 0.32), or patients' survival (log rank test: 4.75, p = 0.09) in the cases of RCC examined (Figure 2 ). Statistically significant correlation was found between MT intensity of staining and histological grade, where higher intensity of staining was found in cases presenting high histological grade (χ 2 = 13.63, p = 0.03). However, no such correlation was found in the case of clear cell RCC (χ 2 = 4.75, p = 0.57). Figure 2 Cancer-specific survival of patients according to intensity of staining for MT (no staining, green line; mild/moderate staining, blue line; intense staining, red line). No statistically significant difference was detected (p = 0.09). Non statistical correlation was found among MT extent of staining and histological types (χ 2 = 2.54, p = 0.86), stage (χ 2 = 7.12, p = 0.52) and grade (χ 2 = 6.24, p = 0.39) of RCC cases examined. Statistically significant inverse correlation was found between MT extent of staining and patients' survival (log rank test: 6.59, p = 0.037) (Figure 3 ). Again, this was not observed in case all other histological types but clear cell RCC were excluded (log rank test 3.36, p = 0.186). Figure 3 Survival curves of patients according to MT extent of staining (0–25% of cells, green line; 26–50% of cells, blue line; >50% of cells, red line). Statistically significant inverse correlation was found between extent of staining and patients' survival (p = 0.037). Discussion MT expression has been observed in different types of human tumors [ 18 , 22 ], including neoplasias of the urogenital tract [ 23 - 28 ]. However, the biological mechanisms underlying MT over expression in tumors, or the consequences of this over expression are not currently well understood. MT is highly expressed in fetal life [ 10 ]; the reappearance of this fetal characteristic in tumors suggests its participation in cellular growth and differentiation [ 16 , 18 , 19 ]. Developing and adult kidneys consistently express MT-I and MT-II mRNA [ 29 ] and the corresponding protein [ 28 ], while the MT-III isoform is also expressed in developing renal tissue, adult proximal tubule and renal cell carcinoma cell lines [ 8 , 9 ]. The MT-0 isoform is absent in adult kidneys but it can be found in nonneoplastic tissue from renal and transitional cell carcinoma [ 30 ]. The isoform specific expression of MT in RCC has not been so far investigated. Using a monoclonal antibody that reacts to both MT-I and MT-II, we demonstrated positive immunoreaction in 49% of our RCC cases, a percentage close to the 55.7% reported by Tüzel et al [ 24 ], using the same antibody. Zhang and Takenaka [ 24 ] used another commercially available monoclonal antibody with unspecified specificity towards different MT isoforms and found positive immunoreaction for the MT protein in 33% of their cases, while such report is not included in the study of Izawa et al [ 23 ] who used a polyclonal antibody prepared by their own laboratory. The cytoplasm and nucleus of normal and malignant cells may both express MT but there is no conclusive data on the functional significance of their subcellular distribution. The majority of the cases in our study expressed MT in both cytoplasm and nucleus, the expression being cytoplasmic only in one third of the cases. Analogous pattern has been reported in other studies [ 23 - 25 ] while the membranous staining observed in some of our cases as well as in two previous series [ 24 , 25 ] could be explained by the nature of clear cell carcinoma. Differential subcellular expression of MT may be related to either cell proliferation or the induction of apoptosis [ 31 , 32 ]. Recently, Kondo et al [ 33 ] pointed out the importance of subcellular distribution of MT in drug resistant-prostatic cancer cells, in which the nuclear MT expression rather the cytoplasmic counterpart seems to predominantly confer resistance to cisplatin. On the other hand, nuclear pattern of MT immunostaining has already been correlated with cellular response to stress stimuli [ 34 ]. The function of MT to protect the cell from apoptosis could be an explanation for MT over expression observed in high grade cases of RCC [ 24 ], as MT is not etiologically correlated with the apoptotic process. In certain tumors MT over expression has been associated with unfavorable prognostic characteristics such as advanced stage and poor differentiation [ 18 ]. In our study, MT intensity of staining did not correlate with stage while it showed an inverse correlation with histological grade. Our results are in accordance to those of other investigators [ 23 , 25 ], who also found an inverse relationship between MT immunoreactivity and tumor grade. In contrast, Zhang and Takenaka [ 24 ] reported positive associations between MT expression and tumor grade. The inverse relationship between MT immunohistochemical expression and tumor grade may suggest a role of MT in cellular growth and differentiation, and reflect alterations of intracellular processes leading to a gradual decline of Zn storage and to the subsequent decrease in MT expression. Although no association was found between MT staining intensity and survival, the reduced extent of MT expression significantly correlated with prolonged survival as reported elsewhere [ 25 ]. The extent of MT expression may offer an additional, prognostic factor in patients suffering from RCC. MT concentrations might also prove useful in predicting the efficacy of a particular cancer treatment protocols. Several types of transformed cells enriched for MT have been shown to exhibit greater resistance to chemotherapeutic agents [ 17 ]. In this context, the thiolate sulfur of the cysteine residues are thought to act as sacrificial scavengers for radicals and alkylyating agents [ 10 ]. Thus, MT might serve as a Cu and Zn reservoir, where their supply may negatively affect the growth of tumor cells. MT could also participate in tumorigenesis by sequestering and then donating these essential bivalent cations to proteins of tumor cells in order to meet metabolic requirements [ 35 ]. Conclusions The current data on the expression of MT in RCC cases examined emphasize the necessity to investigate larger numbers of patients with RCC comparing the staining profile of different MT isoforms with other clinico-pathological parameters and survival status of patients. Currently, it is unknown whether the presence of MT in renal carcinoma cells is related to the induction or inhibition of apoptosis or plays an active role in cell proliferation. Since cytokines may also induce MT expression and immunotherapy is the only, albeit with limited efficacy, available treatment for RCC, the intensity and extent of MT immunostaining should be studied in correlation to the immune status of RCC patients before and after immunotherapy. Thus, it will be possible to elucidate the potential role of MT in renal cell carcinogenesis, as well as its clinical usefulness as a tumor marker and as a tool for selecting patients for adjuvant immunotherapy.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545945.xml
548508	Mutagenicity testing with transgenic mice. Part II: Comparison with the mouse spot test	The mouse spot test, an in vivo mutation assay, has been used to assess a number of chemicals. It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484). It is however rather insensitive, animal consuming and expensive type of test. More recently several assays using transgenic animals have been developed. From data in the literature, the present study compares the results of in vivo testing of over twenty chemicals using the mouse spot test and compares them with results from the two transgenic mouse models with the best data base available, the lacI model (commercially available as the Big Blue ® mouse), and the lacZ model (commercially available as the Muta™ Mouse). There was agreement in the results from the majority of substances. No differences were found in the predictability of the transgenic animal assays and the mouse spot test for carcinogenicity. However, from the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo .	Background This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test. The assessment of the potential genotoxicity of chemicals in vivo is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing, in vitro data must be confirmed by testing in in vivo conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity [ 1 ]. In the mid 1980s, the mouse spot test [ 2 ] was suggested as a complementary in vivo test to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens [ 3 ]. The mouse spot test has been used to assess a number of chemicals (see e.g. Additional file 1 , see separate file). It is at present the only in vivo mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484 [ 4 ]). However to achieve an acceptable sensitivity, a large number of animals are necessary and it is therefore an expensive type of test and seldom used. More recently assays using transgenic animals have been developed for testing in vivo gene mutagenicity. The two transgenic mouse models with the best data base available are the lacI model (commercially available as the Big Blue ® mouse), and the lacZ model (commercially available as the Muta™ Mouse). The present study compares the results of in vivo testing of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models. Descriptions of test systems Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated in utero at gestation day 9–11 with the test substance. The exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. The in utero exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls [ 2 , 4 ]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) [ 5 ]. Gene mutagenic but also clastogenic effects are detected by this test system. Transgenic mouse models The transgenic mutation test systems the lacI model (Big Blue ® mouse), and the lacZ model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test Methods Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used. Results and Discussion Comparison of the mouse spot test with transgenic mouse model systems In the literature search chemicals have been identified that had been tested using the spot test and the Muta™ mouse assay (n = 20) or the Big Blue ® mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in Additional file 1 . The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate, N -ethyl- N -nitrosourea, N -methyl- N '-nitro- N -nitrosoguanidine, N -methyl- N -nitrosourea, 4-nitroquino-line-1-oxide, N -nitrosodiethylamine, N -nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and N -propyl- N -nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene). The positive results obtained with the Big Blue ® mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo[ a ]pyrene, 1,3-butadiene, cyclophosphamide, N -ethyl- N -nitrosourea, N -methyl- N -nitrosourea, N -nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems. With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in in vitro gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of in vivo studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice. Although no data on carcinogenicity on mice is available on N -propyl- N -nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats [ 113 ] and in vitro gene mutation assays revealed clearly positive results. The following substances did not show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see Additional file 1 ). These are therefore discussed in more detail here; for references see Additional file 1 . Table 1 Characteristics of the Muta™ mouse assay and the Big Blue ® mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test Term# Calculation* for the mouse spot test Calculation* for Muta™ and/or Big Blue ® mouse combined ** Sensitivity 84% (16/18) 79% (15/18) Specificity 0 (0/0) 0 (0/0) Positive predictability 100% (16/16) 100% (15/15) Negative predictability 0 (0/2) 0 (0/3) Overall accuracy 84% (16/18) 79% (15/18) # Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in the STS Positive predictivity = % of positive results in the STS that are carcinogens Negative predictivity = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results : carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used *: judged as positive in transgenic assays if positive in one of the two test systems For methylmethanesulfonate, the weak positive results were judged as positive. Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test). 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay [ 19 ] but negative results in the mouse spot test [ 12 , 13 ]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two in vitro test systems indicated gene mutagenic activity supporting results in the transgenic assay [ 15 - 18 ]. 2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay [ 28 ] but negative results were obtained in the mouse spot test [ 29 ]. This negative result in the mouse spot test is in contrast to all other in vivo gene mutation assays on rodents and insects which revealed positive results [ 27 ]. Furthermore, gene mutagenic activity was detected in in vitro test systems and carcinogenic effects were observed in long-term studies on mice [ 27 ]. The results in the Muta™ mouse assay are in accordance with these data. Di-(2-ethylhexyl)phthalate Negative results in the mouse spot test [ 51 ] are in agreement with the negative Big Blue ® assay [ 11 ]. Furthermore no gene mutagenic or questionable activity was reported in in vitro tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed. Hydrazine This substance induced mutagenic effects in the mouse spot test [ 72 ] but negative results were observed in the Muta™ mouse assay [ 71 ]. Other in vivo as well as in vitro test systems revealed gene mutagenic effects [ 70 ]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay [ 71 ]. Studies on other in vivo genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive [ 20 ]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure. Methyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse [ 19 , 57 , 75 - 77 ] and negative results in the Big Blue ® mouse [ 63 - 65 , 78 ]. In the mouse spot test this carcinogenic substance is mutagenic [ 3 ] as well as in other gene mutation assays in vitro and in vivo [ 73 , 74 ]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. [ 19 ] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects [ 1 ]. Mitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure [ 93 ] but positive results were obtained with the mouse spot test [ 2 , 3 ] and other gene mutation assays in vitro and in vivo with this carcinogenic substance [ 90 - 92 ]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay [ 93 ]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained. Trichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay [ 117 ], the mouse spot test was positive [ 3 ], but this result is possibly related to contaminations with epoxides [ 116 ]. Further in vitro and in vivo assays on gene mutation resulted in weak positive, questionable, or negative effects [ 116 ]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. [ 117 ]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele [ 118 ], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions [ 1 ]. Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue ® mouse assay combined) and the mouse spot test are documented in Table 1 . Data on 18 substances (see Additional file 1 ) are available on carcinogenicity in mice and mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test). Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems. Advantages and disadvantages of both test systems Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired [ 1 ]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150–200 melanoblasts and each melanoblast has 4 coat colour genes under study [ 2 , 5 ]. In the transgenic Big Blue ® mouse, for example, 30–40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of each cell of the animal [ 1 ]. Other factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig [ 2 ] suggested that 30–40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control [ 5 ] and at least two dose groups are used (OECD guideline 484 [ 4 ]). Therefore, the mouse spot test is an expensive type of in vivo test. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender [ 119 - 121 ]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic [ 2 ]. A comparison of both test systems is presented in Table 2 . Table 2 Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue ® and Muta™ mouse assays Mouse spot test [2-5] Transgenic mouse mutation assay [1, 122] Age restriction Exposure restricted to embryos at gestation day 9–11 Usually less than 3 months Toxicokinetics and metabolism Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution Target tissue Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs Type of mutation Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions Dependency of effects on application route Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected Target gene/cell 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk Number of animals Animal consuming test system Not more than 5 animals per gender per dose necessary Specificity of test system Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity Characterisation of mutations by molecular methods Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the "molecular signature" of a particular mutagen by DNA sequence analysis with standardized methods Possibility of parallel investigation of several genetic endpoints No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other in vivo genotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange Endogenous versus foreign target gene The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no "normal" mutational target Costs Expensive type of in vivo test Less expensive Conclusions Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations in vivo in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo . Author's contributions UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript. Supplementary Material Additional File 1 Results in the transgenic mouse assay versus mouse spot test Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548508.xml
555565	Patterns of lung cancer mortality in 23 countries: Application of the Age-Period-Cohort model	Background Smoking habits do not seem to be the main explanation of the epidemiological characteristics of female lung cancer mortality in Asian countries. However, Asian countries are often excluded from studies of geographical differences in trends for lung cancer mortality. We thus examined lung cancer trends from 1971 to 1995 among men and women for 23 countries, including four in Asia. Methods International and national data were used to analyze lung cancer mortality from 1971 to 1995 in both sexes. Age-standardized mortality rates (ASMR) were analyzed in five consecutive five-year periods and for each five-year age group in the age range 30 to 79. The age-period-cohort (APC) model was used to estimate the period effect (adjusted for age and cohort effects) for mortality from lung cancer. Results The sex ratio of the ASMR for lung cancer was lower in Asian countries, while the sex ratio of smoking prevalence was higher in Asian countries. The mean values of the sex ratio of the ASMR from lung cancer in Taiwan, Hong Kong, Singapore, and Japan for the five 5-year period were 2.10, 2.39, 3.07, and 3.55, respectively. These values not only remained quite constant over each five-year period, but were also lower than seen in the western countries. The period effect, for lung cancer mortality as derived for the 23 countries from the APC model, could be classified into seven patterns. Conclusion Period effects for both men and women in 23 countries, as derived using the APC model, could be classified into seven patterns. Four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. Factors other than smoking might be important, especially for women in Asian countries.	Background Worldwide, over one million people die of lung cancer each year [ 1 ]. In the US, lung cancer is the most common cause of cancer deaths in both sexes [ 2 ], and mortality rates in women have risen 500% since 1950 [ 3 ]. In the European Union countries, although age-standardized mortality rates have decreased for most cancer sites, lung cancer mortality rates have significantly risen in women [ 4 ]. A rising death rate from lung cancer has also been observed in Taiwan. Between 1971 and 2001, age-standardized lung cancer mortality rates per 100 000 per year in Taiwan have increased sharply, from 12.66 to 32.93 among men and from 7.83 to 14.94 among women [ 5 ]. Today, in Taiwan, lung cancer is the leading cause of cancer death in women and the second leading cause in men [ 5 ]. Epidemiological studies have shown that cigarette smoking is the major cause of lung cancer in both sexes [ 6 - 8 ]. However, smoking habits do not seem to be the main explanation of the epidemiological characteristics of female lung cancer mortality in Asian countries [ 9 - 13 ], where the prevalence of smoking is relatively low but lung cancer mortality rates are relatively high. Factors other than smoking habits might contribute to the variability in lung cancer mortality. Long-term geographical trends in cancer mortality can provide useful information to assist etiological research. However, Asian countries are often excluded from studies of geographical differences in trends in lung cancer mortality. In order to clarify the changing patterns of lung cancer mortality worldwide, we examined lung cancer trends from 1971 to 1995 among men and women for 23 countries including four from Asia – Taiwan, Japan, Singapore, and Hong Kong. In addition, we plotted the pattern of mortality rate in these countries by using the age-period-cohort (APC) model. Methods We used data from the World Health Organization (WHO) and Taiwan to analyze secular trends from 1971–1995 in lung cancer mortality in both men and women. Mortality data provided by WHO were relatively incomplete in some countries, so we analyzed data from 22 countries and Taiwan. The twenty-two countries – Hong Kong, Singapore, Japan, Portugal, Poland, Italy, Cuba, Spain, Hungary, France, Greece, Finland, United States, England and Wales, Netherlands, Belgium, Canada, Australia, New Zealand, Denmark, Norway and Sweden – are members of WHO. The data for Taiwan came directly from the Office of Statistics, Department of Health in Taiwan. Since rates for the under 30-year age group are often based on few deaths, and rates for the over 80-year group might be affected by competitive death effects, only rates for the age range 30 to 79 were considered, so as to ensure adequate reliability of the estimates. Lung cancer mortality rates between 1971 and 1995 were analyzed in five consecutive five year periods (1971–1975, 1976–1980, 1981–1985, 1986–1990, and 1991–1995) and in five year age groups. Statistical methods Age-standardized mortality rates (ASMR) were calculated using the world population for 1976 as the reference [ 14 ]. Percent changes in the ASMR were calculated as [(ASMR 1991–1995 - ASMR 1971–1975 ) / (ASMR 1971–1975 )] × 100. In order to apply the APC model, the matrix of age-specific death rates was calculated for each 5-year calendar period (from 1971–1975 to 1991–1995) and age group (from 30–34 to 75–79). The effect of period of death in the APC model was evaluated by a log-linear Poisson model with a modified method as described by Osmond and Gardner [ 15 ]. Briefly, the estimate of period effect results from minimizing the weighted sum of the Euclidean distances from the three possible two-factor models (age/period; age/cohort; period/cohort). The weights used in the minimization process were based on the goodness-of-fit measures of each two-factor model. In this study, these were taken as the inverse of the deviance statistics. The sum of period effects were constrained to be zero. These "effects" can be interpreted as logarithms of "relative" risks. These relative risks were estimated separately for men and women. A computer program written in the SAS/IML language [ 16 ] was developed to perform the above calculations. Results Age-standardized mortality rates from lung cancer per 100 000 population per year in 23 countries for 1971 to 1995 are listed for men in Table 1 and for women in Table 2 . Trends in the ASMR varied by sex. From 1971 to 1995, in men, the rates progressively increased in nine countries (Portugal, Hungary, Taiwan, Spain, Poland, Japan, Norway, France and Greece), progressively decreased in two countries (England and Wales and Finland) and increased then declined in the others. In women, rates increased between 1971–1975 and 1991–1995 in 23 countries except for Hong Kong, Cuba, and Spain, with the highest increasing rate observed in the Netherlands (223.46%). Table 1 Age-standardized mortality rate (per100 000 person years) from lung cancer in males in 23 countries, 1971–1995 ASMR Country 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 Percent increase* Rank $ Portugal 31.88 40.51 45.96 54.40 59.48 86.57 1 Hungary 97.10 116.05 138.49 161.34 180.91 86.31 2 Taiwan 32.08 39.80 49.95 54.24 58.44 82.17 3 Spain 56.92 68.84 80.68 93.80 101.49 78.30 4 Poland 93.91 113.65 135.11 152.23 158.34 68.60 5 Japan 38.88 46.55 53.40 56.96 59.44 52.87 6 Norway 45.20 53.24 62.61 65.43 67.36 49.03 7 France 74.81 87.45 93.16 99.32 100.08 33.77 8 Greece 79.55 93.63 99.23 103.77 105.44 32.54 9 Italy 94.59 110.47 123.02 125.63 117.10 23.80 10 Hong Kong 94.96 118.63 116.24 116.89 110.13 15.97 11 Canada 96.68 108.38 116.23 118.13 107.71 11.42 12 USA 109.60 117.87 121.16 120.33 115.23 5.13 13 Denmark 101.45 109.36 116.83 113.21 103.74 2.26 14 Sweden 48.34 52.24 50.10 48.56 48.21 -0.27 15 Singapore 92.34 114.24 115.29 102.90 91.47 -0.95 16 Belgium 146.33 163.41 166.59 155.70 144.87 -1.00 17 Cuba 75.86 76.35 76.59 76.16 72.90 -3.90 18 Netherlands 151.10 162.14 160.49 148.73 129.40 -14.36 19 Australia 99.14 100.74 100.11 90.81 80.86 -18.44 20 New Zealand 99.81 104.55 100.52 93.07 79.31 -20.54 21 England and Wales 159.59 152.45 136.82 120.40 101.21 -36.58 22 Finland 142.74 142.51 126.97 105.58 90.20 -36.81 23 percent increase (%) = 100 × (ASMR 1991–1995 - ASMR 1971–1975 ) / (ASMR 1971–1975 ) $ Rank by percent increase Table 2 Age-standardized mortality rate (per100 000 person years) from lung cancer in females in 23 countries, 1971–1995 ASMR Country 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 percent increase Rank $ Netherlands 8.42 11.06 15.46 20.80 27.24 223.46 1 Norway 8.86 11.02 15.61 21.21 26.42 198.24 2 Denmark 21.25 29.28 40.86 49.78 58.41 174.91 3 Canada 17.94 25.44 34.93 43.53 49.13 173.81 4 USA 24.96 33.85 43.41 51.50 56.51 126.46 5 Hungary 16.75 19.34 22.99 29.11 36.83 119.89 6 Sweden 11.36 13.47 16.96 20.68 24.50 115.72 7 Poland 11.40 13.66 16.41 19.68 22.90 100.83 8 Australia 14.91 19.70 23.61 26.47 28.47 90.99 9 Belgium 10.74 12.31 13.79 16.45 19.86 84.90 10 New Zealand 22.58 26.88 30.83 36.53 38.00 68.29 11 France 7.07 7.44 8.42 9.97 11.89 68.19 12 Finland 8.71 11.35 12.39 13.48 14.54 67.02 13 Taiwan 16.03 19.55 23.13 25.69 26.43 64.94 14 Italy 10.69 12.12 13.51 15.09 16.23 51.86 15 England and Wales 30.45 35.75 40.06 43.52 42.84 40.68 16 Portugal 6.74 7.14 7.97 8.56 9.43 39.97 17 Japan 11.90 13.60 14.97 15.35 15.73 32.25 18 Greece 12.64 13.57 13.21 13.86 14.28 12.97 19 Singapore 29.66 34.34 37.07 36.01 31.25 5.36 20 Hong Kong 44.18 48.99 48.01 48.32 43.14 -2.34 21 Cuba 27.87 26.63 26.90 27.77 27.03 -3.02 22 Spain 8.54 7.99 7.32 7.01 7.58 -11.27 23 * percent increase(%) = 100 × (ASMR 1991–1995 - ASMR 1971–1975 ) / (ASMR 1971–1975 ) $ Rank by percent increase Table 3 shows the sex ratio (male:female) of the ASMR for lung cancer for five consecutive five- year periods in 23 countries. The sex ratio was greater than one in each five-year period, indicating that the ASMR from lung cancer was higher in men than in women. Among the 23 countries, the trend in the sex ratio gradually decreased for the whole period in most countries except for Spain, France, Italy, Poland, Greece, Portugal, Hungary, Cuba and the Asian countries. For example, in 1971–1975, the highest sex mortality ratio was seen in the Netherlands with a sex ratio of 17.95, the ratio then gradually decreasing to a value of 4.75 by 1991–1995. The change in the sex ratio of mortality in the Netherlands might be due to the increase and then decrease in male lung cancer mortality and simultaneously to the increase in female lung cancer mortality. On the other hand, the ratio gradually increased in Spain. This might be due to Spain having the the fourth highest increase in male lung cancer mortality accompanied by a progressive decrease in female lung cancer mortality from 1971–1975 through 1986–1990, followed by a slight increase. Table 3 Sex ratio of the age-standardized mortality rate from lung cancer in 23 countries, 1971–1995 Ratio Country 1971–1975 1976–1980 1981–1985 1986–1990 1991–1995 Mean Range Rank* Belgium 13.62 13.27 12.08 9.46 7.29 11.15 6.33 1 Netherlands 17.95 14.67 10.38 7.15 4.75 10.98 13.20 2 Finland 16.40 12.56 10.25 7.83 6.20 10.65 10.20 3 Spain 6.66 8.62 11.02 13.39 13.39 10.62 6.73 4 France 10.58 11.76 11.06 9.97 8.42 10.36 3.34 5 Italy 8.85 9.12 9.11 8.33 7.21 8.52 1.91 6 Poland 8.24 8.32 8.23 7.73 6.91 7.89 1.41 7 Greece 6.30 6.90 7.51 7.49 7.39 7.12 1.21 8 Portugal 4.73 5.67 5.77 6.36 6.31 5.77 1.63 9 Hungary 5.80 6.00 6.03 5.54 4.91 5.66 1.12 10 Australia 6.65 5.11 4.24 3.43 2.84 4.45 3.81 11 Norway 5.10 4.83 4.01 3.09 2.55 3.92 2.55 12 England and Wales 5.24 4.26 3.42 2.77 2.36 3.61 2.88 13 Canada 5.39 4.26 3.33 2.71 2.19 3.58 3.20 14 Japan 3.27 3.42 3.57 3.71 3.78 3.55 0.51 15 New Zealand 4.42 3.89 3.26 2.55 2.09 3.24 2.33 16 Denmark 4.77 3.74 2.86 2.27 1.78 3.08 2.99 17 Sweden 4.26 3.88 2.95 2.35 1.97 3.08 2.29 18 Singapore 3.11 3.33 3.11 2.86 2.93 3.07 0.47 19 USA 4.39 3.48 2.79 2.34 2.04 3.01 2.35 20 Cuba 2.72 2.87 2.85 2.74 2.70 2.78 0.17 21 Hong Kong 2.15 2.42 2.42 2.42 2.55 2.39 0.40 22 Taiwan 2.00 2.04 2.16 2.11 2.21 2.10 0.21 23 Rank by mean The mean values of the sex ratio of the ASMR from lung cancer in Taiwan, Hong Kong, Cuba, Singapore, and Japan were 2.10, 2.39, 2.78, 3.07, and 3.55, respectively, with a range of 0.17 to 0.51 over the five-year periods. These values were not only relatively constant over time, but were also lower than seen in the western countries. For example, the lowest sex ratio of 2.10 was seen in Taiwan, with the sex ratio remaining a relatively constant value over the entire period. Only age is adjusted when the ASMR is calculated. However, both age and cohort effect are adjusted in the APC model. The period effects for males and females from the APC model applied to the data from the 23 countries could be classified into seven patterns: 1) an increasing trend in both sexes, seen in Taiwan (Figure 1 ), Norway, Japan, Hungary, and Portugal; 2) a sharply increasing trend in women, with little change seen in men seen in USA (Figure 1 ), Sweden, Poland, Italy, Canada, Belgium, Denmark and France; 3) a sharply increasing trend in women, and a sharply decreasing trend in men, seen in New Zealand (Figure 1 ), Finland, Australia and Netherlands; 4) a more gradual increasing trend in women, but a sharply declining trend in men, seen only in England and Wales (Figure 1 ); 5) a decreasing trend in both sexes, seen in Singapore (Figure 1 ) and Hong Kong; 6) a decreasing and then a gradually increasing trend in women, but a sharply increasing trend in men, seen in Spain (Figure 1 ) and Greece; and 7) a relatively steady trend in both sexes, seen only in Cuba (Figure 1 ). In most countries, the long-term trend in the period effect as derived from the APC model was similar to the trend of ASMR. It is worth noting that the trend in the ASMR for female lung cancer increased and then declined in Singapore and Hong Kong. After adjusting for the cohort effect, however, a decreasing trend in the period effect was observed in Singapore and Hong Kong. Figure 1 Secular trend in the relative risk (RR) of dying from male and female lung cancer, 1971–1995, based on analyses using the age-period-cohort model in Taiwan, England and Wales, New Zealand, USA, Singapore, Spain and Cuba Discussion We found that the sex ratio in lung cancer mortality varied over time and geographically. After adjusting for age and cohort effects, seven patterns could be identified using the APC model, indicating that some countries had a similar trend in lung cancer mortality. Koo and Ho [ 17 ] indicated that smoking was a strong risk factor in the west and worldwide where there were high rates of smoking in men. We appreciate that lung cancer mortality rates for a given year depend on smoking habits over a period before that year; however, it is not possible to get data on smoking prevalence before 1975 from WHO. Therefore, based on the smoking prevalence obtained from World Health Organization (Table 4 ), the first, second, third, and fifth highest sex ratios (male:female) of smoking prevalence among the 23 countries were in Taiwan, Hong Kong, Singapore, and Japan, respectively. However, the sex ratios of ASMR from lung cancer in the four Asian countries were significantly lower than in the western countries. That is, the four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. This fact is of particular interest. Table 4 Smoking prevalence in males and females, and their sex ratio, in 23 countries Country Prevalence Male Female Male: female ratio Rank Data source $ Taiwan 55.1 3.3 16.7 1 Adult (18 years & older), 1996 Hong Kong 27.1 2.9 9.3 2 Adult (15 years & older), 1998 Singapore 26.9 3.1 8.7 3 Adult (18–64 year olds), 1998 Portugal 30.2 7.1 4.3 4 Adult (15 years & older), 1995–1996 Japan 52.8 13.4 3.9 5 Adult (15 years & older), 1998 Poland 39.0 19.0 2.1 6 Adult, 1998 Italy 32.2 17.3 1.9 7 Adult (14 years & older), 1998 Cuba 48.0 26.3 1.8 8 Adult (15 years & older), 1995 Spain 42.1 24.7 1.7 9 Adult (16 years & older), 1997 Greece 46.0 28.0 1.6 10 Adult, 1994–1998 Hungary 44.0 27.0 1.6 11 Adult (18 years & older), 1998–1999 France 39.0 27.0 1.4 12 Adult (18 years & older), 1997 Finland 27.0 20.0 1.4 13 Adult (15–64 year olds), 1999 USA 27.6 22.1 1.3 14 Adult (18 years & older), 1997 Netherlands 37.0 30.0 1.2 15 Adult (15 years & older), 1998 Belgium 31.0 26.0 1.2 16 Adult (15 years & older), 1999 Canada 27.0 23.0 1.2 17 Adult (15 years & older), 1999 Australia 27.1 23.2 1.2 18 Adult (16 years & older), 1995 New Zealand 26.0 24.0 1.1 19 Adult (15 years & older), 1998 Denmark 32.0 30.0 1.1 20 Adult (14 years & older), 1998 England and Wales 29.0 28.0 1.0 21 Adult (16 years & older), 1996 Norway 33.7 32.3 1.0 22 Adult (16–74 year olds), 1998 Sweden 17.1 22.3 0.8 23 Adult (16–84 year olds), 1998 Rank by Male: female ratio $ Data were obtained from the World Health Organization Dietary fat consumption has been found to be positively related to lung cancer mortality [ 18 - 20 ]. Our data from Japan, Taiwan and Cuba women (on Tables 2 and 5 ) also indicated that the percent increase of fat consumption was positively related to the percent increase of ASMR by using the Spearman's rank correlation coefficient. Further study of factors other than smoking, like fat intake, on lung cancer mortality seems warranted, especially for women in Asian countries (Japan and Taiwan). Table 5 Changes in annual per caput fat consumption in 23 countries Fat Consumption Country 1970 1990 Percent increase Rank & Taiwan 38.0 136.8 260.0% 1 Hong Kong 71.4 $ 135.6 # 89.9% 2 Spain 88.9 137.0 54.1% 3 Portugal 78.6 120.6 53.4% 4 Japan 54.6 79.3 45.2% 5 Greece 101.9 138.4 35.8% 6 Hungary 115.3 153.5 33.1% 7 Italy 114.4 151.0 32.0% 8 France 126.4 161.3 27.7% 9 Cuba 67.6 85.1 25.9% 10 New Zealand 115.4 134.7 16.7% 11 USA 119.6 138.8 16.1% 12 Canada 113.7 127.1 11.8% 13 Australia 117.8 130.6 10.9% 14 Netherlands 132.0 140.9 6.7% 15 Poland 103.9 110.3 6.2% 16 Sweden 116.8 122.6 5.0% 17 Finland 123.6 124.2 0.5% 18 Norway 131.6 127.7 -3.0% 19 England & Wales 141.7 135.8 -4.2% 20 Denmark 140.7 132.6 -5.8% 21 Belgium - - - - Singapore - - - - *Data were obtained from FAO & Rank by percent increase $ :1961, # : 1995 Conclusion Period effects for both men and women in 23 countries, as derived using the APC model, could be classified into seven patterns. The four Asian countries have a relatively low sex ratio in lung cancer mortality and a relatively high sex ratio in smoking prevalence. Factors other than smoking might be important, especially for women in Asian countries. Competing interests The author(s) declare that they have no competing interests. Authors' contributions YPL was responsible for the development of intellectual content and the study design, collected and analyzed the data, interpretation of the results, manuscript drafting and the critical revisions of manuscript. YCH was responsible for the development of intellectual content, interpretation of the results and manuscript drafting. GWL was responsible for data coding and entry and statistical analyses. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555565.xml
516448	Stress, burnout and doctors' attitudes to work are determined by personality and learning style: A twelve year longitudinal study of UK medical graduates	Background The study investigated the extent to which approaches to work, workplace climate, stress, burnout and satisfaction with medicine as a career in doctors aged about thirty are predicted by measures of learning style and personality measured five to twelve years earlier when the doctors were applicants to medical school or were medical students. Methods Prospective study of a large cohort of doctors. The participants were first studied when they applied to any of five UK medical schools in 1990. Postal questionnaires were sent to all doctors with a traceable address on the current or a previous Medical Register . The current questionnaire included measures of Approaches to Work, Workplace Climate, stress (General Health Questionnaire), burnout (Maslach Burnout Inventory), and satisfaction with medicine as a career and personality (Big Five). Previous questionnaires had included measures of learning style (Study Process Questionnaire) and personality. Results Doctors' approaches to work were predicted by study habits and learning styles, both at application to medical school and in the final year. How doctors perceive their workplace climate and workload is predicted both by approaches to work and by measures of stress, burnout and satisfaction with medicine. These characteristics are partially predicted by trait measures of personality taken five years earlier. Stress, burnout and satisfaction also correlate with trait measures of personality taken five years earlier. Conclusions Differences in approach to work and perceived workplace climate seem mainly to reflect stable, long-term individual differences in doctors themselves, reflected in measures of personality and learning style.	Background Sir William Osler (1849–1919), one of the most distinguished physicians of the nineteenth and early twentieth century, recognised that only some doctors are happy in their professional lives: "To each one of you the practice of medicine will be very much as you make it – to one a worry, a care, a perpetual annoyance; to another, a daily joy and a life of as much happiness and usefulness as can well fall to the lot of man."[ 1 ] The modern medical workplace is a complex environment, and doctors respond differently to it, some finding it stimulating and exciting, whereas others become stressed and burned out from the heavy workload. The medical workplace also provides an environment where new skills are continually being learned, both as a result of medical knowledge evolving and because a doctor's work changes, in part due to career development and progression through different jobs. In an important study, Delva et al [ 2 ] used earlier research [ 3 , 4 ] to develop two separate instruments for studying how doctors work, the Approach to Work Questionnaire (AWQ) and the Workplace Climate Questionnaire (WCQ). In Canadian physicians [ 2 , 5 ] the AWQ showed three separate factors, which were called Surface-Rational , Surface-Disorganised , and Deep (see table 1 ). These approaches related to different methods and motivations for continuing medical education. Those with a deep approach preferred independent and problem-based learning and motivation was internal. Surface-rational and surface-disorganised approaches were primarily driven by external motivation, with the preferred mode of continuing education learning being independent for the surface-rational, and in consultations for the surface-disorganised. The WCQ showed three dimensions, called Choice-Independence, Supportive-Receptive , and Workload (see table 1 ), which correlated with the AWQ. Doctors reporting Choice-Independence and Supportive-Receptive work environments had a Deeper approach, whereas those describing an environment dominated by Workload tended to be more Surface-Disorganised. Some doctors are unhappy with their work, which can manifest as stress (usually assessed by the General Health Questionnaire) or burnout, which has three separate components of emotional exhaustion, depersonalisation and reduced personal accomplishment (see table 2 ). Greater stress and burnout in doctors are related to the personality trait of neuroticism or 'negative affectivity' [ 6 ]. The AWQ and WCQ provide a snapshot of a doctor's learning environment and approach to work at one particular time, as also do measures of stress and burnout. A key question, as Deary et al recognised [ 6 ] when considering stress, is the extent to which different approaches to work and the climate of the workplace are consequences of the workplace or of the doctor . At first sight it might seem that the workplace itself has to be the primary force driving both workplace learning and workplace climate. However, it is also possible that approaches to learning and work mainly depend upon pre-existing differences among doctors, differences that may already have manifested earlier in the doctors' careers. The AWQ bears a strong formal similarity to the surface, deep and strategic study habits and learning styles identified by the Study Process Questionnaire (SPQ), which assesses the motivations and approaches used by students in higher education (see table 3 ). The similarity is not accidental since the AWQ was developed by adapting items from Entwistle and Ramsden's Approaches to Study Inventory [ 7 ], which has a similar factor structure to that of the Study Process Questionnaire [ 8 ]. It is therefore expected that there may be significant continuities across approaches to study and approaches to work. In this paper we describe a large cohort of UK doctors, typically aged 29 or 30 at the time of the study, who have been qualified for five or six years, who are practising as SHOs or SpRs in hospital or are in general practice, and who previously had been studied when aged 17 or 18 at application to medical school in autumn 1990 [ 9 ], in their final year at medical school [ 10 ] and as PRHOs [ 11 ]. The main interest here will be in the extent to which a doctor's present approaches to work and their workplace climate, as well as their stress and burnout, relate to earlier measures of study habits and personality at application to medical school and subsequently. Method Participants In the autumn of 1990 a questionnaire was sent to all individuals with European Community postal addresses who had applied to any of the five UK medical schools taking part in the study [ 9 ]; they represented about 70% of all applicants and acceptances for medical school in that year. The response rate was 93%. Students who were accepted for entry in 1991, 1992 or 1993 were followed up in their final year at medical school (1995–1998), when the response rate was 56%, and at the end of their PRHO year (1996–1999), when the response rate was 58%. In 2002 a tracing exercise searched the Medical Register and Medical Directory from 1995 to 2002 to find the addresses of as many doctors as possible who were in the original survey, and who were known not to have died, left medical school during basic medical sciences, or otherwise to be no longer in the survey. For study design see figure 1 . Questionnaire Questionnaires were sent to all individuals with current or recent GMC addresses. The questionnaire consisted of a single folded A3 sheet of paper (4 A4 sides). Included in the present questionnaire (described in the results as '2002') were the 12-item General Health Questionnaire (GHQ) [ 12 ]; an abbreviated version of the Maslach Burnout Inventory (aMBI), which has three sub-scales, Emotional Exhaustion, Depersonalisation, and Personal Accomplishment [ 13 , 14 ]; a three-item scale modelled on the aMBI, which assesses Happiness with a Medical Career [ 15 ]; an abbreviated version of the Study Process Questionnaire, which has three sub-scales of Surface, Strategic and Deep learning [ 16 , 17 ]; an abbreviated questionnaire assessing the 'Big Five' personality dimensions of Neuroticism, Extraversion, Openness to Experience, Agreeableness and Conscientiousness [ 15 , 18 ]; and abbreviated versions of the Approach to Work Questionnaire (aAWQ) and the Workplace Climate Questionnaire (aWCQ) [ 19 ], each of which has three sub-scales, and for which a detailed description is provided in the Supplementary Information (see Additional file: 1 ) [ 2 ]. The GHQ, aMBI and personality questionnaire had also been administered previously in the PRHO survey, and the SPQ had been administered in the Applicant and Final year surveys. Procedure Questionnaires, along with a postage-paid return envelope, were posted at the beginning of December 2002. Two reminders were sent to non-respondents. Although the official closing date was 25 th March 2003, a few questionnaires were returned up until the end of August 2003. Statistical analysis used SPSS version 10.5, and structural equation modelling used LISREL 8.52. Results The tracing exercise looked for 2,912 individuals thought to have completed basic medical sciences and entered a clinical course. Eighty-nine had never been on the UK Medical Register, and either had failed finals, had never registered, or had emigrated. Of 2,823 individuals who were traced, 2,754 doctors were on the 2002 Register, 7 returned to the Register during 2002, and 64 were on an earlier Register. Of 2,823 questionnaires sent, 176 were returned by the Post Office as undeliverable, 10 doctors were travelling and hence uncontactable, and2 had died. Of the remaining 2,635 doctors, 1,668 returned questionnaires, giving a response rate of 63.3%. There was no evidence of response bias (see Supplementary Information see Additional file: 1 ). Respondents The mean age of respondents on 1 st December 2002 was 30.4 years (SD 1.86, range 28.3 – 49.2). There was substantial variation in the scores on the aAWQ and the aWCQ, and the factor structures of the aAWQ and aWCQ were similar to those reported elsewhere [ 19 ] (see Supplementary Information see Additional file: 1 ). There was also substantial variation on the measures of stress, burnout and satisfaction with medicine as a career, with 21.3% of doctors (345/1617) reporting GHQ scores of 4 or more, the conventional level of 'caseness'. Approaches to work and learning were correlated with climate in the workplace, and as in the Delva et al study, the highest correlations were for a surface-disorganised approach correlating with high workload, and a deep approach correlating with a supportive-receptive environment and with choice-independence (table 4 ). Approaches to work Table 4 shows correlation of the stress measures with approaches to work and study habits. The largest correlations were of a surface-rational approach with a strategic learning style, and a deep approach to work with a deep learning style. In each case the correlations were not only highly significant when study habits were measured in the final year at medical school, six or seven years earlier, but were also very significantly correlated with study habits measured at selection, twelve years earlier. Correlations of approaches to work and stress, burnout and satisfaction with medicine were generally small, and generally were only with measures taken in 2002, and not with measures taken as a PRHO, five or six years earlier. The sole exception was that a surface-disorganised approach correlated with high stress as measured by the GHQ, both in 2002 and with stress when the doctors were PRHOs. Workplace climate Table 5 shows correlations between the workplace climate and study habits, stress, burnout and satisfaction with medicine. In contrast to the associations with approaches to work, the workplace climate showed only small correlations with study habits, but showed strong correlations with stress, burnout and satisfaction with medicine. In particular, high stress in the PRHO year showed very significant correlations with measures in 2002 of a perceived high workload, a less supportive-receptive environment, and less choice-independence. In addition, emotional exhaustion both in 2002 and in the PRHO year were related to a high perceived workload in 2002. Personality Table 6 shows the correlations of approaches to work and workplace climate with the 'Big Five' measures of personality, measured both in 2002 and also measured five to six years previously when the doctors were PRHOs. The surface-disordered approach to work is associated with high neuroticism and low conscientiousness, the PRHO correlations also being highly significant in each case. Neuroticism, both in 2002 and as a PRHO, is also associated with a perceived high workload (although in contrast to its prediction of a surface-disordered approach, conscientiousness is not a significant correlate of workload). The deep approach to work and learning is associated with being extravert and with greater openness to experience, and again the measures taken six years earlier are predictive. Finally a supportive-receptive work climate is associated with greater reported agreeableness, both in 2002 and six years earlier as a PRHO. There were no substantial correlations between personality and the surface-rational approach to work or choice-independence in work climate. Multiple regressions Tables 4 to 6 show a large number of correlations, which are not always straightforward to interpret, both because they are numerous and because many variables are themselves inter-correlated. Multiple regression was used to clarify the relationships (for technical details see Supplementary Information see Additional file: 1 ). Each individual measure of the aAWQ and aWCQ was regressed on the measures of study habits at application (n = 3) and in the final year (n = 3), of stress and burnout during the PRHO year (n = 4) and in 2002 (n = 4), and of personality in the PRHO year (n = 5) and in 2002 (n = 5). Alpha for entry was set at p < 0.0001 in view of the large sample size and the number of independent variables. The variables that were significant are shown in tables 4 , 5 and 6 and 3 in italics. Of particular interest are variables that show not only show significant contemporaneous correlations but also significant correlations when measured five or more years previously. A surface-disorganised approach to work is predicted by surface learning in medical school and by higher neuroticism scores and lower conscientiousness (see tables 4 and 6 ). The surface-rational approach to work is predicted by strategic learning in medical school, and by less openness to experience and higher conscientiousness. The deep approach to work is predicted by a deep approach to learning at medical school, by greater extraversion, by greater openness to experience, and by lower emotional exhaustion. A workplace climate dominated by a high workload is predicted by higher stress and emotional exhaustion measures five years earlier, and by lower openness to experience (see tables 5 and 6 ). A supportive-receptive workplace is predicted by lower stress and depersonalisation, and a higher sense of personal accomplishment when measured previously, and by a more agreeable personality. Choice-independence in the work environment is predicted only by lower previous measures of stress. Stress, burnout and satisfaction with medicine Although in the previous analyses, stress and burnout have been used as predictors of approaches to work and workplace climate, they are also important outcome measures in their own right. Table 7 shows the correlations of the five 'stress-related measures' (GHQ, the three burnout measures and satisfaction) with measures of learning style and personality, in each case measured on two separate occasions. Personality correlates with each of the measures, as do study habits. Because of the complex inter-correlations between the dependent variables, multiple regression was used, as before, to find the most important relationships (for technical details see Supplementary Information see Additional file: 1 ). Doctors who are most stressed showed higher levels of neuroticism, both currently and previously, and those reporting most emotional exhaustion also had higher neuroticism levels, as well as being more introvert. High levels of depersonalisation related to lower levels of agreeableness. A greater sense of personal accomplishment related to previous deep approaches to study and learning, as well as to being more extravert. Overall satisfaction with medicine as a career related to lower levels of neuroticism. Path analysis The complex relationships described by the various correlations are best analysed and described by means of path analysis or causal modelling [ 20 ], which analyses the entire set of correlations between variables, using plausible assumptions about causality and removing non-significant paths. The path diagram, which was analysed using LISREL 8.52 [ 21 ], is shown in figure 2 . Measures to the left can causally influence measures to their right. Based on the time-lagged correlations reported previously, we assumed that stress causes different approaches to work, and we also assumed that approaches to work cause differences in workplace climate rather than vice-versa. (Nevertheless, we acknowledge that the causation may well be reciprocal, as suggested by the originators of the scale [ 2 , 19 ]; further longitudinal data will be required to test that hypothesis). Study habits are temporally and causally prior to stress, approaches to work and workplace climate. Personality, being a trait, was prior to all other measures. For technical details see the Supplementary Information (see Additional file: 1 ). Although several of our variables are measured at different time points, we have chosen not to present a model in which each variable has been included on each occasion that it is measured, as the resulting diagram becomes unmanageably complex. Although the path diagram in figure 2 is complex at first sight, the paths are readily interpretable. The diagram divides into two broad sections, with the measures of learning style and approach to work at the bottom, and stress at the top. Here we have simplified the model by omitting the closely correlated measures of burnout, and only including paths with t-values greater than 3.6. Estimates of all the paths are available in the Supplementary Information (see Additional file: 1 ). Stress in our model is caused by personality differences, being greatest in those having high neuroticism scores, low extraversion scores, and low conscientiousness scores. It is unrelated to learning style. Learning styles at medical school relate to different personality measures, in particular showing no relationship to neuroticism. Deep learning is highest in extraverts who are open to experience, whereas strategic learning is highest in highly conscientious individuals with low openness to experience. Surface learning style is higher in introverts who are low in openness to experience. These findings are similar to those of others [ 22 ]. Approaches to work are mainly but not entirely driven by learning styles. A deep approach to work occurs in extraverts who are open to experience and have a deep learning style. The surface-rational and surface-disorganised approaches to work are both greater in those with a surface learning style. However, a surface-disorganised approach occurs in individuals with higher neuroticism scores, in those with lower conscientiousness scores, and in those who have been stressed, whereas the surface-rational approach to work occurs in strategic learners and in those who are low in openness to experience. Workplace climate has a range of influences. High perceived workload occurs in those with a surface-disorganised approach to work, who have been stressed and are more neurotic. In contrast, choice-independence and a supportive-receptive environment both occur in individuals who have not previously been stressed, the choice-independence approach occurring in those with a deep approach to work, whereas the supportive-receptive approach occurs in those who have higher scores on the personality trait of agreeableness. Discussion Many doctors at the age of 30 are unhappy in their jobs, and a fifth of our sample reached the conventional GHQ criterion of psychiatric 'caseness'. In contrast, many doctors reported high levels of personal accomplishment, choice and independence in their work environment, satisfaction with medicine as a career, and intellectual and emotional satisfaction from their work. That is not new; Sir William Osler in 1905 contrasted doctors "whose stability of character and devotion to duty make one proud of our profession" with those who find it difficult to keep "the flame alive, smothered as it is apt to be by the dust and ashes of the daily routine" [ 1 ]. In 2001, Richard Smith asked "Why are doctors so unhappy?" and concluded that "The most obvious cause of doctors' unhappiness is that they feel overworked and undersupported" [ 23 ]. Certainly many doctors in our study report a high workload and a work climate that is neither supportive nor receptive, and those doctors also report more stress, burnout and dissatisfaction with medicine as a career. It is tempting therefore to conclude, as did an article in a special edition of BMJ Careers devoted to "Doctors' Wellbeing", that excessive workload and absence of support are directly caused by poor working conditions: "the way in which the NHS is run generates stress for members of the workforce every day" [ 24 ]. However, such an interpretation is not straightforward in general [ 25 ]. It is particularly difficult for the doctors in our study because the study is longitudinal, and workload and lack of support correlate with stress and burnout reported five or six years earlier , when the doctors were PRHOs and carrying out entirely different jobs. High perceived workload and poor support are therefore determined as much by doctors themselves as by specific working conditions. That view was expressed in another article in the special edition of BMJ Careers : "A critical element contributing to the stress that many conscientious doctors experience is internal..." [ 26 ]. A similar conclusion was reached in a previous study of ours when these doctors were PRHOs, and multi-level modelling showed that stress is not a characteristic of jobs but of doctors, different doctors working in the same job being no more similar in their stress and burnout than different doctors in different jobs [ 11 ]. If differences in reported workload are partly explained by differences among doctors, what in turn explains those differences? Doctors reporting a high workload also have what Delva et al [ 2 ] describe as a surface-disorganised approach to work, which in turn is correlated with being a surface learner at application to medical school, a dozen years previously. Surface-disorganised doctors are also high on the personality trait of neuroticism and low on the trait of conscientiousness; and again those correlations are with measures taken six years earlier when the doctors were PRHOs. Doctors reporting a work climate low in support were lower on the personality scale of agreeableness in the measures collected when they were PRHOs. Some doctors may be stressed and burned out, but what predicts those others who are happy in their work? Doctors reporting high satisfaction with medicine as a career have a deep approach to work, and that approach is more common in those who also had a deep learning style when they applied to medical school. Satisfaction with medicine also relates directly to the personality traits of greater extraversion and lower neuroticism, and the deep approach to work correlates with greater extraversion and more openness to experience. Doctors who describe their colleagues as receptive and supportive score more highly on the personality trait of agreeableness; and as in many other correlations reported here, that correlation is stable across time – those who are more agreeable at the age of 24 have a more receptive and supportive work environment when aged 30. An overview of our findings is that approaches to work are predicted by earlier measures of study habits and learning styles, whereas perceived work climate, and its pathologies such as stress and burnout, are predicted mainly by personality. Although unfortunately our study did not measure personality during selection, the high stability of the Big Five measures across the life-span [ 27 - 29 ] (and across our two measures six years apart), as well as their heritable component [ 30 ], means that we have little doubt that personality at selection would also have been predictive, particularly given that a similar pattern of correlations was found in a different cohort of doctors in mid-career [ 15 ]. Other studies on very different groups of students have also found, like us, that both strategic and deep learning correlate with conscientiousness, and that deep learning also correlates with extraversion and openness to experience [ 22 , 31 ]. Our study has, for various reasons, not looked at academic performance in relation to study habits, learning styles and personality, although previous work of ours has found clear correlations between learning styles and examination performance [ 32 ]. In contrast we have not found any correlation of undergraduate or postgraduate academic achievement with personality [ 15 ], and although some studies have found correlations of conscientiousness with academic achievement [ 33 ], this does seem to vary according to the learning context [ 34 , 35 ]. Although we will be looking at this question again in more detail in a further analysis, it does seem probable that personality mostly has an indirect effect upon academic achievement via approaches to learning [ 31 , 36 ]. If, as William Wordsworth said, "the child is father to the man", then the seeds of subsequent job satisfaction and dissatisfaction in doctors may be visible in the personality, motivations and learning styles of medical school applicants. This argument may provide some justification for using such measures in selection, particularly given the general association of job performance and satisfaction with personality [ 37 ] and motivation [ 38 ], and learning styles with personality [ 22 ] . However, just as genes are not destiny, so neither personality nor learning style is destiny. Nurture interacts with nature [ 39 ], the environment building upon the genes, and the genes using what is provided by the environment; the poetic complement to William Wordsworth is therefore Alexander Pope, who said, "This education forms the common mind: Just as the twig is bent, the tree's inclined." Extreme introverts can, with sufficient insight, preparation and appropriate training become effective public speakers, less conscientious individuals can learn to be more organised and efficient, and those who are more neurotic can transcend their anxieties (and indeed neuroticism may be beneficial if sublimated into a professional concern for detail in critical situations, rather than merely being undifferentiated personal anxiety). . Formal education, particularly effective formal education [ 40 ], can also alter study habits and learning styles, which are less fixed and 'trait-like' than personality measures [ 17 ]. Intercalated degrees increase deep and strategic learning and decrease surface learning at medical school [ 41 ], making it likely that they also encourage surface-rational and deep approaches to work. Deep and strategic learning also relate to the clinical experience gained by medical students [ 32 ], making it possible that greater patient involvement during undergraduate clinical training, rather than mere reliance on textbook learning to pass exams, a characteristic of surface learners, will also reduce surface-disorganised approaches to work. Conclusions Longitudinal data suggest that personality and learning style are not merely correlates of approaches to work, workplace climate, stress, burnout and satisfaction with a medical career, but are causes , events later in time being predicted by events earlier in time [ 35 ]. Doctors with greater stress and emotional exhaustion, who were less satisfied with medicine as a career, had higher neuroticism scores and were more likely to be surface-disorganised. Lower conscientiousness on the personality measure also predicted greater stress. Extraverts reported more personal accomplishment and were more satisfied with medicine. The personality measure of agreeableness predicted a more supportive-receptive work environment. These results imply that differences in approach to work and workplace climate in our study result from differences among doctors themselves, as much as they do from differences in working conditions. Competing interests None declared. Authors' contributions The cohort study was designed by ICMcM. The present follow-up was designed by ICMcM and EP, who also prepared the questionnaire. AK was responsible for day-to-day running of the study, and for data entry and cleaning. ICMcM was primarily responsible for data analysis and for writing the first draft of the paper. ICMcM, EP and AK were all involved in preparing the final draft of the paper. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional file 1 Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC516448.xml
526212	Cloning of a novel inhibin alpha cDNA from rhesus monkey testis	Background Inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion. Inhibin B is produced by testicular Sertoli cells and is the primary circulating form of inhibin in most adult male mammals. Inhibin B is comprised of the inhibin alpha subunit disulfide-linked to the inhibin/activin betaB subunit. Here we describe the cloning of the cDNAs encoding these subunits from adult rhesus monkey testis RNA. Methods The subunit cDNAs were cloned by a combination of reverse transcriptase polymerase chain reaction (RT-PCR) and 5' rapid amplification of cDNA ends (RACE) RT-PCR from adult rhesus monkey testis RNA. Results Both the inhibin alpha and betaB subunit nucleotide and predicted protein sequences are highly conserved with other mammalian species, particularly with humans. During the course of these investigations, a novel inhibin alpha mRNA isoform was also identified. This form, referred to as rhesus monkey inhibin alpha-variant 2, appears to derive from both alternative transcription initiation as well as alternative splicing. rmInhibin alpha-variant 2 is comprised of a novel 5' exon (exon 0), which is spliced in-frame with exon 2 of the conventional inhibin alpha isoforms (variant 1). Exon 1 is skipped in its entirety such that the pro-alpha and part of the alpha N regions are not included in the predicted protein. rmInhibin alpha -variant 2 is of relatively low abundance and its biological function has not yet been ascertained. Conclusion The data show that the predicted inhibin B protein is very similar between monkeys and humans. Therefore, studies in monkeys using recombinant human inhibins are likely to reflect actions of the homologous ligands. In addition, we have observed the first inhibin alpha subunit mRNA variant. It is possible that variants will be observed in other species as well and this may lead to novel insights into inhibin action.	Background The inhibins are dimeric gonadal protein hormones that negatively regulate pituitary FSH synthesis and secretion [ 1 , 2 ]. Inhibins are comprised of an α subunit (inhibin α) and one of two inhibin β subunits (inhibin βA or inhibin βB). In adult male mammals, inhibin B (α-βB dimer) appears to be the primary circulating form of the hormone, whereas females produce both inhibin A and B and do so in discordant fashion across the reproductive cycle [ 3 - 15 ]. One exception to this general pattern is in rams, where inhibin A appears to be the primary circulating form [ 16 ]. Historically, investigations of inhibin action have relied principally upon recombinant preparations of inhibin A because inhibin B has not been available in sufficient quantities to permit in vivo studies of its role in the negative feedback regulation of gonadotropin secretion [ 17 , 18 ]. Because inhibin B is the biologically relevant ligand in male primates, this has placed some constraints on our understanding of inhibin action in these animals. For this reason, we cloned the inhibin B subunit cDNAs from adult monkey testis as a requisite first step to producing recombinant monkey inhibin B. In the course of cloning the monkey inhibin α subunit, we identified a novel transcript, which has not been observed in other species. In this paper, we describe the new transcript called rhesus monkey inhibin α-variant 2. Methods RNA extraction Total RNA was extracted from frozen testis samples of two adult male rhesus monkeys ( Macaca mulatta ) (#1861 and #2333) using Trizol following the manufacturer's instructions (Invitrogen, Carlsbad, CA). RNA was dissolved in diethyl pyrocarbonate-treated H 2 O and quantified by spectrophotometry. Animals were treated in accordance with institutional and federal guidelines. Reverse transcriptase polymerase chain reaction (RT-PCR) Contaminating genomic DNA was removed from RNA samples using RQ1 DNase (Promega) following standard protocols. Four μg of DNased RNA (from #1861) was reverse transcribed into cDNA using 100 ng random hexamer primers and 100 U MMLV-RT (Promega). Four hundred ng of cDNA was subjected to PCR to amplify part of the αN domain and the entirety of the mature domain (αC) of the inhibin α subunit using the following primer set: 5'-CCYTTCCTGGTGGCCCACACT (forward) and 5'-TTAGATACAAGCACAGTGYTG (reverse) (see primers A and B in Fig. 3 ). Reactions were subjected to 35 cycles of 94C for 30 sec, 55C for 30 sec, and 72C for 30 sec. No amplified products were observed in H 2 O or RT- controls (data not shown). The amplified 465 bp product was ligated into pCR3.1 (Invitrogen) following the manufacturer's instructions. Recombinant clones were screened by colony hybridization using the gel purified PCR product as probe. Plasmids were purified from hybridizing clones and sequenced using DyeTerminator Cycle sequencing (ABI). All hybridizing clones corresponded to inhibin α. Figure 3 Rhesus monkey inhibin α gene structure. Schematic representation of the genomic organization of the inhibin α subunit in rhesus monkey. Boxed regions reflect exons and the intervening straight line is the intron (the 2 kb is an estimate based on the 2051 bp intron in humans). Black boxes reflect 5' and 3' UTRs. White boxes reflect sequences encoding the signal peptide (in inhibin α-variant 1) or a domain of unknown function (inhibin α-variant 2). The other shaded regions correspond to the pro-α, αN, and αC domains of the inhibin α prepro-hormone and are labeled in the figure. Inhibin-α variant 1 is the canonical inhibin α mRNA described previously in other species and is produced through splicing of exons 1 and 2. Inhibin α-variant 2 is produced through splicing of the novel exon 0 and exon 2. In this latter form, the entirety of exon 1 is removed, in addition to intron 1. An A to G transition in the monkey genomic sequence (relative to the human sequence) at the end of exon 0 appears to introduce a GT 5' splice donor site, which is absent in other species. This allows the splicing event observed in inhibin α-variant 2 when an upstream transcription start site is utilized. This variant is not predicted to exist in other species and also appears to be rare in monkey. The approximate positions of the primers used in different RT-PCR analyses are shown. A and B refer to the forward and reverse primers, respectively, designed to amplify the final 465 bp of the open reading frame. C and D are the outer and inner gene specific reverse primers used in the 5' RACE procedure. The full-length monkey inhibin α cDNA was amplified by RT-PCR from monkey testis RNA as described using a primer (5'-ATGGTGCTGCCCCTACTGCT) directed against the putative start of translation as determined by 5' rapid amplification of cDNA ends (RACE) (see below) and the reverse primer described above. Three prominent bands of approximately 1100, 700, and 400 bp were amplified. The top band was of the predicted size and was purified, cloned and sequenced. The identities of the other two bands have not yet been determined. The mature region of the inhibin βB subunit cDNA was amplified from monkey (#1861) testis RNA by RT-PCR as described for the α subunit using the following primer set: 5'-AGCTGGCCGTGGTGCCBGTGTT (forward) and 5'-TCAGGCGCAGCCGCACTCCTC (reverse). The resulting 455 bp product was gel purified and sequenced directly. 5' RACE RT-PCR 5' RACE was performed on monkey (#1861) testis RNA using RNA ligase mediated (RLM)-RACE reagents following the manufacturer's instructions (Ambion, Austin, TX). The primary PCR was performed using the Outer Adapter Primer (Ambion) and the following gene specific primer: 5-GGCAGGTTTGGTGGGATGTGCA (Fig. 3 , primer C). The secondary PCR was performed on 4 μl of a 1:100 dilution of the primary PCR reaction using the Inner Adapter Primer and the following nested gene specific primer: 5'-GGAAGGAGATGTTCAGTGCTAC (Fig. 3 , primer D). For both PCR reactions, the following reaction conditions were used with AmpliTaq (Perkin-Elmer): 35 cycles of 94C for 30 sec, 55C for 30 sec, and 72C for 1.5 min. Three prominent amplicons were observed in the secondary PCR reaction. No bands were observed in the primary PCR or in any of the negative controls (data not shown). A pool of the different RACE products was ligated into pCR2.1 (Invitrogen). Plasmids were isolated from recombinant clones screened by α complementation and were sequenced as described. Northern blot Twenty μg of total RNA prepared from testes of two adult rhesus monkeys were run on a 1% MOPS-formaldehyde agarose gel. RNA was transferred to Hybond N+ charged nylon membrane by capillary action using 20X SSC. The blot was first probed with a 32 P-labeled (Ready-to-go; Amersham Pharmacia) cDNA corresponding to the last 465 bp of the coding sequence of monkey inhibin α. The blot was hybridized overnight at 42C in 50% formamide, 5X SSC, 1X Denhardt's, 20 mM NaPO 4 (pH 6.8), 1% SDS, and 100 μg/ml denatured salmon sperm DNA using a modified sandwich method [ 19 ]. Following washes in 2X SSC/0.1% SDS at RT and 70C, the blot was exposed to X-ray film (Kodak) overnight with an intensifying screen at -85C. The blot was subsequently stripped and re-probed with a 32 P-labeled cDNA corresponding to 256 bp of monkey inhibin α exon 1. Hybridization and washing conditions were as described for the first probe. Results Cloning of the rhesus monkey inhibin α cDNA The inhibin α cDNA was cloned from rhesus monkey testis using a combination of RT-PCR based approaches. First, the cDNA encoding the last 20 amino acids of αN and the entirety of the mature (αC) domain was amplified by RT-PCR using adult monkey testis RNA as starting material. The resulting PCR product was cloned and sequenced. BLASTN of the non-redundant database showed highest sequence identity (97%) to the human inhibin α cDNA. Within the 402 bp encoding the αC domain, sequence identity was also 97% and the predicted amino acid sequence was 98% conserved (Fig. 1 ). Within the 134 amino acid αC domain there are three non-conservative differences between human and rhesus monkey (L4P, S72P, and Y86P; the first letter refers to the amino acid in human and the number denotes the residue in the αC domain); however, all occur at residues that vary between the mammalian inhibin α subunits sequenced thus far (Fig. 1 ). Proline at position 4 of rhesus monkey is also observed in pig, horse, cow and sheep. The proline at position 72 is observed in all mammalian species examined, except human. Finally, the proline at residue 86 is leucine in all non-human mammalian species examined thus far and is tyrosine in human. Figure 1 Inhibin α amino acid sequence alignment. Alignment of the inhibin α mature domain (αC) amino acid sequence in several mammalian species. Differences from the human sequence are bolded and underlined. Differences between the human and monkey sequences are indicated by arrows. To clone the full-length cDNA, we used 5' RACE to amplify the remainder of the αN and the pro-α regions. Three prominent RACE products were amplified. The longest (919–927 bp) and shortest (642 bp) were cloned and sequenced. The intermediate sized amplicon has not yet been definitively characterized. The long product was within the expected size range and was somewhat heterogeneous in that the clones had 5' ends that extended to differing extents. This likely reflects differences in transcription start sites, but all were within 8 bp of each other. The 5' untranslated region (UTR) ranged from 105 to 113 bp, which is slightly shorter than the 144 bp described in humans (GenBank acc.# NM_002191). When combined with the original PCR fragment, the contiguous sequence contained an open reading frame of 1098 bp predicted to encode a 366 amino acid prepro-hormone, consistent with the size of the human prepro-inhibin α. To confirm expression of a transcript containing this uninterrupted open-reading frame, PCR primers were designed against the start and end of the translation and the predicted 1101 bp fragment (including the stop codon) was amplified. DNA sequencing confirmed its identity. A monkey placental EST (CB548960) overlapped with and confirmed the sequence of the final 183 bp of the monkey inhibin α open reading frame and included an additional 188 bp of 3' UTR (not including the poly A+ tail). Thus, the mRNA encoding the inhibin α subunit would be predicted to be approximately 1.4 kb. Northern blot analysis of monkey testis RNA using a probe directed against sequence within αC domain (exon 2 probe) hybridized to an mRNA of approximately 1.7 kb (Fig. 2A ). The slight size discrepancy may result from a long polyA+ tail, the use of alternative polyadenylation sequences (although a consensus AAUAAA sequence appears 21 nt upstream of the polyA+ tail in the EST), and/or alternative transcriptional start sites. An additional transcript of about 4 kb was also detected with this probe (top arrow in Fig. 2A ). The identity of this less abundant transcript has not yet been ascertained. The monkey inhibin α sequence from the end of the longest 5'RACE product through the end of the open reading frame has been deposited in GenBank (GenBank Acc. #AY574369). Figure 2 Inhibin α mRNA expression in monkey testis. Northern blots showing inhibin α mRNA expression in adult monkey testes. Equal amounts of total RNA from two adult males were run on a MOPS-formaldehyde gel. RNA was transferred to a nylon membrane and hybridized consecutively with 32 P-labeled cDNA probes corresponding to 465 bp of exon 2 (A) and 256 bp of exon 1 (B) of monkey inhibin α. Both probes detected transcripts of 1.7 and 4 kb (top two arrows). The exon 1 probe also detected a 0.4 kb transcript (bottom arrow in B). Molecular weight standards (in kb) are shown at the left of each panel. Hybridization patterns were the same in both animals. Novel inhibin α mRNA in monkey testis The short RACE product when cloned and sequenced was determined to correspond to a novel variant of the inhibin α subunit. In humans (and other species), inhibin α has been described as a two exon gene (Fig. 3 ). Exon 1 encodes the 5' UTR, pro-α, and 85 bp of the αN region. The remainder of αN, αC and the 3'UTR are contained within exon 2. The 546 bp at the 3' end of the short RACE product corresponded exactly to sequence within monkey exon 2 (based on the human nomenclature). The 96 bp at the 5' end of the amplicon did not, however, correspond to the exon 1 sequence determined in the long RACE product. Upon BLASTN search, this 96 bp sequence was determined to show highest identity (91–97%) with human (GenBank acc.# AF272341), mouse (GenBank acc.# M95526), pig (GenBank acc.# AF510728), cow (GenBank acc.# S72864), and rat (GenBank acc.# M32754) inhibin α proximal promoter sequences (Fig. 4A ). These data suggested that transcription of the short RACE product was initiated in what is conventionally thought of as inhibin α promoter (5' flanking sequence) in all species described to date. Figure 4 DNA sequence alignment of novel exon 0 in monkey inhibin α. A) Alignment of the novel exon 0 in inhibin α-variant 2 with inhibin α promoter sequences from Human (GenBank acc.# AF272341), mouse (GenBank acc.# M95526), pig (GenBank acc.# AF510728), cow (GenBank acc.# S72864), and rat (GenBank acc.# M32754). Note that the numbering is relative to the 96 bp of the monkey exon 0 and does not reflect the numbering in the GenBank entries. Bolded and underlined bases reflect differences from the monkey sequence and spaces (-) are added where needed to facilitate the alignment. The non-consensus cAMP responsive element (CRE), which is important for basal and FSH stimulated expression of inhibin α, is boxed and is conserved in all species. The arrows denote the start sites of the human (h, GenBank acc.# CB997542), mouse (m1, GenBank acc.# BY303064; m2, GenBank acc.# BI082792), and pig (p, GenBank acc.# BP457576) ESTs referred to in the text. B) Alignment of the end of monkey exon 0 and the start of exon 1 with human inhibin α genomic sequence. The AT dinucleotide in human is GT in monkey, thereby introducing a novel 5' splice donor site. The bold and underlined base reflects a difference from the monkey sequence. We used this 96 bp sequence to screen the expressed sequence tag (EST) database to determine whether or not this portion of the inhibin α gene was included in transcripts in other species. BLASTN showed significant homology to four entries from three species: human placenta (GenBank acc.# CB997542), mouse dpc 14.5 Rathke's pouch (GenBank acc.# BY303064), mouse mammary tumor (GenBank acc.# BI082792), and pig ovary (GenBank acc.# BP457576). None of the ESTs extended as 5' as the monkey short RACE sequence, but one mouse EST (BY303064) started 15 bp 3' of where monkey RACE product began (Fig. 4A ). In all cases, the EST sequences where contiguous with previously described 5' UTRs in exon 1 of the various species. Therefore, these ESTs appear to define alternative transcription initiation sites in the inhibin α gene and ostensibly increase the length of the 5' UTRs, but do not alter the exon-intron structure of the gene nor do they alter the open reading frame of the mRNA. This contrasts with what we observed in monkey. We aligned both the short and long (see above) RACE product sequences to human genomic sequence derived from a BAC clone in GenBank (acc.# AC009955). The 96 bp unique to the short RACE product terminated 12 bp 5' of where the longest RACE product began (Fig. 4B ). We noted that the first two bp of this intervening sequence was AT in human. We hypothesized that if the adenine in the first position in human was guanine in monkey, this would provide a 5' splice donor site (GT; [ 20 ]) and might explain how exon 1 sequence was skipped in its entirety in this transcript. We designed PCR primers corresponding to sequences flanking the intervening region and amplified genomic DNA extracted from monkey testis. Resulting amplicons of the predicted size were gel purified and sequenced directly. The results confirmed that the first two bp of the intervening sequence were GT in monkey, and therefore potentially provided a novel 5' splice site (Fig. 4B ). The same 3' splice acceptor used in the long transcript also appears to be used in the short transcript, such that exon 2 is spliced identically in both cases (Fig. 3 ). We propose to call the unique sequence in the short RACE product exon 0. The resulting transcript reflects the splicing together of exon 0 and exon 2 (Fig. 3 ). Exon 1 and the intron are removed in the process. The long RACE product reflects transcription initiation from a downstream (more common?) site and is produced through splicing together of the canonical exon 1 and exon 2 (Fig. 3 ). We propose to call the transcript identified in the short RACE product rhesus monkey inhibin α-variant 2 (GenBank acc.# AY574370) and the canonical form inhibin α-variant 1. The short RACE product was initiated from a reverse primer directed against sequence within exon 2 (Fig. 3 , primer D). By virtue of its positioning, this primer excluded the last 287 bp of the open reading frame in exon 2. To confirm that inhibin α-variant 2 extended at least as far as the stop codon in exon 2, we used RT-PCR to amplify a contiguous sequence from the 5' end of exon 0 to the end of the ORF in exon 2. A faint band was amplified, but was of insufficient abundance to clone or directly sequence. We therefore performed nested PCR on this amplicon using primers directed against exon 0 (15 bp 3' of the first primer) and exon 2 (170 bp 5' of the first primer; primer C in Fig. 3 ). A band of the predicted size was amplified and directly sequenced following gel purification. The product corresponded to inhibin α-variant 2, indirectly confirming that the entirety of the open reading frame in exon 2 is contained within this transcript. The ORF of inhibin-α variant 2 is 888 bp, potentially encoding a protein of 296 amino acids. A putative AUG start codon is observed 38 bp from the 5' end. However, the surrounding sequence does not conform to the consensus Kozak sequence [ 21 ]. The next AUG is observed 129 bp 3' of the first, within the αN encoding portion of exon 2. This potential start site also fails to conform to the consensus Kozak sequence. Therefore, it is not clear which, if either, of these codons may be used to initiate translation of inhibin α-short. The putative protein contains 19 novel amino acids (encoded by exon 0) at its N-terminus followed in-frame by amino acids 90–366 of inhibin α variant 1. The N-terminal 19 amino acid peptide does not encode a signal sequence nor does it possess significant homology to sequences in the public databases. The amino acids from exon 2 encode the majority of αN and the entirety of αC (see Fig. 3 ). The northern blot in Fig. 2A was probed with a cDNA corresponding to exon 2, which is contained in both inhibin α-variants 1 and 2. Two transcripts were detected. To determine whether the transcripts might encode these two alternative forms, we stripped the blot and re-probed it with an exon 1 specific probe (Fig. 2B ). Both transcripts were again detected, indicating that both contained exon 1 sequence and, by extension, that neither transcript encoded inhibin α-variant 2 (which lacks exon 1). This is perhaps not surprising in light of the difficulty we experienced in amplifying inhibin α-variant 2 by RT-PCR and is consistent with the notion that it is a relatively low abundance mRNA. Surprisingly, a smaller transcript of ~0.4 kb was also detected with the exon 1 probe. These data suggest that yet another inhibin α transcript may be expressed in monkey testis. The identity of this mRNA species is currently unknown; however, it is predicted to be truncated and contain some or all of exon 1 sequence. Cloning of the rhesus monkey inhibin βB cDNA The mature domain of inhibin βB is highly conserved across all species investigated to date. We used RT-PCR to amplify this region of the cDNA in rhesus monkey (GenBank acc.# AY574371). Not surprisingly, the 115 amino acid domain shared 99% sequence identity with several other mammalian species, including human (Fig. 5 ). The one amino acid difference was a non-conservative threonine to alanine substitution at position 75 of the mature domain. This amino acid is also divergent (proline) in rat and mouse. Figure 5 Inhibin βB amino acid sequence alignment. Alignment of the inhibin β B mature domain amino acid sequence in several mammalian species. Differences from the human sequence are bolded and underlined. An arrow indicates the one amino acid difference between the human and monkey sequences. Discussion In this report, we describe the cloning of the inhibin B subunit cDNAs from testis of the adult rhesus monkey. The results indicate that both the inhibin α and inhibin βB subunits are highly conserved with other mammalian species, particularly within the mature domains of the prepro-hormones. For inhibin α, monkey and humans share 131 out of 134 amino acids. The three differences are all non-conservative; however, all occur at residues that vary across mammalian species. The human and monkey βB mature regions share 114 of 115 amino acids. Again, the one difference is non-conservative but occurs in a residue that varies across mammalian species. Collectively, these data suggest that the mature inhibin B is nearly identical in the human and rhesus macaque. As a result, current assays developed to measure human inhibin B are expected to accurately measure native monkey inhibin B. In addition, because rh-inhibin A and rh-inhibin B are equipotent at suppressing FSH secretion by monkey gonadotrophs in primary culture (Winters and Plant, unpublished observations), it seems reasonable to assume that the FSH suppressing potency of rh-inhibin A may be comparable to that of native monkey testicular inhibin B. If this assumption is substantiated when recombinant monkey inhibin B becomes available, then it will allow the physiological significance of previous and future studies using rh-inhibin A administration in male monkeys to be placed into perspective. In the course of cloning the inhibin α subunit, we identified at least one alternative inhibin α mRNA, which we call rhesus monkey inhibin α-variant 2. The canonical inhibin α subunit mRNA, rhesus monkey inhibin α-variant 1, is comprised of two exons. Monkey inhibin α-variant 2 is produced through a combination of alternative transcription initiation and alternative splicing. As a result, a novel exon (exon 0) is used in place of exon 1 observed in inhibin α-variant 1. Both transcripts incorporate exon 2. 5' RACE indicated that transcription of inhibin α-variant 2 initiates about 108 bp 5' to the start of inhibin α-variant 1 (exon 1). As a result, the short variant contains an additional sequence at its 5' end that is conventionally referred to as inhibin α promoter or 5' flanking sequence. In monkey, an A to G transition (relative to the human sequence) in the genomic sequence upstream of the conventional transcription start site (in exon 1) leads to the introduction of a novel 5' splice donor site. As a result, exon 1 and the intron are removed from the pre-mRNA and a new exon (exon 0) is spliced to exon 2. The putative protein encoded by this variant is predicted to contain the majority of the αN and the entirety of the αC domains. At its N-terminus, however, it lacks a signal sequence as well as the pro-α region. Thus, if produced, it is unlikely that the protein would be secreted. We have not yet tried to express the protein to see if it is indeed synthesized in mammalian cells and where it may be trafficked. The putative translation initiation codon does not conform to the consensus sequence so there is some question about the efficiency with which this variant may be translated. In addition, its low abundance (at least at the mRNA level) also calls into question its functional significance. At this point, we do not know to what extent inhibin α-variant 2 may be expressed in other species, nor have we examined its expression in monkey ovaries. However, this isoform may be unique to monkey because of the A to G transition (relative to human) in the genomic sequence. An alignment of the relevant "promoter" sequences in human, mouse, rat, and cow indicates that the splice donor (GT) in monkey is AT in human and GG in the other three species (not shown). Moreover, screening of the EST database with the monkey inhibin α-variant 2 unique sequence (exon 0) identified very few clones and in each case merely extended the 5' UTR in these species. That is, where monkey inhibin α-variant 2 skipped exon 1 entirely, the mouse, human, and pig EST sequences were contiguous with exon 1. Perhaps the most important aspect of the identification of monkey inhibin α-variant 2 is that in this primate and perhaps in other species, transcription can be initiated further 5' than previously considered. As a result, sequences previously characterized as promoter may actually be 5' UTR, at least in some mRNA species. For example, a mouse EST contains sequence in its 5' UTR previously identified as a conserved non-consensus CRE in the inhibin α promoter [ 22 ] (Fig. 4A ). Because this EST contains the CRE sequence, its transcription may not be cAMP dependent. Conclusions Cloning of the inhibin B cDNAs from rhesus monkey testis indicates that the mature inhibin B is highly conserved in monkeys and humans. Therefore, the results in monkeys obtained with recombinant human inhibins may accurately reflect results that would be obtained with recombinant homologous ligands. The characterization of the inhibin subunit cDNAs in monkeys will greatly facilitate the production of macaque inhibins and will permit a direct test of this hypothesis. In addition, a novel inhibin α mRNA isoform was isolated in this investigation. This is the first example of an inhibin α mRNA variant described in any mammalian species. The results of both northern blot (Fig. 2 ) and RT-PCR analyses indicate that additional inhibin α mRNA variants also exist. Future studies will not only more thoroughly characterize these variants, but will examine their expression in other species. Moreover, functional analyses may highlight heretofore-unknown aspects of inhibin biology and function. Authors' contributions DJB participated in the design of the study, performed all of the molecular biological experiments and analyses, and drafted significant portions of the manuscript. TKW participated in the design of the study and critically revised the manuscript. TMP provided the animal tissues, participated in the design of the study, and drafted sections of the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526212.xml
526206	Patterns of use, dosing, and economic impact of biologic agent use in patients with rheumatoid arthritis: a retrospective cohort study	Background Variability in dosing and costs of biologics among patients with rheumatoid arthritis (RA) is of interest to healthcare descision-makers. We examined dosing and costs among RA patients newly treated with infliximab or etanercept under conditions of typical clinical practice. Methods Integrated pharmacy and medical claims data were obtained from 61 U.S. health plans. RA patients newly treated with infliximab or etanercept between July 1999–June 2002 were selected. A maintenance number of infliximab vials was determined after the "loading period" (2–3 infusions); those with ≥ 2 occurrences of an increase in vials or an interval between infusions of <49 days were considered to have had escalated. For etanercept patients, escalation was based on ≥ 2 instances of increased average daily dose. Multiple logistic regression analyses were conducted to assess variables associated with dose escalation. RA-related costs at one year post-initiation also were examined; comparisons were made using generalized linear models. Results A total of 1,548 patients were identified (n = 598 and 950 for infliximab and etanercept respectively). Infliximab recipients were somewhat older (50.5 vs. 46.6 years for etanercept). Nearly 60% of infliximab patients increased their dose at one year, compared to 18% for etanercept. Infliximab patients who escalated dose incurred a 25% increase in mean one-year costs ($20,915 vs. $16,713 for no increase; p < 0.0001). Costs among etanercept patients did not substantially differ based on dose escalation ($14,482 vs. $13,866 respectively). Conclusions Infliximab is associated with higher rates of dose escalation relative to etanercept, which contributes to substantially higher one-year medical costs.	Background Rheumatoid arthritis is a costly and debilitating autoimmune disorder that is characterized by joint pain, stiffness, and impaired functionality. Symptoms arise from the inflammation and degradation of the synovial membrane, causing progressive disability in joint function [ 1 ]. As the disease progresses, patients require more frequent invasive procedures (e.g., joint injections, synovectomy) as well as the eventual replacement of affected joints. Consequently, the economic costs of RA are considerable, as the estimated direct and indirect costs of related care in the US totals $19 billion annually [ 2 ]. Because there is no known cure for RA, the goal of therapy is to treat the disease's symptomatology while attempting to slow or halt its overall progression. Pharmacotherapy is the cornerstone of treatment where symptoms may be treated with various combinations of nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and narcotic analgesics. In addition, disease-modifying antirheumatic drugs (DMARDs) are sometimes administered in an effort to alter the disease's progression. The effectiveness of DMARDs, however, is offset by the high levels of toxicity experienced by some patients taking these medications, and is problematic for long-term therapy [ 3 ]. In addition to the use of various DMARDs either alone or in combination with other therapies, several new DMARDs have recently been introduced for the treatment of RA. These include two biologic agents, etanercept (Enbrel ® , Amgen, Inc./Wyeth, Inc., Thousand Oaks, CA and St. David's, PA) and infliximab (Remicade ® , Centocor Inc., Malvern, PA) which provide anti-rheumatic activity by inhibiting tumor necrosis factor (TNF), another important mediator of an inflammatory response. The use of such agents in combination with the DMARD methotrexate has been shown to be clinically superior to methotrexate alone in controlled clinical trials [ 4 - 12 ]. The results of recent observational studies examining the effectiveness of infliximab indicate that increased or more frequent dosing (i.e., beyond what is mentioned in the product labeling) may provide additional benefits for patients with rheumatoid arthritis [ 13 , 14 ]. However, the costs associated with use of biologic agents is already far greater than other DMARDs. Therefore, economic considerations may impact physicians' willingness to prescribe as well as commercial insurers' placement of biologic agents in the sequence of care. Given the wide disparity in costs between therapy alternatives and the potential impact of dose escalation on these costs, it is essential to examine the total costs of RA-related care associated with each form of therapy from a perspective of typical U.S. clinical practice. In this study, the direct costs of RA-related care were estimated on an annual basis after initial treatment with anti-TNF biologics. Resource utilization and cost estimates were also stratified by dosing status (increase in dose during follow-up vs. no increase), using retrospective claims data from commercial insurers in the US. Methods Data source Medical and pharmaceutical service claims were obtained from the PharMetrics Patient-Centric Database, and spanned the period from January 1999 to June 2002. At the time of this study, the database contained fully adjudicated service claims from 61 health plans across the US. Inpatient and outpatient diagnoses (ICD-9-CM format) and procedures (CPT-4 and HCPCS formats), as well as standard and mail order prescription records, are included in the data set. Reimbursed payments and charged amounts are available for all services rendered, as well as dates of service for all claims. Additional data elements include demographic variables (e.g., age, gender, geographic region), product type (e.g., HMO, PPO), payor type (e.g., commercial, self-pay), provider specialty, and start and stop dates for plan enrollment. All patients who met the sample selection criteria specified below were included in the analyses. Sample selection Patients with a diagnosis of rheumatoid arthritis (ICD-9-CM 714.XX) who newly started on infliximab or etanercept between July 1999 and June 2001 were initially selected for inclusion in the study sample. A hierarchical procedure was then implemented to stratify patients into treatment cohorts according to their first utilization of a particular medication at a specific point in time. For example, a patient initially receiving infliximab and later receiving etanercept would be classified as an "infliximab" patient for the duration of the study period. An index date for each therapy was established based on the first occurrence of a claim. Patients with no claims activity for the index therapy for six months prior to the index date were deemed "newly started". Those patients not continuously enrolled during the six-month pretreatment and 12-month follow-up periods were excluded from all analyses. Additionally, patients must have had at least five infusions or prescriptions for their index medication. Patients 65 and older who were not enrolled in a Medicare "risk" plan (i.e., a commercial plan that agrees to undertake full financial risk for a Medicare beneficiary) were excluded from each of the analyses, as such patients may not have had fully visible utilization and cost values due to coordination of medical benefits. All medical and pharmaceutical claims spanning the period January 1, 1999 to June 30, 2002 were then extracted for eligible patients in the data set. Measures The primary measures of interest in this evaluation were the frequency and economic impact of an escalation in biologic dose. Dose escalation was assessed for patients new to infliximab and etanercept during the study period described above. Infliximab and etanercept doses reported at the third infusion/prescription respectively were considered to be the maintenance dose levels. Subsequent utilization was then examined to determine dose escalation. Dose escalation for patients initiating infliximab was based on at the presence of least two occurrences of an increase in the number of vials reported on the infusion claim. The standard period (as indicated on the prescribing information for infliximab) between infusions following the third infusion is eight weeks. Therefore, patients with two infusions within seven weeks on two or more occasions were also considered to have an increase in dose. Dose escalation for patients initiating etanercept therapy was determined according to a change in the average daily dose (expressed in terms of mg per day); average daily dose was calculated based on data from pharmacy claims, using the following formula: metric strength(25 mg)*quantity dispensed (in vials)/days supplied Patients having two or more prescriptions with a higher average daily dose than that reported on their maintenance dose were considered to have an increase in dose. Dose escalation results were reported on an overall basis and stratified by age (<18, 18–34, 35–44, 45–54, 55–64, and 65 years and over respectively), geographic region (East, South, Midwest, West), calendar year of biologic therapy initiation (1999, 2000, or 2001), and quartile of pre-index RA-related costs. In addition to dose escalation, the demographic and clinical characteristics of the sample also were assessed and stratified among patients who did and did not escalate their dose. Characteristics of interest included age, gender, health plan type, geographic region, physician specialty as of the index date, presence of selected pre-index medications, procedures, and comorbid diagnoses, co-diagnosis of Crohn's disease, and pre-index total (i.e., RA-related and unrelated) healthcare costs. During follow-up, the numbers of prescriptions or infusions of biologic therapy were tracked, as were the costs of all appropriate medical interventions, inpatient, outpatient, and pharmacy services. Costs were tallied for both RA-related and unrelated services and medications. Costs were deemed to be RA-related based on the presence of a relevant diagnosis, medication claim, or procedure (See Appendix ' Additional file 1 ' for ICD-9-CM, CPT-4, and GPI drug codes). Analyses Based on the sample described above, a series of analyses were conducted as follows: 1. Dose Escalation – compared the rate of dose escalation at one year among patients initiating etanercept or infliximab therapies; 2. Predictive Model for Dose Escalation – identified RA patients most likely to experience an increase in dose at one year, isolating specific medical, pharmaceutical and demographic characteristics that served as predictors for patients increasing drug utilization; and 3. Comparison of Annual Costs – compared costs between etanercept and infliximab stratified by whether or not the patient escalated their dose. The proportion of patients escalating dose was compared between patients receiving etanercept and infliximab using a chi-square test or Fisher's Exact Test (for cell sizes less than five). In addition, a multiple logistic regression model was applied to identify characteristics that were most predictive of patients experiencing an escalation in dose (including the index biologic therapy). The selection of predictors for inclusion in the models began with a univariate analysis of each variable to determine the frequency of the observations associated with each treatment cohort. An initial model run was performed using the following variables: age group, gender, region, biologic therapy group, plan type, prescribing specialty, RA-related costs during the six month pre-index period, non-RA related costs during the pre-index period, maintenance dose, dummy variables (1 = present, 0 = absent) for the receipt of other RA related medications during the pre-index period, and selected comorbidities. A stepwise method was employed to produce the final model specification using an "entry" level of α = 0.15 and a "stay" level of α = 0.05. The amount of costs (reimbursed amounts paid by health plans) for all services previously described were calculated on an annual per patient basis for each cohort. Total RA-related, unrelated, and overall costs during the one-year follow-up period were compared controlling for differences in age, gender, pre-index RA related costs and other appropriate variables between the cohorts. A generalized linear model using a gamma distribution was used to control for these differences. Results Patient demographics and clinical characteristics Clinical and demographic characteristics of the study sample (N = 1,548) are presented in Table 1 . Overall, approximately one-third of patients (31%) were aged 55 or older. However, infliximab use was more concentrated among older patients, as 37% of patients on this therapy were 55 or older; compared to 27% of etanercept patients. Not surprisingly, females had a greater representation than males in the study sample, accounting for nearly three quarters of the study population (74%). Rates were similar for the infliximab and etanercept groups respectively (76.4% and 72.1%). Most patients in the sample were members of an HMO or PPO product; however, the use of infliximab was much lower in the HMO group compared to etanercept (30% vs. 45% respectively) and substantially higher among PPO patients (49% vs. 35% respectively). The use of other RA-related medications prior to biologic use differed numerically by treatment group. NSAIDs were used more frequently by etanercept users relative to infliximab (42% vs. 26% respectively), as were Cox-II inhibitors (35% vs. 27% respectively) and leflunomide (26% vs. 19% respectively). The rate of pretreatment methotrexate use was similar among the etanercept and infliximab groups (55% and 56%). Utilization of joint aspiration procedures was numerically higher during the pre-index period for patients in the infliximab group relative to the etanercept sample (35% and 28% respectively). Additionally, pre-index RA related costs also were somewhat higher among infliximab patients ($3,916 vs. $3,585). Dose escalation Patients who initiated infliximab therapy experienced significantly higher rates of dose escalation during the first year of follow-up relative to patients who were initiated on etanercept (58% vs. 18%; p < 0.001) (Table 2 ). When stratified by pre-index costs, patients initiating infliximab therapy had consistently higher rates of escalation relative to patients on etanercept therapy, although the rate of dose escalation generally increased with pre-index costs; the rate of escalation at one year for patients with the lowest pre-index costs was 50% for the infliximab cohort compared to 17% for patients initiating etanercept (p < 0.001); corresponding rates were 62% and 21% in the highest cost group (p < 0.001). When stratified by year of therapy initiation, age, and geographic region, rates of escalation were significantly higher among patients initiating infliximab therapy relative to the etanercept cohort across all groups. The rate of escalation increased by calendar year for patients receiving infliximab, but declined among etanercept users. Interestingly, while the rate of dose escalation increased with increasing age in the etanercept group, this rate declined in the infliximab group as age increased (beyond age 35); for example, 66.4% of those aged 35–44 in the infliximab group increased their dose, versus 39.3% in patients aged 65 and older. Finally, rates of dose escalation varied considerably by region, with the highest rates observed in the South and Midwest. Predictive model for dose escalation Modeled dose escalation results for patients who initiated infliximab or etanercept therapy are presented in Table 3 . The type of biologic therapy was by far the most significant predictor of dose escalation, as patients starting infliximab were over 6 times more likely to increase dose than patients starting on etanercept (Transformed Odds Ratio [OR] = 6.38; p < 0.0001). Patients who were members of an HMO were less likely to have an increase in dose than those who did not. RA patients with a listed comorbid diagnosis of Crohn's disease were less likely to escalate dose (OR = 0.48; p = 0.0477) than those without, while patients utilizing Cox II therapy were significantly more likely to experience an increase in dose over the course of the year (OR = 1.36; p = 0.0175). Patients in the West were much less likely to experience an increase in dose. Patients in the Northeast were 1.92 times more likely to increase dose (p = 0.0215), while patients in the South and Midwest were 1.87 and 1.89 times more likely to dose escalate (p = 0.0102 and 0.0063 respectively). Age and gender were not significant predictors of dose escalation, although the likelihood of dose escalation increased by 4% with every additional $1,000 of RA-related pretreatment costs. In an effort to better understand the factors associated with infliximab dose escalation, an additional model was conducted among patients initiating infliximab only. Results are presented in Table 4 . In this model, patients who belonged to an HMO were significantly less likely to have an increase in dose at one year relative to patients with other coverage (OR = 0.68; p = 0.0372). Patients utilizing methotrexate during pretreatment were more likely to escalate dose than those without (OR = 1.48; p = 0.0216). There was a trend towards significance in terms of an age effect, as infliximab users between the ages of 35–44 were more likely to escalate dose relative to younger patients (OR = 1.94; p = 0.0682). Comparison of annual costs Overall, costs for patients initiating infliximab therapy were numerically higher than for patients in the etanercept group ($19,144 vs. $13,977). Much of the cost difference was due to the difference in drug costs ($13,470 vs. $10,159 respectively). In addition, infliximab patients had higher costs for physician management visits ($691 vs. $381), ancillary services ($1,511 vs. $866), and hospitalizations ($2,277 vs. $1,322). Use of alternative biologic therapy (i.e., use of etanercept in a patient starting on infliximab and vice versa) was minimal, as illustrated by extremely low average annual costs for these alternative strategies. Average annual RA-related, unrelated, and total costs are also stratified by whether patients underwent dose escalation in Table 5 . Patients in the infliximab group who experienced an increase in dose had significantly higher RA-related costs relative to those who remained at maintenance levels ($20,915 vs. $16,713; p < 0.0001). This difference was primarily manifested in lower pharmacy costs for patients not escalating dose; for example, annual infliximab costs were 60% higher for patients escalating dose ($15,998 vs. $10,000; p < 0.001). Ancillary costs were also higher for patients with an increase in dose ($1,601 vs. $1,387). However, RA-related hospitalization costs were lower for patients who had an increase in dose ($1,516 vs. $3,323). Discussion In an effort to better understand the differences in dosing patterns and costs among RA patients on biologic therapy, a retrospective analysis of pharmacy and medical claims for patients new to biologic therapy was undertaken. Dosing frequency and quantity was examined, as were RA-related costs at one year after therapy initiation. Dosing guidelines suggest that etanercept patients receive two 25 mg vials a week; the use of higher doses has not been studied. The recommended dosing for infliximab is 3 mg/kg of body weight for the first dose, and then at two and six weeks and every eight weeks thereafter. Patients experiencing an inadequate response may increase dose to 10 mg/kg; or they may receive treatment as frequently as every four weeks [ 15 ]. The flexibility in these guidelines appears to be necessary, as infliximab patients in our study were much more likely to experience a dose escalation than patients on etanercept. There also appeared to be a strong relationship between the utilization of Cox-II inhibitors as well as pretreatment RA-related costs and dose escalation, indicating that disease severity may play a role in the decision to increase dose. Recent evidence suggests, however, that the relationship between dose escalation and disease activity is nonlinear. In a recent examination of infliximab and etanercept use in Sweden, improvement in disease activity levels following infliximab dose escalation was similar to that observed among infliximab patients not escalating dose as well as etanercept recipients [ 16 ]. Lastly and most importantly, differences in RA-related cost among patients new to infliximab and etanercept therapy ($19,144 vs. $13,977) were manifested mainly in the treatment costs ($13,470 vs. $10,159). Management and ancillary services accounted for most of the remaining difference. The difference in treatment costs may be attributed to the higher rate of dose escalation among the infliximab group. These patients had treatment expenses that were ~60% higher than patients who did not dose escalate ($15,998 vs. $10,000), while infliximab patients who did not dose escalate had costs similar to patients in the etanercept group. There was little difference in drug therapy costs among etanercept patients who experienced an increase in dose and those with no change ($10,427 vs. $10,100). These findings highlight the differences in treatment patterns and associated costs among patients new to etanercept and infliximab. Our study was subject to some important limitations. First, as this was a retrospective analysis of claims data, results were based on amounts billed to health plans. As a result, the unit of measurement for infliximab is billed whole vials. For example, if 1.2 vials were administered to a patient, 2 vials would be billed to the health plan. Therefore, these results may not reflect the true amount of infliximab utilized and may in fact under- or overstate the rate of dose escalation – for example, a patient who increases from 1.2 to 1.7 vials will be shown to have utilized 2 vials in both instances; in contrast, a patient moving from 1.9 to 2.1 vials will appear as having moved from 2 to 3 vials. In addition, information regarding body mass and/or patient weight was not available. As stated above, infliximab dosing levels may range from 3 mg/kg to 10 mg/kg. Dosing changes resulting from weight changes alone were therefore undetectable. Also, one method of estimation of dose change for infliximab was based on two infusions within seven weeks on two or more occasions. It is possible that some patients may have had these non-standard queuing times simply as a result of scheduling availability, and not as a result of dose escalation. Clear estimates of dose increase due to increased frequency may only be obtained through a more controlled observational study. Furthermore, no information is available in this administrative database regarding the reason for dose escalation – lack of efficacy, increase in symptoms, other reasons. Our major focus for this study was therefore to simply document that standard dosing assumptions regarding infliximab may lead to erroneous conclusions regarding its cost, given the high level of escalation seen in this and other studies. In addition, the database lacks clinical detail on levels of disease severity as well as other potentially important variables (e.g., working status) for consideration of the full clinical and economic impact of dose escalation. As with all retrospective study, we cannot rule out the possibility that differences in disease progression and/or severity between patients who do and do not escalate dose may have influenced our findings. Nevertheless, our results remained statistically significant even after controlling for observable differences between groups, indicating that any selection bias would likely only affect the magnitude, not the direction, of our findings. Finally, while the data used represent final, adjudicated claims in a health plan setting, it is possible that the data elements used are subject to coding or misclassification error. Nevertheless, if such an error rate exists, it is likely not a systematic phenomenon – that is, there is no reason to expect that coding errors would disproportionately affect the infliximab or etanercept samples in our study. Conclusions Despite the limitations noted above, we believe our study has important implications. While, the results of recent observational studies suggest that both infliximab and etanercept are highly effective in clinical practice [ 17 ], our findings suggest that patients with rheumatoid arthritis who initiate infliximab therapy are much more likely to experience an increase in dose over the course of one year relative to patients who initiate etanercept. This increase in dose leads to significantly higher pharmacy costs, as well as increases in many other RA-related medical costs. While there may be clinical factors in the decision to pick one route of therapy administration over another, public and private payers alike should carefully consider the economic implications of coverage decisions when targeting appropriate candidates for anti-TNF therapy. Competing interests All authors were employed by PharMetrics, Inc. at the time of this analysis, which was conducted based on an unrestricted research grant from Abbott Laboratories, Inc. No other competing interests are declared by any author, including stocks or other holdings, other financial interests, or non-financial interests. Authors' contributions TG was involved in the conception and design of the study, oversaw and provided quality assurance on all study output, and drafted the methods and results sections of the manuscript. DS was responsible for the design and conduct of all descriptive and statistical analyses. DO was responsible for the conception and design of the study, drafting of the background, discussion, and conclusions sections of the manuscript, and all formal correspondence regarding the study. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 File consists of ICD-9-CM codes, CPT-4 codes, GPI drug codes to describe the various diagnoses, type of drugs and procedures. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526206.xml
545776	Transcriptome analysis of haploid male gametophyte development in Arabidopsis	A transcriptome analysis of male gametophyte development in Arabidopsis uncovers distinct temporal classes of gene expression and opens the door to detailed studies of the regulatory pathways involved.	Background Development of eukaryotic cells towards particular cell fates is regulated by complex and dynamic changes in gene expression. These changes, when monitored on a genome-wide scale, provide a detailed framework for the analysis and modeling of cellular development. To monitor patterns of gene expression it is important to be able to isolate cells at precise stages along a developmental pathway. Well-developed procedures for cell culture and single-cell PCR techniques have allowed genome-wide changes in gene expression to be monitored during animal cell differentiation [ 1 - 4 ]. Transcriptomic studies of single cell types in plants have focused on diploid sporophytic cell types, including undifferentiated cell suspensions [ 5 , 6 ], leaf epidermal and mesophyll cells [ 7 ], stomatal guard cells [ 8 ] and cultured mesophyll cells [ 9 , 10 ]. These studies have provided valuable information about gene expression in single cell types; however, their coverage of the transcriptome has been limited and/or hampered by low RNA yields from individual cells, requiring mRNA preamplification steps that can bias the complementary RNA (cRNA) [ 11 , 12 ]. Moreover, such studies have not involved the use of the most comprehensive tools for monitoring gene expression that are now available for Arabidopsis - which include the Affymetrix ATH1 gene arrays. Recently, a significant advance in transcriptome analysis of plant cell types has been achieved through fluorescence-activated cell sorting of cell-type marked and protoplasted root cells using Affymetrix ATH1 micorarrays [ 13 ]. This has provided a near-comprehensive transcriptomic view of cell-fate determination at three developmental stages in five different domains of the root apex. In contrast to such enabling technologies and procedures developed for sporophytic cell types there have been no studies that provide a genome-wide perspective of cell fate determination and differentiation during haploid gametophyte development. The haploid male gametophyte generation of flowering plants has a simple and well-defined pathway of development and consists of two- or three-celled pollen grains that deliver two sperm cells via the pollen tube to the embryo sac at fertilization. The highly reduced cell lineage and functional specialization of the male gametophyte are thought to be key factors in the reproductive fitness and evolutionary success of flowering plants. Moreover, pollen ontogeny provides an attractive model of cellular development in which to dissect the regulation of cell growth and division, cellular differentiation and intercellular communication (for reviews see [ 14 - 17 ]). Recent progress in understanding of molecular and cellular aspects of pollen development has emerged from genetic studies that have identified mutants in Arabidopsis that affect all phases of pollen development [ 18 - 29 ]. In parallel, cDNA libraries and databanks have been obtained for sperm cells in maize, lily, tobacco and Plumbago zelanica [ 30 - 33 ]. Despite such advances there is limited information about developmental changes in gene expression associated with particular phases of male gametophyte development. Our objective was to develop procedures to enable the isolation of populations of microspores and developing pollen grains at precise developmental stages in Arabidopsis and to analyze changes in gene expression from unicellular microspores to mature differentiated pollen grains. A particular advantage of the male gametophyte generation is that developing microspores and pollen grains are symplastically isolated. This facilitates access to viable cell populations at different stages of haploid development without contaminating sporophytic cells. Some initial progress has been made towards the definition of the male gametophytic transcriptome of Arabidopsis using serial analysis of gene expression (SAGE) [ 34 ] and Affymetrix AG microarrays that harbor probes for approximately 8,000 different genes [ 35 , 36 ]. These studies have provided valuable insight into the complexity of gene expression in mature pollen and the extent of overlap between male gametophytic and sporophytic gene expression (reviewed in [ 37 ]). However, these studies monitored the expression of only 30% of the annotated genes in Arabidopsis and analyzed mRNA populations only in mature differentiated pollen grains. These studies do not, therefore, provide a developmental perspective of gene expression during development and differentiation of the male gametophyte. Here we describe spore isolation procedures for Arabidopsis and the use of Affymetrix ATH1 Genome Arrays to analyze transcript expression profiles throughout four successive stages of male gametophyte development in Arabidopsis . Isolated spore populations were large enough to enable RNA extraction for direct microarray hybridization without any preceding amplification step that could lead to bias in expression signals between stages or between genes within individual stages. Progression from proliferating microspores to terminally differentiated pollen was characterized by large-scale repression and the activation of a unique collection of late-program genes during pollen maturation. Putative male gametophyte-specific genes and distinct clusters of coexpressed genes are identified, including key groups of regulatory factors including cell cycle, transcription and translation factors. Bioinformatic and experimental data are used to address the importance of transcription and translation during pollen germination and tube growth Results Isolation and characterization of developing spores Transcriptome profiling throughout microgametogenesis in Arabidopsis required the introduction of a procedure for the isolation of homogeneous populations of viable spores at precisely defined stages of development. The method was based on centrifugation of isolated mixed spores in a Percoll step-gradient [ 38 , 39 ]. Large homogeneous spore populations at three developmental stages were collected: uninucleate microspores (UNM), bicellular pollen (BCP) and immature tricellular pollen (TCP). In addition, a homogeneous mature pollen grain (MPG) population was isolated from open flowers according to Honys and Twell [ 35 ]. Microscopic examination of isolated spore populations revealed no contaminating sporophytic cells and little or no other cellular debris (Figure 1a ). Vital staining revealed more than 90% viable spores at each stage (data not shown). The purity of spore populations was evaluated by DAPI staining (Figure 1b-e ). The UNM population was the most homogeneous, containing 95% uninucleate microspores, 2.5% tetrads and 2.5% bicellular pollen. The BCP population was 77% pure, but also contained some tetrads (3.5%), microspores (12%) and tricellular grains (7.5%). The TCP population comprised 88% tricellular pollen and 12% bicellular pollen. The MPG population was 100% pure with approximately 2% aborted pollen. Developmental changes in the male gametophytic transcriptome Arabidopsis ATH1 Genome Arrays were used to explore the dynamics of gene expression throughout male gametophyte development in comparison with sporophytic tissues. Microarrays were hybridized with cRNA probes made from total RNA purified from isolated spores. Hybridization data from two biological replicates derived from independently grown populations of plants were compared. Only genes with a positive hybridization signal and a detection call value of 1 in both experiments were scored as expressed. Microarray data from each pair of replicates were highly correlated, with correlation coefficients of 0.986 (UNM), 0.972 (BCP), 0.991 (TCP) and 0.971 (MPG). Complete microarray data are publicly available at the European Arabidopsis Stock Centre (NASC) microarray database [ 40 ]. Sporophytic ATH1 Genome Array datasets were downloaded from the NASC website [ 41 ]. This provided transcriptome data for seedlings at open cotyledon stage (COT, stage 0.7 [ 42 ]), leaves (LEF, stage 6.0), petiole (PET, stage 3.9), stems (STM, stage 6.1), roots (ROT), root hair zone (RHR, stage 1.02), and suspension cell cultures (SUS). Genes that were consistently expressed in replicate sporophytic datasets were identified using the same algorithm used for gametophytic data. We have previously confirmed and validated the expression pattern of 15 putative pollen-specific genes identified using Affymetrix AG arrays by reverse transcription-PCR analysis [ 35 ]. Similarly we validated the current ATH1 datasets by RT-PCR analysis in two separate experiments that included analysis of 41 genes encoding predicted glycosylphosphotidylinositol-anchored proteins (GAPs) [ 21 ] and 16 cation/proton exchanger proteins [ 43 ]. In both experiments the expression patterns of all genes tested that were identified as pollen-expressed, or pollen-specific by ATH1 analysis were confirmed by RT-PCR. The ATH1 Genome Array harbors oligonucleotide probes representing 22,591 genes based on the Arabidopsis Genome Initiative annotation. This represents 80.7% of the most recent estimate of 28,000 protein-coding genes in Arabidopsis [ 44 ]. Of these, 13,977 genes gave a consistently positive expression signal in at least one stage of male gametophyte development, representing 61.9% of the unigene targets on the microarray. The majority of these were expressed in the two earliest developmental stages; 11,565 in microspores and 11,909 in bicellular pollen (Figure 1f ). After pollen mitosis II, there was a sharp decline in the number of diverse transcripts to 8,788 in tricellular pollen and 7,235 in mature pollen. To identify genes expressed preferentially or specifically in developing male gametophytes, hybridization data was compared with sporophytic ATH1 datasets (COT, LEF, PET, STM, ROT and RHR; see Additional data file 1). Transcripts with a consistent positive expression signal in at least one stage of male gametophyte development and a zero signal in any sporophytic dataset were considered male gametophyte-specific. In total, 1,355 specific transcripts were identified, representing 9.7% of the male gametophytic transcriptome. The number of male gametophyte-specific transcripts ranged from 857 (BCP) to 625 (MPG). Thus, in contrast to the decline in the total number of diverse transcripts expressed, the representation of male gametophyte-specific transcripts increased, from 6.9% and 7.2% at UNM and BCP-stages to 8.0% and 8.6% at TCP and MPG-stages respectively. Analysis of the distribution of transcripts among three abundance classes: high (up to 10-fold less than the maximum signal), medium (10- to 100- fold less) and low (more than 100-fold less) (Figure 1f ), revealed a decrease in the proportion of transcripts forming the high-abundance class during development from 20% to 12%. On the contrary, there was sharp increase in the proportion of mRNAs forming the low-abundance class after pollen mitosis II from 4% (UNM) to 14% (MPG). Moreover, 55% of low-abundance transcripts at MPG stage represented repressed mRNAs expressed more abundantly at earlier stages. Thus, the dramatic decrease in the number of transcripts expressed between bicellular and tricellular stages is paralleled by redistribution of mRNA from the high to the low abundance classes. These changes may be associated with reduced cellular activities and cell differentiation processes together with preferential expression of certain classes of genes during pollen maturation. This finding is in accord with the over-representation of cytoskeleton, cell-wall and signaling-related genes that comprise 26% of the high-abundance transcripts at MPG stage. In particular, the average expression signals of cytoskeleton, cell-wall and signaling-related transcripts were increased by 3.1, 3.7 and 2.3-fold, respectively, compared with the UNM stage. Scatter-plot analysis was used to examine in more detail the complexity of the mRNA decline after PMII. The expression levels of individual genes were normalized using a scale of 0 to 100. Genes coexpressed in pairs of datasets were plotted using a logarithmic scale and a correlation coefficient ( R value) calculated (Figure 2 ). There was an extremely high correlation ( R = 0.96) between the transcriptomes of UNM and BCP, the two earliest developmental stages (Figure 2a ). These stages are closely related, with a moderate increase in the expression of a number of genes at BCP stage. The profiles of the two latest developmental stages, TCP and MPG, were also very similar (Figure 2c , R = 0.858), but with greater deviation than the early stages. The scatter plot of TCP and MPG revealed the shift between extreme mRNA abundance classes as described above. This was more evident when bicellular and tricellular stages were compared (Figure 2b ). The scatter of gene expression values and the low correlation ( R = 0.541), provide evidence that the major quantitative shift in transcriptome size between BCP and TCP stages is not simply the result of repression, but also involves the activation of new groups of genes associated with pollen maturation. The lack of correlation ( R = 0.194) between gene expression profiles in uninucleate microspores and mature pollen (Figure 2d ), also reflects the pronounced change in cell status from proliferating microspore to terminally differentiated pollen. The relationship between cell proliferation activities and transcriptome profiles was examined by comparison of early UNM and late MPG stages with a publicly available suspension cell culture dataset. These comparisons demonstrated that the microspore transcriptome was significantly more similar to that of cell suspensions ( R = 0.474) than to mature pollen ( R = 0.194). This is also in accord with the lack of correlation between transcriptome profiles of mature pollen and cell suspensions ( R = 0.13). Co-regulated clusters of gametophytic genes To identify gametophytic genes that may form co-regulated clusters, all 13,977 male gametophyte-expressed genes were hierarchically clustered using EPCLUST clustering and analysis software. Application of a threshold value of 0.05 resulted in the definition of 39 gene clusters covering all phases of male gametophyte development (Figure 3 ; see also Additional data files 1 and 2). Cluster 37 contained 735 early genes (5.3% of all gametophytic genes) with positive expression signals only at UNM stage. Transcriptome data reflect steady-state mRNA profiles that result from the combination of transcription and mRNA turnover rates. In this regard, some transcripts grouped in early cluster 37 may be inherited through meiosis and/or from the tetrad stage. The majority of male gametophyte-expressed genes (52%) were grouped into four clusters (25, 27, 29 and 35) comprising early expressed genes repressed after PMII. Several large gene clusters collectively containing 1,899 genes (13.6%) were associated with pollen maturation. These were activated or upregulated between BCP and TCP stages, forming clusters 5, 7, 11, 13, 18-24, 26, 28, 38 and 39. In contrast, a discrete set of 298 genes forming cluster 17 was upregulated only after TCP stage. In total, 3,342 late genes (24%), forming clusters 1-3, 6, 8 and in particular, cluster 17, encode proteins that are likely to function during post-pollination development. Expression of regulatory genes throughout male gametophyte development We focused our further analysis on three key categories of genes with likely regulatory significance in male gametophyte development; core cell-cycle genes, transcription factors and core translation factors (Figure 4 ). Core cell-cycle genes [ 45 ] were defined according to TAIR [ 46 ]. Genes comprising Arabidopsis transcription factor families were derived by compilation of data available at The Ohio State University Arabidopsis Gene Regulatory Information Server [ 47 ], data published in [ 48 ] and databases homology searches. Recent annotations of the MADS-box and bHLH transcription factor gene families were defined according to [ 49 , 50 ], respectively. Core translation factors [ 51 ] were defined according to TAIR [ 46 ]. Core cell-cycle genes Among 61 core cell-cycle genes, 55 genes were present on the ATH1 GeneChip and 45 (82%) of these were expressed in the male gametophyte (see Additional data file 1). Representative(s) of all families and subfamilies were expressed. The majority of gametophytic core cell-cycle genes showed similar expression profiles (Figure 4a ), with a decline in mRNA abundance after UNM stage to zero (or low levels) at TCP and MPG stages. This pattern is consistent with the termination of proliferation of the microspore and generative cell before pollen maturation. Putative transcription factors We identified 1,594 genes encoding putative transcription factors that were divided into 34 gene families (see Additional data file 1). Their representation on the ATH1 GeneChip was 1,350 (85%). Of these, 608 (45%) were expressed in the male gametophyte, including 54 (15.7%) that were male gametophyte-specific. There were distinct differences in the representation of large transcription factor families (with over 25 members) in the gametophyte. Among those over-represented were the p-coumarate 3-hydroxylase (C3H) family (67% of family members present on the ATH1 GeneChip), the CCAAT family (64%), C2H2 zinc finger proteins (57%), the WRKY family (53%), the bZIP family (51%), the TCP family (50%) and the GRAS family (50%). In contrast, the AUX/IAA (20%), HSF (33%), bHLH (34%), NAC (34%), AP2-EREBP (35%), HB (36%), R2R3-MYB (37%), MADS (37%) and C2C2 zinc finger (37%) gene families were all under-represented. The dominant expression pattern of transcription factor genes reflected the general repression of mRNA diversity between BCP and TCP stages (Figure 4b ). Besides a limited number of constitutively expressed genes, two major transcription factor gene groups could be distinguished. One contained a major group of early-expressed genes and the second a smaller group of genes that were more abundantly expressed late during pollen maturation. The same general tendency was apparent when the profiles of individual transcription factor families were analyzed (exemplified by the C3H family, Figure 4d ). Several gene families comprised predominantly early-expressed genes. These were the NAC, WRKY, TCP, ARF, Aux/IAA, HMG-box and Alfin-like gene families (Figure 4c-e , Additional data file 3). Complete lists of transcription factor gene families and their expression profiles are presented in Additional data files 1 and 3. Core translation factors Among 100 annotated core translation factor genes, 82 were present on the ATH1 GeneChip and 75 (91%) of these were expressed in the male gametophyte (see Additional data file 1). The vast majority of translation factor genes belonged to the early group and these were strongly expressed (Figure 4g ). Reflecting the constitutive requirement for protein synthesis, only six genes showed male gametophyte-specific expression. These were: AtPAB3 (At1g22760), AtPAB6 (At3g16380), AtPAB7 (At2g36660), AteIF2 - B3 (At3g07920), AteIF4G -like (At4g30680) and AteIF6 - 2 (At2g39820). There was a striking over-representation of poly(A)-binding (PAB) proteins among the male gametophyte-specific genes; seven out of eight PAB genes were male gametophyte-expressed, three of which were specific. Moreover, two of these gametophyte-specific PAB genes were among the few late pollen genes encoding translation initiation factors (Figure 4h ). Integrating transcriptomic and experimental data The rapid decline of mRNAs encoding translation initiation factors after bicellular stage and the parallel de novo synthesis of a new set of late pollen transcription factors, suggested storage of translation factors and ongoing transcription after pollen germination. Therefore we investigated the dependence of Arabidopsis pollen germination and tube growth on transcription and translation. Pollen was cultured with increasing concentrations of actinomycin D and cyclohexmide to examine the importance of transcription and translation, respectively. Actinomycin D had only moderate effects on both pollen germination and tube growth even at high concentrations (Figure 5a ). Similar results were observed when another transcription inhibitor, cordycepin, was used (data not shown). In contrast, cycloheximide had a dramatic effect on pollen tube growth (Figure 5b ). The presence of 0.1 μg/ml cycloheximide only inhibited pollen germination by 40%, but pollen tube growth was inhibited by 90%. At higher concentrations, 40% of pollen was still able to germinate, but further pollen tube growth was blocked. We conclude that active pollen tube growth is strictly dependent upon protein synthesis, and that pollen germination and tube growth are relatively independent of transcription. Discussion To identify patterns of gene expression involved in Arabidopsis male gametophyte development, we compared the transcriptomes of isolated spores at four discrete developmental stages using ATH1 microarrays. ATH1 microarrays harbor probe sets for 22,591 annotated genes [ 52 ]. Of these, 61.9% (13,977 genes) gave positive hybridization signals in at least one stage of male gametophyte development. A comparable proportion of active genes was reported for isolated root cells which expressed 10,492 genes (46%) on ATH1 microarrays [ 8 ]. Moreover, in similar studies of animal cell development, 53% of 13,179 arrayed genes were found to be expressed during early murine adipocyte differentiation [ 1 ]. As the proportion of known genes embedded on the ATH1 array is 80.7%, we estimate the total number of genes expressed throughout Arabidopsis male gametophyte development to be more than 17,000. Similarly, the total number of genes expressed at individual developmental stages is estimated to be 14,300 at UNM stage, 14,800 at BCP stage, 10,900 at TCP stage and 9,000 at MPG stage. Previous gene-by-gene approaches identified only 21 different genes expressed during Arabidopsis male gametophyte development (for a review see [ 16 ]). Moreover, only three of these genes were shown to be expressed at microspore stage [ 53 - 55 ]. The data sets reported here include more than 11,000 microspore-expressed genes, representing a 3,600-fold increase in knowledge of gene expression in haploid microspores. Two recent studies of the Arabidopsis mature pollen transcriptome using Affymetrix 8K AG arrays led to the identification of 992 and 1,584 pollen-expressed mRNAs, respectively [ 35 , 36 ]. Results obtained with ATH1 and AG arrays are considered comparable and largely independent of the different probe sets used [ 56 ]. However, there was a significant discrepancy in the number of incorrectly annotated genes between both arrays, with 6.3% of probe sets on the AG array being incorrectly annotated in comparison with only 0.4% on the ATH1 array [ 56 ]. Therefore, results from ATH1 arrays are more accurate as well as more comprehensive. Accordingly, the use of the more complete ATH1 array and more accurate microarray normalization protocols led to an increase in the estimated total number of genes expressed in mature pollen from around 3,500 [ 35 ] to around 9,000 (this study). The proportion of these genes that are considered male-gametophyte specific is strongly dependent on the choice of the set of reference sporophytic datasets. In the work reported here, the availability of more comprehensive sporophytic datasets and the application of more stringent criteria therefore led to a decrease in the estimated number of putative pollen-specific genes from around 1,400 [ 35 ] to around 800 (this study). This number could be reduced further if cell-type-specific expression within an organ limits detection of overlap with pollen expression. Our data highlight the extensive overlap between sporophytic and gametophyte gene expression and reveal the subset of the transcriptome that is strongly enhanced or specifically expressed during male gametophyte development. Considering all stages of microsporogenesis the total number of putative male-gametophyte-specific genes was 1,355 with the proportion of specific genes increasing from 6.9% at UNM-stage to 8.6% at MPG-stage. Among the male-gametophyte-specific genes identified there was an increase in the collective proportion of cell-wall, cytoskeleton, signaling and transport-related genes from 22% at UNM stage to 34% in MPG stage. This reflects the increasing functional specialization of mature pollen in preparation for a dramatic change in the pattern of cell growth during pollen germination and pollen tube growth. Developmental analysis of transcriptome data revealed two striking features, a sharp reduction in transcript diversity after BCP stage and a major shift in mRNA populations between BCP and TCP stages. The decline in mRNA diversity after BCP stage is associated with terminal differentiation as well as the documented phenomenon of protein storage in pollen (see [ 57 ], and this study). Moreover, this large-scale repression associated with termination of cell proliferation after PMII is accompanied by the selective activation of new groups of genes that are likely to function during pollen maturation and post-pollination development. It is interesting that the expression profiles of UNM stage and BCP stages are similar despite the presence of two different cell types in pollen grains at BCP stage - the larger vegetative cell and the smaller generative cell. Given the limited volume of cytoplasm associated with the generative cell, developmental changes in gene expression in the gametic or male germline cells are likely to be masked by the predominant contribution of the vegetative cell cytoplasm. Therefore, our male gametophytic gene expression profiles largely reflect the passage of the microspore through cell division and changes in gene expression associated with the differentiation of the vegetative cell. Large-scale changes in gene expression occur between BCP and TCP stages, and therefore do not coincide with asymmetric division of the microspore. UNM expression patterns persist into the bicellular stage, which is consistent with experiments that demonstrate that vegetative cell fate is specified independently of cell division at pollen mitosis I [ 58 ]. In contrast, generative cell fate appears to be dependent on asymmetric division at pollen mitosis I [ 25 , 58 ]. Sperm-cell cDNAs and databanks recently established in maize, lily, tobacco and Plumbago zelanica [ 30 - 33 ] provide valuable gametic gene-expression data in other species. Although our data do not provide direct information about gametic gene expression in Arabidopsis , further development of cell gamete isolation sorting [ 36 ] would allow genome-wide identification of generative- and sperm-cell-specific genes in comparison with the datasets generated here. Hierarchical cluster analysis provided detailed evidence for the dramatic switch between early and late developmental programs. We identified 39 gene clusters that could correspond to co-regulated genes. These included early clusters, several clusters of late genes, those with constitutive expression profiles and clusters showing transient expression with peaks at BCP or TCP stages. The large size of cluster 29 (4,464 genes) documents the homogeneity in expression profiles of most early genes. In contrast, late gene clusters included a significant number of genes with similar profiles between BCP and TCP stages, followed by expression profiles that deviated between TCP and MPG stages. Cluster 1, and in particular cluster 17, contained genes strongly upregulated in TCP and MPG, with likely functions in post-pollination events. The differential fate of certain late gene clusters is likely to be a feature of their requirement during pollen maturation or post-pollination events. Our analysis revealed completely different expression profiles of transcription factors when compared to core translation factors. The majority of core translation factors belonged to the early-group genes with few that were male gametophyte-specific. This may be expected, given that many genes are involved in general cellular activities. However, genes encoding PAB proteins did not follow the general trend. Seven out of eight Arabidopsis PAB mRNAs were gametophytically expressed. Three PAB genes ( PAB3 , PAB6 and PAB7 ) appeared to be male gametophyte-specific and PAB5 was preferentially expressed in pollen. Moreover, PAB3 and PAB5 are the most abundant early and constitutive PAB mRNAs and PAB6 and PAB7 belong among the few late core translation-factor genes. Although these data suggest-specific expression, our data do not rule out expression in other sporophytic tissues, particularly in flowers. Indeed, previously published expression data confirmed the expression of these PABs in other reproductive tissues together with pollen [ 59 ]. Conversely, transcription factors showed more diverse spectra of expression profiles including early, constitutive and late. There was a considerable variation in the expression profiles of individual transcription factor families. The most over-represented was the C3H family, members of which are known to have roles in lignin and other phenylpropanoid pathways in plants [ 60 ]. Although sporopollenin synthesis is believed to be under strict sporophytic control (see [ 16 ]), the diversity of gametophytic C3H transcription factors might suggest a function for these genes in regulating chemical interactions between phenylpropanoid precursors secreted by the tapetum. One candidate is the At1g74990 gene encoding a putative RING finger protein, which is abundantly and preferentially expressed at UNM and BCP stages. The majority of core translation factors belonged to the early gene clusters. In contrast, a significant number of transcription-factor genes were strongly expressed during pollen maturation. These data alone did not obviously support the fact that pollen germination and early tube growth in many species are largely independent of transcription, but vitally dependent on translation [ 61 ]. Similarly, we found that Arabidopsis pollen germination and tube growth were relatively independent of transcription, and that active pollen-tube growth, and to a lesser extent pollen germination, were dependent upon protein synthesis. It is known for some plant species that mRNAs and rRNAs accumulate during pollen maturation and are stored for use during pollen germination [ 62 , 63 ]. Our results show that Arabidopsis pollen is charged with a diverse complement of stored mRNAs that could be used to support pollen germination and pollen tube growth. Moreover, the early synthesis of mRNAs encoding translation factors strongly suggests that these are preformed and stored in mature pollen grains to support rapid activation upon hydration and germination. We also suggest that some abundant late transcription factors could regulate maturation-associated genes or act as repressors of inappropriate transcription in growing pollen tubes. Conclusions The key impact of this work is that it provides a genome-wide view of the complexity of gene expression during single cell development in plants. Analysis of the male gametophytic transcriptome provides comprehensive and unequivocal evidence for the unique state of differentiation that distinguishes the developing male gametophyte from the sporophyte. Male gametogenesis is accompanied by large-scale repression of gene expression that is associated with the termination of cell proliferation and the selective activation of new groups of genes involved in maturation and post-pollination events. Development is associated with major early and late transcriptional programs and the expression of about 600 putative transcription factors that are potential regulators of these developmental programs. This wealth of information lays the foundation for new genomic-led studies of cellular functions and the identification of regulatory networks that operate to specify male gametophyte development and functions. Materials and methods Plant material and spore isolation Arabidopsis thaliana ecotype Landsberg erecta plants were grown in controlled-environment cabinets at 21°C under illumination of 150 μmol/m 2 /sec with a 16-h photoperiod. Isolated spores from three stages of immature male gametophyte were obtained by modification of the protocol of Kyo and Harada [ 38 , 39 ]. After removal of open flowers, inflorescences from 400 plants were collected and gently ground using a mortar and pestle in 0.3 M mannitol. The slurry was filtered through 100 μM and 53 μM nylon mesh. Mixed spores were concentrated by centrifugation (50 ml Falcon tubes, 450 g , 3 min, 4°C). Concentrated spores were loaded onto the top of 25%/45%/80% Percoll step gradient in a 10-ml centrifuge tube and centrifuged (450 g , 5 min, 4°C). Three fractions were obtained containing: (1) microspores mixed with tetrads; (2) microspores mixed with bicellular pollen; and (3) tricellular pollen (Figure 1 ). Fraction 2 was diluted with one volume of 0.3 M mannitol loaded onto the top of a 25%/30%/45% Percoll step gradient and centrifuged again under the same conditions. Three subfractions of immature pollen were obtained: (2.1) microspores; (2.2) microspores and bicellular pollen mixture; and (2.3) bicellular pollen. Spores in each fraction were concentrated by centrifugation (eppendorf tubes, 2,000 g , 1 min, 4°C) and stored at -80°C. The purity of isolated fractions was determined by light microscopy and 4',6-diaminophenylindole (DAPI) staining according to [ 25 ]. Viability was assessed by fluorescein 3',6'-diacetate (FDA) treatment [ 58 ]. Mature pollen was isolated as described previously [ 35 ]. Pollen tubes were cultivated in vitro for 4 h according to [ 21 ]. Pollen was scored as germinated when pollen tubes were longer than half a pollen grain diameter. Pollen-tube growth was scored by counting those with tubes longer than two pollen grain diameters. RNA extraction, probe preparation and DNA chip hybridization Total RNA was extracted from 50 mg of isolated spores at each developmental stage using the RNeasy Plant Kit (Qiagen) according to the manufacturer's instructions. The yield and RNA purity was determined spectrophotometrically and using an Agilent 2100 Bioanalyzer at the NASC. Biotinylated target RNA was prepared from 20 μg of total RNA as described in the Affymetrix GeneChip expression analysis technical manual. Double-stranded cDNA was synthesized using SuperScript Choice System (Life Technologies) with oligo(dT) 24 primer fused to T7 RNA polymerase promoter. Biotin-labeled target cRNA was prepared by cDNA in vitro transcription using the BioArray High-Yield RNA Transcript Labeling Kit (Enzo Biochem) in the presence of biotinylated UTP and CTP. Arabidopsis ATH1 Genome Arrays were hybridized with 15 μg labeled target cRNA for 16 h at 45°C. Microarrays were stained with streptavidin-phycoerythrin solution and scanned with an Agilent 2500A GeneArray Scanner. Data analysis Sporophytic data from public baseline GeneChip experiments used for comparison with the pollen transcriptome were downloaded from the NASC website [ 41 , 64 ]. The list of dataset codes was as follows: COT (three replicates), Cornah_A4-cornah-wsx_SLD_REP1-3; LEF (three replicates), A4-LLOYD-CON_REP1-3; PET (three replicates), Millenaar_A1-MILL-AIR-REP1-3; STM (two replicates), Turner_A-7-Turne-WT-Base1-2_SLD; ROT (two replicates), Sophie_A1-Fille-WT-nodex_SLD, Sophie_A5-Fille-WT-nodex_SLD; RHR (two replicates), Jones_A1-jones-WT1, SLD, Jones_A1-jones-WT2_SLD; SUS (three replicates), A1-WILLA-CON-REP1-3. All gametophytic and sporophytic datasets were normalized using freely available dChip 1.3 software [ 65 ]. The reliability and reproducibility of analyses was ensured by the use of duplicates or triplicates in each experiment, the normalization of all 26 arrays to the median probe intensity level and the use of normalized CEL intensities of all arrays for the calculation of model-based gene-expression values based on the Perfect Match-only model [ 66 , 67 ]. A given gene was scored as 'expressed' when it gave a reliable expression signal in all replicates. Expression signal value '0' means that the detection call value was not 'present' in all replicates provided. All raw and dChip-normalized gametophytic datasets are available at the Institute of Experimental Botany AS CR website [ 68 ]. Although a RT-PCR validation of microarray data was not performed specifically for the purpose of this publication, our confidence in the quality of the data presented is based on our previously published RT-PCR validation of the expression of 70 genes [ 21 , 35 , 41 ]. Microsoft Excel was used to manage and filter the microarray data. For annotation of genes present on the ATH1 Array, the Arabidopsis Genome Annotation Release 3.0 published by The Institute for Genomic Research [ 52 ] was used. Genes were sorted into functional categories created according to data mined from the Munich Information Center for Protein Sequences Arabidopsis thaliana Database [ 69 ], Kyoto Encyclopedia of Genes and Genomes [ 70 ] and TAIR [ 46 ]. Hierarchical clustering of expressed genes was performed using expression-profile data clustering and analysis software EPCLUST [ 71 ], with correlation measure based distance and average linkage clustering methods. Additional data files The following additional data is available with the online version of this article: Additional data file 1 is an Excel file containing the following items. The table Data contains the complete transcriptomic datasets used. Data were normalized using dChip 1.3 as described in Materials and methods. Expression signal value '0' means that the detection call value for particular gene was not 'present' in all replicates provided. In the column 'Cluster', the appropriate cluster for each male gametophyte-expressed gene is shown. The table Clusters gives the number of genes comprising all 37 clusters of genes coexpressed during male gametophyte development. The table Cell-cycle data lists core cell-cycle genes showing their expression values in male gametophytic datasets. Genes were defined according to [ 21 ]. The chart shows expression profiles of male gametophyte-expressed core cell-cycle genes. The table Transcription data lists transcription-factor genes, showing their expression values in male gametophytic datasets. Genes comprising Arabidopsis transcription factor families were derived by compilation of data available at the Ohio State University Arabidopsis Gene Regulatory Information Server [ 47 ], data published in [ 22 ] and database homology searches. MADS-box and bHLH gene families were defined according to [ 23 ] and [ 24 ], respectively. The table Translation data lists core translation-factor genes showing their expression values in male gametophytic datasets. Genes were defined according to the FIAT database [ 51 ]. The chart shows expression profiles of male gametophyte-expressed core translation-factor genes. The Transcription table summarizes transcription factor gene families showing the number of genes expressed during male gametophyte development. Additional data file 2 lists a complete set of 39 clusters of genes coexpressed during male gametophyte development. Clusters were determined using EPCLUST software with a threshold value of 0.05. The list of genes comprising each cluster is given in Additional data file 1 . Additional data file 3 gives the expression profiles of male gametophyte-expressed transcription factors sorted into individual gene families. Expression data are given in Additional data file 1 . Supplementary Material Additional data file 1 The complete transcriptomic datasets used Click here for additional data file Additional data file 2 The complete set of 39 clusters of genes coexpressed during male gametophyte development Click here for additional data file Additional data file 3 The expression profiles of male gametophyte-expressed transcription factors sorted into individual gene families Click here for additional data file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545776.xml
509296	Grafting the Way to the Systemic Silencing Signal in Plants	Grafting is a powerful but complex means to study the spread of RNA silencing	Grafting is an ancient technique used by farmers and gardeners to combine desired attributes of the rootstock with those of the donor plant shoot, or scion. Grafting essentially saved European wine making: when the insect Dactylosphera vitifoliae devastated European grapewine varieties over the course of the late 1800s and early 1900s, the varieties were saved by grafting them onto resistant rootstocks from the New World. Since then, these rootstocks have been used to maintain the susceptible Old World cultivars. But grafting is also an excellent tool for scientists studying systemic signals traveling between the rootstock and distal parts of the plants, and vice versa. For example, two important studies ( Palauqui et al. 1997 ; Voinnet et al. 1998 ) used grafting to demonstrate the spreading of RNA silencing in plants. However, it was a subsequent paper ( Crete et al. 2001 ) that followed up on certain inconsistencies in the grafting results that pointed to subtleties important for both experimental design and understanding systemic signaling in plants. RNA silencing (termed posttranscriptional gene silencing in plants, quelling in fungi, and RNA interference in animals) refers to the phenomenon whereby specific gene transcript levels are reduced in the presence of a related RNA. From studies of RNA silencing in several systems, much is now known about the mechanisms involved ( Matzke 2002 ; Mlotshwa et al. 2002 ), but the systemic spreading in plants is still a bit of a mystery. Posttranscriptional gene silencing spreads systemically throughout the individual plants in a very characteristic manner reminiscent of viral spread. This has led to the hypothesis of a systemic silencing signal that is produced in the tissues where silencing is initiated and is then transmitted to the distant parts of the plant where it can initiate silencing in a sequence-specific manner. The sequence specificity of the silencing strongly implies that the signal is a nucleic acid, most likely an RNA, but the identity of the signal remains unknown. Silencing spreads mainly in the direction from carbon source to carbon sink, that is, from tissues such as leaves that export the sugar products of photosynthesis, to tissues such as roots that import these products, and it can take up to several weeks until it is established in the whole plant ( Palauqui et al. 1997 ; Palauqui and Vaucheret 1998 ; Voinnet et al. 1998 ; Sonoda and Nishiguchi 2000 ). As expected, the discovery of this process triggered a quest for the “systemic inducer” of the process: a signal that travels through the plant and is able to initiate silencing in a remote location within the plant. Grafting was an obvious tool to use in the quest for this signal, as it allowed silencing source and sink tissues to be of different origin. Palauqui et al. (1997) were the first to unambiguously demonstrate that silencing spreads from a silenced rootstock to a nonsilenced scion. They used as a stock a transgenic tobacco carrying an additional copy of the endogenous nitrate reductase gene, Nia . Some of the transgenic lines generated always showed higher levels of Nia transcripts than the wild type—as expected from the presence of an additional gene—and were termed class I lines. However, other transgenic lines had undergone silencing for both the endogenous and exogenous Nia genes and those were termed class II lines. Pallaqui et al. (1997) found that silenced class II rootstocks were able to silence class I scions. This was true even in a “sandwich graft,” where a wild-type (nontransgenic) segment was grafted between the silenced stock and the nonsilenced scion. Spreading of silencing was unidirectional from stock to scion. Though not explicitly stated, it was implied that it took more than 3 wk after grafting for systemic silencing to occur in the scion. The reported rate of transmission was 100%. Related experiments by Voinnet et al. (1998) used scions that transgenically expressed green fluorescent protein grafted onto plants with established silencing of the same transgene. There, too, silencing spread through the graft to the nonsilenced scion, even when a wild-type section was grafted between transgenic rootstock and scion. Unlike the Nia transgene, which has an endogenous counterpart in the wildtype plant, green fluorescent protein has no homolog in nontransgenic lines. Therefore, silencing spreading in the wild-type “spacer” in the sandwich grafts could not be assisted by an endogenous sequence. Rather, the systemic signal must have traveled all the way to the scion and induced gene silencing there. The establishment of systemic silencing took 4 wk in the “direct” grafts and 6 wk in the sandwich grafts. However, silencing spread to the scion only in some of the grafts: in ten out of 16 direct grafts and five out of 11 sandwich grafts. I found these papers were very important not only for what they proved—the existence of a systemic signal of silencing—but also because they gave an unequivocal answer to the scientific questions they posed, using relatively simple methodology. Although excited by these successful examples of the transmission of silencing, I kept coming back to two questions: (1) what prevents transmission in some of grafts, and (2) why does it take longer to transmit silencing to the scion than it takes systemic silencing to reach the most remote parts of an intact plant? When we started working on the silencing signal ourselves, we repeated some of the above experiments but found somehow lower efficiencies in the initiation of silencing in the scions. We soon realized that our results were influenced by the developmental stage of our scions. A paper from Jeff Meins's laboratory in Switzerland shed light on some aspects of the grafting puzzle. Researchers there introduced additional chitinase genes using bolistics, in sense or antisense orientation under the control of a strong promoter (35S) into chitinase transformant lines of tobacco that never exhibited spontaneous gene silencing ( Crete et al. 2001 ). In lines bombarded late in plant development, triggering of silencing was rarely observed. However, when the transgene was introduced earlier in development, a large portion of the lines showed a substantial decrease and eventually full suppression of the chitinase mRNA levels. Lines that showed silencing were used as rootstocks and nonsilencing lines were used as scions in three types of grafting experiments. In the first type of grafting experiment, called top grafting, a 5-cm scion cut into a wedge at the bottom was inserted into the vascular ring at the cut surface of a 50-cm-high rootstock ( Figure 1A ). In the second type of experiment, reciprocal transverse grafts of 50-cm-tall plants were exchanged between class I and class II plants ( Figure 1B ). Finally, the third type of experiment involved plug grafts, which were made by exchanging transverse plugs of stems cut with a 5-mm-diameter cork borer from an internode approximately in the middle of a 50-cm plant ( Figure 1C ). Figure 1 Grafting Method May Influence the Spreading Efficiency of the Silencing Signal (According to Crete et al. 2001 ) (A) Top grafting, the most effective in transmitting the silencing signal. (B) Reciprocal transverse graft. (C) Plug graft. Surprisingly, only top grafting resulted in scions that were systemically silenced by a rootstock signal. Furthermore, even transmission after top grafting was less effective than expected; in one stock/scion combination only 27 out of 71 grafts exhibited transmission of the silencing signal. The authors also found that antisense-induced silencing was never transmitted to the scion. These findings do not answer the many questions about the mechanisms underlying systemic silencing, but they point us in certain directions. The individual parts of a whole plant are, in terms of import and export, in an equilibrium that changes with development. When grafting takes place, how this equilibrium is altered depends on the individual “parts” that contribute to the “new” whole plant. In addition, there is now indirect evidence ( Fagard and Vaucheret 2000 ) that what is source tissue and what is sink tissue in terms of sugar transport affects what is source and what is sink in terms of the systemic silencing signal. Taking into account the above findings and choosing the right combination of stock/scion, we have managed to significantly increase the efficiency of graft-transmitted silencing, a prerequisite for continuing the search for the systemic signal. From the grafting experiments to date, it is now evident that the transporting capacity of the vascular tissue bypass that is formed at the graft junction does not fully reach the level of the original vascular tissue. The basis for these restrictions is not known. In a way, the graft interface functions as an unintentional filter. If the specificities of this “filter” were known, it would help us comprehend some transmission inconsistencies. Keeping in mind these limitations, grafting remains an invaluable tool in the search for the systemic silencing signal.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC509296.xml
524363	Relationship between CRP and hypofibrinolysis: Is this a possible mechanism to explain the association between CRP and outcome in critically ill patients?	Background- Endothelial cell dysfunction may be implicated in the development of multiple organ failure (MOF) by a number of mechanisms. Among these, altered fibrinolysis promotes fibrin deposition, which may create microvascular alterations during inflammation. Elevated concentrations of C-reactive protein (CRP), especially when these persist over time, are correlated with an increased risk of MOF and death. CRP may inhibit fibrinolysis by inducing plasminogen activator inhibitor-1 (PAI-1) release from human aortic endothelial cells. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels. In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis in intensive care patients with and without sepsis. Methods- We studied the association of inflammation and abnormal fibrinolysis in intensive care unit (ICU) patients with (n = 11) and without (n = 21) sepsis. The inflammatory response was assessed by serum concentration of C-reactive protein (CRP), a marker of the acute phase reaction, which increase rapidly in the inflammatory response, and the plasma fibrinolytic capacity was evaluated by the Euglobulin Clot Lysis Time (ECLT), determined by a new semi-automatic method. Results- ECLT was significantly higher in septic than non-septic patients (1104 ± 439 vs 665 ± 275 min; p = 0.002) and was significantly correlated with CRP concentration (R 2 = 0.45; p < 0.001). In a multivariate analysis, CRP was the strongest predictor of ECLT (R 2 = 0.51, F = 25.6, p < 0.001). In addition, the overall ICU length of stay was significantly correlated with CRP (R 2 = 0.264, p = 0.003) and ECLT (R 2 = 0.259, p = 0.003). Conclusion- In critically ill patients a significant correlation thus exists between plasma fibrinolytic capacity and serum CRP levels. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. This finding is compatible with a link between inflammation and abnormal fibrinolysis, and may explain the negative prognostic value of CRP in critically ill patients.	Background Endothelial cells have a key role in the control of vascular permeability and vessel tone, coagulation and fibrinolysis, and inflammatory response [ 1 ]. There is an increasing body of evidence supporting the critical role of the vascular endothelium in the pathogenesis of multiple organ failure (MOF) in critically ill patients [ 2 ]. Endothelial dysfunction/or activation is associated with an imbalance in hemostatic functions. Endothelial cells are responsible for the release of tissue plasminogen activator (t-PA) and contribute to the release of plasminogen activator inhibitor-1 (PAI-1). Inhibition of the fibrinolytic system amplifies the pathogenic role of fibrin deposition during severe inflammation [ 3 ]. Multiple factors, including lipoproteins, cytokines, and inflammatory proteins can modulate the endothelial cells to produce t-PA and PAI-1 [ 4 ]. In infected patients, elevated concentrations of serum CRP are correlated with a risk of MOF and death [ 5 ], especially when these persist over time [ 5 ]. However, the possible biological involvement of CRP in the development of MOF and death is unknown. CRP can act directly on endothelial cells, inducing, for example, the expression of intercellular adhesion molecule (ICAM)-1 [ 6 ] and the production of inflammatory cytokines such as interleukin- (IL)-6 [ 7 ]. It may also inhibit fibrinolysis by inducing PAI-1 release from human aortic endothelial cells [ 8 ]. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels [ 9 ]. In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis as measured by ECLT in intensive care patients with and without sepsis. Material and methods Subjects After approval by the A. Vésale hospital ethics committee, we studied 32 ICU patients with severe sepsis (n = 11) or other diagnoses (n = 21). Infection definition required isolation of a microorganism from a normally sterile body site, concurrent with accompanying signs and symptoms of sepsis and decision of antibiotic therapy. Criteria for severe sepsis included signs of at least one organ dysfunction attributed to sepsis [ 10 ]. All patients (septic and non-septic) were enrolled in the first day of ICU admission to limit the delay of inflammatory response. The exclusion criteria were: antibiotics treatment in the non-septic group except for surgical prophylaxis, red blood cell transfusion in the last 72 h, active hemorrhage, hematological disorders, recent cytotoxic chemotherapy, burns, cardiogenic shock, cirrhosis, pregnancy. The simplified acute physiologic score (SAPS II score) [ 11 ] was determined in each patient during the first 24 hours after admission. Blood samples Blood samples were obtained during the first 24 hours of sepsis or on the first day of admission for non-septic patients. Serum samples were collected in vacuum tubes without anticoagulant. Plasma samples were harvested in citrated vacuum tubes and put in melting ice. Whole blood was collected on EDTA-treated tubes. CRP was evaluated by antibody-binding and turbidity measurement on SYNCHRON LX ® . Fibrinogen was determined by thrombin time on a STA ® automate (STAGO). Leukocyte and platelet counts were determined on a hemocytometer (CELL-DYN4000 ® , ABBOTT). All tests were performed on blood obtained from the same venipuncture. Plasma fibrinolytic capacity The Euglobulin Clot Lysis Time (ECLT), which is the most common test used to estimate the plasma fibrinolytic capacity, represents the balance between t-PA and PAI-1 activities [ 12 ]. ECLT was measured on fresh plasma the same day as other parameters by a method described elsewhere [ 13 ]. Briefly, we designed a completely computerized, semi-automatic, 8-channel device for measurement and determination of fibrin clot lysis (Lysis Timer, EREM, Belgium). The lysis time is evaluated by a mathematical analysis of the lysis curve and the results are expressed in minutes (range: 5 to 9999). The efficiency scores of the method are <4% in intra-assay and <7% in inter-assay. Statistics We used SigmaStat ® software package (Jandle Scientific). The data are presented as mean ± SD. Correlation between variables was analyzed using a Pearson correlation test. A multivariate analysis was used with stepwise backward selection of the explicative variables. Sepsis was considered as a dichotomous variable while all other data were considered as continuous (CRP, fibrinogen, leukocyte, monocyte and platelet counts). ECLT was the dependent variable. A probability level of p < 0.05 was considered as statistically significant. Results The major cause of severe sepsis (9 patients) or septic shock (2 patients) was pneumonia (8 patients); angiocholitis was the cause in 1 patient, and in 2 patients the cause was not identified. In 8 patients (7 with pneumonia and 1 with angiocholitis), the infection was due to a Gram negative bacteria. Only 1 patient had documented bacteremia. Non-septic patients were admitted for postoperative surveillance (7 patients), intracerebral hemorrhage (3 patients), heart failure (3 patients), drug intoxication (3 patients), or aggravated chronic obstructive pulmonary disease (5 patients). As expected, inflammatory parameters such as white blood cells and CRP levels, the SAPS II score and ECLT were higher in the septic than the non-septic population (Table 1 ). In an univariate analysis (in all patients), the ECLT was strongly correlated with serum CRP concentrations (R 2 = 0.45; p < 0.001) with no perceptible threshold (Fig 1 ). Surprisingly, there was no relationship between the SAPS score and ECLT (R 2 = 0.08; p = 0.15). In multivariate analysis, ECLT was best predicted by the CRP level (R 2 = 0.51; F = 25.6; p < 0.001) and not significantly by sepsis or the fibrinogen concentration. Interestingly, the ICU length of stay was significantly correlated with CRP (R 2 = 0.264, p = 0.003) and ECLT (R 2 = 0.259, p = 0.003) in all patients, and in the survivors (R 2 = 0.13, p = 0.05 and R 2 = 0.3, p = 0.003, respectively). Table 1 Population characteristics Sepsis (n = 11) Non-sepsis (n = 21) p value Age, years 68 ± 19 67 ± 17 0.95 SAPS II 47 ± 11 25 ± 14 0.001 ICU stay, days 11.6 ± 7.8 6.7 ± 6.8 0.07 Death, n (%) 2 (18) 3 (14) 0.57 Leukocytes (×10 3 cells/μl) 10.4 ± 4.5 10.9 ± 3.7 0.74 Monocytes (×10 3 cells/μl) 549 ± 225 711 ± 398 0.24 Platelets (×10 3 cells/μl) 207 ± 157 234 ± 94 0.54 Fibrinogen (mg/dl) 657 ± 123 445 ± 132 <0.001 CRP (mg/dl) 24.2 ± 10.5 7.6 ± 6.1 <0.001 ECLT (min) 1104 ± 439 665 ± 275 0.002 Mean ± SD; p value (t-stest). SAPS : Simplified Acute Physiologic Score; ICU : Intensive Care Unit; CRP : C-Reactive Protein; ECLT : Euglobulin Clot Lysis Test Figure 1 Correlation between serum CRP levels and Euglobulin Clot Lysis Time. Discussion Many studies have demonstrated that sepsis is associated with endothelial cell dysfunction and promotes coagulation activation [ 14 ]. Although levels of t-PA antigen increase in sepsis, fibrinolysis is inhibited by increased levels of PAI-1. ECLT is, therefore, an interesting test, because it represents the balance between t-PA and PAI-1 activities. Previously considered as an imprecise method, we have been able to improve the precision and reproducibility of the test with a new semi-automatic device [ 13 ]. Inhibition of the fibrinolytic system may contribute to MOF, as fibrin can activate endothelial cells leading to a disorganization of the monolayer and the release of inflammatory cytokines [ 4 ]. Increased CRP levels are associated with worse outcomes and MOF in ICU patients [ 3 ]. The role of CRP on fibrinolysis is unknown in vivo . Our data suggest that CRP could itself be involved in the processes leading to endothelium dysfunction. The observed relationship does not prove a direct biological link between increasing CRP and hypofibrinolysis; however, indirect arguments exist in support of the concept. Several in vitro studies have reported the direct effects of CRP on endothelial cells [ 6 - 8 ]. In vivo , Cleland et al. [ 15 ] reported a relationship between serum CRP levels and the forearm blood flow response to N G -monomethyl-L-arginine (L-NMMA), reflecting endothelial dysfunction. Bisoendial et al. reported that the administration of CRP in volunteers impairs the fibrinolytic balance [ 9 ]. In addition, CRP has a strong prognostic value in acute coronary syndromes [ 16 ]. In a non-selected population with no inflammatory syndrome (CRP below 1.5 mg/dl, n = 160), we also observed that ECLT was significantly correlated with serum CRP levels [ 17 ]. CRP could also act indirectly on endothelial cells via the action of monocytes and the release of tumor necrosis factor-α (TNF-α). TNF-α is a strong inducer of PAI-1 production in-vitro and in-vivo . This mechanism seems to be important in sepsis, as high plasma levels of PAI-1 are associated with poor outcome [ 18 ]. Moreover, an association between CRP and TNF-α has also been described [ 19 ]. CRP can induce the monocyte release of cytokines such as IL-1β, IL-6, and also TNF-α through Fc receptors (γRI/CD64, FcγRIIa/CD32) [ 20 ]. CRP also has essential biological functions. No polymorphism of either the gene coding sequence or of the protein itself has been described in humans [ 21 ]. Also, high levels of human CRP protect against lethal infection. Transgenic mice capable of produce human CRP are protected against lethal infection by Gram positive and negative bacteria, Szalai et al [ 22 , 23 ]. This work is a pilot study. We have chosen to include patients in the first day of ICU admission to limit the possible rapid effect of inflammatory response on fibrinolysis, especially in septic patients. Indeed, this particular patient population has inflammatory reaction before signs of severe sepsis and thus before their admission to ICU. In fact, we have studied ECLT test at the onset of the organ dysfunction and the inflammatory reaction. Other studies with serial measurement of ECLT in patients who developed nosocomial ICU infections are needed to study the time course of these events. Moreover, we could not definitively exclude that all patients in the non-septic groups were non infected. For example, some non-septic patients with decompensated COPD may have minor infections, despite the negative microbiology cultures and the absence of antibiotic therapy. Viral infections were also possible in some patients. Moreover, it would be of great interest to determine the interactions between CRP and ECLT with IL-6, TNF-α and endothelium dysfunction markers such as soluble thrombomodulin and soluble von Willebrand factor. Conclusion Despite accumulating evidence that the inflammatory and coagulation systems are activated in sepsis, little is known about the mechanisms that ultimately lead to organ dysfunction and death. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. Prospective studies including the time course of CRP and hypofibrinolysis would provide additional information about this relationship. Authors' contributions KZB: Laboratory analysis, writing of the manuscript and design of the study. MP: patients recruitment and design of the study. DB: coordination and design analysis of the results. MG: design of the study. PC: laboratory analysis. JLV: design of the study and analysis of the results. CR: design of the study and analysis of the results. YB: patients recruitment. MV: statistical analysis and coordination.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524363.xml
548285	HIV, malaria and beyond: reducing the disease burden of female adolescents	In sub-Saharan Africa the highest overlap between malaria and HIV infections occurs in female adolescents. Yet control activities for these infections are directed to different target groups, using disparate channels. This reflects the lack of priority given to adolescents and the absence of an accepted framework for delivering health and health-related interventions to this high-risk group. In this paper it is argued that female adolescents require a continuum of care for malaria and HIV – prior to conception, during and after pregnancy and that this should be provided through adolescent services. The evidence for this conclusion is presented. A number of African countries are commencing to formulate and implement adolescent-friendly policies and services and disease control programs for malaria and HIV will need to locate their interventions within such programs to ensure widespread coverage of this important target group. Failure to prioritize adolescent health in this way will seriously limit the success of disease control programs for malaria and HIV prevention.	Background The current generation of adolescents – over one billion – is the largest in history [ 1 ] and, far from representing a picture of health [ 2 ], many will suffer untimely disease and death. HIV and malaria are responsible for much of the disease burden affecting female adolescents, who suffer disproportionately from these combined infections relative to other age groups. This is due to a high HIV incidence during the period when many adolescents become pregnant for the first time – an event which greatly increases their susceptibility to Plasmodium falciparum malaria [ 3 ]. Biological interactions between malaria and HIV in pregnancy complicate therapy, which can also be compromised by inappropriate adolescent health-seeking behaviour. The public health importance of HIV and malaria synergism is only now emerging. It has led to a recommendation by the World Health Organization (WHO) that the two disease programs should collaborate to ensure integrated service delivery, especially within the framework of reproductive health services [ 4 ]. A case has also been made for linking disease control programs as a more efficient and effective way for lowering the malaria and HIV disease burden [ 5 ]. Nevertheless, it is difficult to seen how these goals will be attained unless adolescents are accorded a much higher priority by disease control programs. In this paper we consider the justification, as well as the challenges, of using common approaches to improve delivery of HIV and malaria interventions to female adolescents. HIV and malaria burden in pregnant and non-pregnant adolescents Prevalence estimates for HIV infection [ 1 ] for 35 sub-Saharan African countries, for young males and females (15–24 years), are shown in Figure 1 . Estimations are often derived from sentinel surveillance of women attending antenatal care (ANC) and tend to overestimate prevalence in adolescents due to the selection of sexually active [ 6 ] and less-educated groups [ 7 ]. Accepting this bias, in every country, listed HIV prevalence is two to three times higher among females than males. Having an older male partner is associated with a modest increased HIV risk [ 8 ] but probably does not explain a sex difference that occurs uniformly across all countries and cultures and at all levels of HIV endemicity. In Rakai, Uganda, only 12.4% of HIV cases in 15–19 year olds was attributed to relationships with men 10 years older [ 9 ]. Other sexually transmitted infections (STIs) increase susceptibility to HIV but do not account for the sex difference [ 10 ]. Host biological factors may be critical [ 11 ]. Sexual maturation takes several years to complete, but menarche is delayed amongst socio-disadvantaged girls, and many will be sexually active before biological maturation is complete. Interventions that delay the onset of sexual activity could reduce exposure at a time of peak biological susceptibility to HIV and highest risk of malaria in pregnancy. Figure 1 Female and male HIV prevalence among young people 15–24 years of age in sub-Saharan Africa. Compiled from UNFPA 1 Malaria is an important cause of adolescent hospital admissions in many sub-Saharan African countries with stable malaria transmission [ 12 ]. Malaria mortality in African adolescents (both sexes) aged 10–14 years has been estimated at over 45,000 deaths per annum [ 13 ]. The highest prevalence of P. falciparum infection after childhood is also in young, mostly adolescent, women. Data from Malawi (Table 1 ) shows that non-pregnant and pregnant adolescents had significantly higher parasite rates than women above 19 years. Primigravidae also have a markedly increased susceptibility to malaria [ 3 ]. Young age and nulliparity in the same individual lead to greatly increased malaria risk. This partly relates to reduced acquisition of acquired malaria immunity in young individuals [ 14 ] as well as the lack of parity-specific malaria immunity acquired during the first pregnancy [ 15 ]. Risk factors for symptomatic malaria in adolescents have not been studied, but both symptomatic and asymptomatic malaria infection will contribute to the development of adolescent and newborn anaemia [ 16 ]. HIV infection increases P. falciparum prevalence during pregnancy and this is marked during the first pregnancy [ 17 , 18 ], which in Africa, is nearly always in adolescence. Table 1 Malaria prevalence in non-pregnant and pregnant adolescents and adults in the Shire Valley, Malawi Adolescent Adult Non-Pregnant Pregnant * Non-Pregnant Pregnant* % (n) % (n) % (n) % (n) 41.4(29)† 45.9 (122)‡ 20.3 (74) 35.4 (345) * At delivery. Includes women who had received anti-malarials during pregnancy † Difference with non-pregnant adults χ 2 = 4.8; p = 0.028 ‡ Difference with pregnant adults χ 2 = 4.3; p = 0.04 HIV and malaria interventions during adolescence For both HIV and malaria there are effective interventions for disease control. HIV and malaria frequently occur in the same African populations and the same individuals, yet control activities are directed to different target groups, using different channels. Adolescent HIV prevention, with its focus on behavioural change, has taken place largely outside conventional health settings. Few HIV strategies have been directed at pregnant adolescents and/or their partners – an omission noted by a WHO Technical Consultation on married adolescents [ 19 ]. In some African countries, by the age of 19 years, half of all female adolescents were in marital or permanent consensual unions [ 9 ] that will almost certainly lead to pregnancy. In contrast, malaria control efforts for women have focused on pregnancy, but not on adolescents and certainly not on adolescents before they become pregnant. As a result, in many sub-Saharan African settings, adolescents are often parasitaemic and anaemic when they first become pregnant. The problem is that currently no overall strategy for adolescent health has been approved for most African countries. The burden of infection and disease among adolescents is of sufficient magnitude that a way must be found to provide services to this population. The existence of an appropriate strategy would allow both HIV and malaria interventions to be delivered within a common framework. In traditional cultures, menarche often marks the transition from childhood to adulthood because it signifies reproductive potential. Nevertheless, adolescence is a physical process that takes a number of years and is usually completed by the late teens. This developmental process should be encompassed within health care provision. Pregnancy may punctuate that process, but during it, the adolescent continues to grow physically and mentally [ 20 ]. Strategies to reduce adolescent malaria or HIV should, therefore, provide a continuum of care – before conception, during and after pregnancy – encompassing appropriate information, preventive and curative services. For prevention, adolescents need access to condoms and bednets, information on how to use them and, when and where to go to deal with infection. Services must be available for infection monitoring and treatment, including HIV testing and drug therapy for malaria, anaemia and HIV-related opportunistic infections. System improvements may not be strictly about adolescent health services, but about support for ANC. Adolescents need good access to pregnancy care and this includes attendance for early pregnancy assessment and screening for HIV and other sexually transmitted infections (STIs). ANC presents an opportunity for HIV prevention, support for discordant couples and care and referral for HIV-infected adolescents. Early assessment is important as the HIV positive individual needs to plan for anti-retroviral treatment (ART) at delivery to prevent mother-to-child transmission. It is also important for malaria control in pregnancy. Intermittent preventive antimalarial treatment (IPT) is the key malaria prevention policy that improves birthweight outcomes and reduces pregnancy anaemia [ 21 ]. Its effectiveness depends on the prevalence of HIV infection [ 22 ]. It also depends on adherence to the treatment regimen. Coverage with the standard two dose intermittent sulphadoxine-pyrimethamine preventive malaria treatment was lower in younger adolescents in the Shire Valley, Malawi ≤ 17 years, 35.1% versus 18–19 years, 53.7%; p = 0.028, Verhoeff, personal communication), and more than half of all pregnant adolescents received inadequate antimalarial treatment. This demonstrates the importance of pre-conceptual malaria health education for girls. Adolescent mothers need instruction on how to recognize and respond to infant malaria infection and encouragement for using bednets for themselves and their babies. A major challenge for malaria control is to improve adolescent health prior to the first pregnancy. For non-pregnant adolescents there are several conundrums. On account of drug resistance, malaria combination therapy with artemisinin derivatives is being introduced into an increasing number of sub-Saharan African countries. Artemisinin may be foeto-toxic and should be avoided, except for symptomatic infections with multi-drug resistant malaria, in the first trimester of pregnancy [ 23 ]. Inadvertent use or self-treatment by adolescent girls could frequently occur in early pregnancy, before they recognise their pregnancy. The issue of pregnancy testing adolescent girls before treatment with potentially foeto-toxic drugs will need to be considered. A further issue is that the benefits of IPT extend to pregnant but not nulliparous adolescents who may be anaemic or parasitaemic at the start of their pregnancy. The use of permethrin-treated bednets would protect against anaemia and parasitaemia during the first trimester, but adolescent pregnant girls and their newborns are the least likely to be net users [ 24 ]. Reaching the target population The growing literature on adolescent-friendly health services shows that adolescents have substantial concerns about the delivery of interventions through health services [ 25 ]. Almost universally, adolescents complain of lack of information, confidentiality issues and judgmental attitudes of service providers. There may be barriers of physical inaccessibility, service fees and non-supportive community attitudes. A WHO consultation in Africa concluded that adolescents had "a right to access health services that can protect them from HIV/AIDS and from other threats to their health and well-being, and that these services should be made adolescent friendly" [ 26 ]. A WHO Global Consultation on Adolescent Friendly Health Services stated that government ministries should take appropriate action to ensure service provision for adolescents, taking into account cost, epidemiological factors and national health priorities [ 27 ]. The role of non-governmental organizations and the private sector in spearheading adolescent projects was acknowledged, but alone, these organizations cannot obtain sufficient coverage of the adolescent population to substantially reduce the HIV and malaria burden. Governments and disease control programs must also consider training health care providers and modifying service delivery, so as to reduce barriers to adolescent uptake. The only country in Africa that has scaled up adolescent health services is South Africa [ 28 ]. The South African National Adolescent-Friendly Clinic Initiative has strengthened the public health sector's ability to respond to adolescent health needs by building capacity and establishing good clinical services which, eventually, will be maintained by district and provincial health authorities. It has demonstrated that an adolescent strategy is achievable if there is political will. The South African approach may not be suitable or affordable for lower income countries. Well-focused services, training of key staff at selected sites, outreach preventive services and good quality, adolescent-oriented ANC are potential service reconfigurations that could be used to extend coverage and increase uptake of both HIV and malaria interventions without creating a separate adolescent service. What about young men? Available evidence suggests that young men are not attracted to clinic services which are perceived as "female "spaces [ 29 ]. In Ghana, for example, 76–89% of all adolescent clinic users were female [ 30 ]. Malaria is a reproductive health issue for females, but not for males, and the disease burden of both malaria and HIV infections is much lower in young males. Adolescent females require more health-service based care than males, and this should be reflected in service configuration. Creating an enabling environment to promote adolescent access to HIV and malaria interventions Behavioural change, including appropriate health-seeking practices, is essential for disease reduction. A decline in HIV prevalence amongst adolescents has been reported from Zambia [ 7 ], Uganda [ 31 , 32 ] and Tanzania [ 33 ] and is attributed to changes in sexual behaviour. In Zambia, between 1996 and 1999, urban adolescent males reported less frequent sexual activity, fewer partners and were more likely to have used a condom at the last sexual encounter. Changes in these indices were less marked, or even deteriorated, among rural female adolescents [ 7 ]. Whether behaviour change is the result of HIV programme interventions or other factors is unclear. Recent surveys in sub-Saharan Africa reported that higher female education was associated with lower HIV prevalence and lack of schooling with higher HIV incidence [ 6 ] and lower condom use [ 34 ]. In western Uganda the proportion of illiterate women fell from 41.6% to 24.6% over six years and may have facilitated behavioural changes, but this link has been little investigated. Illiterate females are likely to be young and married, and to be in a situation where they cannot reduce frequency of intercourse, negotiate condom use or delay having children [ 19 ]. In a large pregnancy study in southern Malawi, 73% of 469 nulliparous adolescents were illiterate [ 35 ]. They made significantly fewer ANC visits, were less likely to have a supervised delivery and had a higher risk of low birthweight. Reducing illiteracy and increasing educational and vocational opportunities for female adolescents may be critical enabling actions to promote risk reduction, to enhance their social status and to decrease female vulnerability. [ 36 ] Developing policies to support HIV and malaria control for female adolescents Few developing countries have well-defined policies for delivering care to the non-pregnant or pregnant adolescent, especially for sexual health. Existing malaria guidelines also do not mention adolescents specifically [ 12 ]. An important aspect is the issue of the adolescent's right to sexual and reproductive information and services vis à vis the legitimate rights of parents to act in the best interests of a child, and the health care provider's right to work within the law. These are issues of consent and confidentiality for adolescents considered "minors" and under parental control, or wives under their husband's or husband's family's jurisdiction. When these issues are ignored, adolescents cannot be assured of confidentiality, and health workers are neither bound, nor protected by, the legal system. Rights established in law, or by signed Conventions (eg Convention of the Rights of the Child), may not be implemented. Health policy falls down because it is not explicit about the vulnerability of female adolescents and does not challenge cultural and social norms that can perpetuate the disease burden. Similarly, encouragement for improving female literacy and education may be lacking, reducing the supportive environment for promoting uptake of adolescent health services and safer sex. Advocacy is essential to co-ordinate law, custom and health practice. At another level, it is important to acknowledge that interventions for female adolescents raise ethical issues that are avoided when they are excluded from care. The policy of avoiding treatment of adolescent girls (and adult women) because they might be in the first trimester of pregnancy, raises issues of gender discrimination and has been a serious issue for parasitic disease control programs [ 37 ]. Reducing the necessity for first trimester treatments for malaria can be achieved by increasing efforts to redress the malaria burden prior to pregnancy, but this requires reconfiguring service delivery. HIV-positive adolescents who are at the start of their sexual and reproductive lives might be prioritized to receive anti-retroviral therapy, but their relative poverty and lack of social status may lead to their exclusion from access to this intervention. Conclusions For both HIV and malaria, although interventions are available to reduce the disease burden, adolescents are not prioritized. Effective policies and services are needed to reduce the substantial disease burden they experience from these infections. To secure this goal, concerted action across HIV and malaria disease control programs is required. This will reinforce support for adolescent programs rather than weakening them by multiple separate activities, which can confuse implementation. A schedule of activities is required that is reinforced by other systems (e.g. education) to facilitate embedding it into a way of life. These issues need to be addressed head-on by the adolescent health community to achieve the focus needed, and to establish the realistic steps forward. In the short term, developing services to deliver HIV and malaria interventions can act as a catalyst for addressing broader adolescent health service needs. In the medium-term adolescent-friendly health services provide the required platform for the evaluation and delivery of future vaccines and drug therapies for HIV, malaria [ 38 ] and other diseases. In the longer term an inter-sectoral adolescent strategy provides a basis for breaking the cycle of maternal-child ill health. Authors' Contributions Both authors made substantial contributions to the analysis, interpretation, drafting and revision of this article. Both have agreed to authorship.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548285.xml
544396	Participation of African social scientists in malaria control: identifying enabling and constraining factors	Objective To examine the enabling and constraining factors that influence African social scientists involvement in malaria control. Methods Convenience and snowball sampling was used to identify participants. Data collection was conducted in two phases: a mailed survey was followed by in-depth phone interviews with selected individuals chosen from the survey. Findings Most participants did not necessarily seek malaria as a career path. Having a mentor who provided research and training opportunities, and developing strong technical skills in malaria control and grant or proposal writing facilitated career opportunities in malaria. A paucity of jobs and funding and inadequate technical skills in malaria limited the type and number of opportunities available to social scientists in malaria control. Conclusion Understanding the factors that influence job satisfaction, recruitment and retention in malaria control is necessary for better integration of social scientists into malaria control. However, given the wide array of skills that social scientists have and the variety of deadly diseases competing for attention in Sub Saharan Africa, it might be more cost effective to employ social scientists to work broadly on issues common to communicable diseases in general rather than solely on malaria.	Introduction Malaria control remains ineffective in many endemic areas in spite of efficacious interventions, such as combined antimalarial therapies and insecticide-treated materials. Biological, environmental, political, socio-cultural, economic and behavioural factors influence the transmission of malaria, thus requiring a multidisciplinary and integrated approach to effectively control the spread of malaria [ 1 , 2 ]. In recent years international initiatives, such as Roll Back Malaria, have highlighted the contributions that social scientists bring to a multidisciplinary approach for malaria control. Various studies in Africa have illustrated the benefits of collaborative research between social and biomedical scientists [ 3 - 17 ]. However, social science knowledge and practices are still not fully integrated into malaria research and control programmes, especially when compared to other public health areas such as HIV/AIDS prevention. While it is difficult to do a comparison on the actual number of social scientists involved in HIV/AIDS versus malaria control, a review of the literature illustrates the numerous contributions that social and behavioural research has had in HIV/AIDS control and prevention [ 18 - 27 ]. The early recognition and inclusion of socio-behavioural research was part of the HIV/AIDS prevention strategy at the onset of the pandemic [ 27 ]. Significant funding allowed social scientists to work directly with epidemiologists in HIV/AIDS research. Several factors continue to limit the application of social science research in tropical public health in general, and malaria control in particular. These include: confusion among many non-social scientists and social scientists alike about the variations in social science theory and methods and the different types of contributions that each can make to public health and malaria control activities, a perception among many public health and malaria control professionals that the problems of communicable diseases can be solved with technical and clinical interventions, ignoring the social nature of illness, limited communication among social scientists working in applied health and malaria control, insufficient access to current social science and malaria literature, and the lack of trained social scientists with applied experience in the endemic countries [ 28 , 29 ]. Given the poor academic and professional structures found in many African countries, an increasing number of highly skilled citizens emigrate abroad for further studies but fail to return to their respective country of origin, a phenomenon commonly referred to as 'brain drain' [ 30 - 36 ]. As a result, many African countries now have a vast pool of highly skilled professionals who are permanently living abroad, making little to no contributions to their country of origin [ 37 , 38 ]. While the "brain drain" significantly contributes to the lack of social science capacity in malaria control in Africa, it is unclear the extent to which the current pool of trained social scientists that remain in Africa is being integrated into malaria control. Experience has shown that commissioning a well-trained and field-experienced applied social scientist (i.e. one with both an understanding of the theoretical and applied perspectives of the discipline) can be extremely beneficial for informing malaria programme-related decisions, as well as helping in the development of effective intervention programmes [ 39 , 40 ]. This study was designed to better understand the level of involvement of African social scientists in malaria control, in order to identify potential approaches to facilitating collaborative work among social scientists and malaria control programme personnel and stakeholders. Methods A convenience sample was derived from existing social science networks such as, the Partnership for Social Science and Malaria Control (PSSMC) Network, the Social Science and Medicine Africa Network (SOMA-Net), Pan African Anthropological Association (PAAA), as well as the CHANGE Project database of African social scientists, and the United Nations Development Program, World Bank and World Health Organization's Special Programme for Research and Training (WHO/TDR) database of social scientists trained in the last decade (1992–2000). Snowball sampling was used to elicit names of additional social scientists known personally to the participants and investigators. Social science was broadly defined to include disciplines such as anthropology, sociology, health economics, demography and population studies, development studies and public health. Enrolment criteria included specific social science training within these disciplines, and participants had to be of African origin. Enabling factors were defined as those factors that made it easy to work in malaria control, including factors that first attracted social scientists and those that facilitated entrance into the field. Constraining factors were defined as those factors that impeded work in malaria control and factors that made malaria control unattractive to social scientists. Data collection was organized in two phases. In Phase I, consent forms and questionnaires were sent by email and/or fax to potential participants. Participants could respond directly and/or refer other eligible colleagues. Those who were ineligible or uninterested could decline. For those emails that could not be delivered, three attempts were made to resend the survey and consent form. The questionnaire focused on demographic characteristics, work experiences, factors influencing career choices and sources of career development information. The survey instrument was piloted among five participants from different countries. Based on the feedback from the pilot survey, minor adjustments were made to improve use via email. Email communication was preferred but, in the absence of email, other methods of communication were used. If the survey was not returned within three weeks, attempts were made to follow-up. The survey responses in Phase I provided an overview of the involvement of the participants in malaria related activities but gave only a limited understanding of the specific factors affecting career trajectories. In Phase II, we conducted in-depth phone interviews using a purposive sample derived from Phase I that was selected according to country of origin, sex, academic field (social science discipline) and interest in malaria and applied or operational work experience (both in malaria and non-malaria). A deliberate effort was made to ensure that the sample represented those with or without an interest in malaria, those who actively sought employment in malaria and those who did not. The more descriptive data from Phase II clarified Phase I data. For example, with regards to participants' interest in malaria, some participants in Phase I indicated an interest in malaria while in school, but did not include malaria in their thesis research and did not pursue employment in malaria. The in-depth, open-ended phone interviews in Phase II provided greater detail as to other factors that might have influenced participants' career decisions in malaria control. Open-ended questions focused on key events and times in the career trajectory during which critical decisions were made, current and past work experiences, reasons for selecting specific jobs, technical experiences with proposal writing, challenges and rewards of working in malaria, and suggestions for improving the integration of social science in malaria control. Descriptive statistics were generated using EPI-Info. Qualitative data were transcribed and content analysis was used to develop contextual themes. The study was reviewed and approved by the Institutional Review Board of the Centers for Disease Control and Prevention. Verbatim quotes from the participants are used throughout the text to illustrate contextual points of discussion. Results This study was conducted between May 2002–May 2003. Of the 136 surveys sent out, 40 completed surveys were found to fit our enrolment criteria (Fig. 1 ). Eighteen participants were interviewed in Phase II. Figure 1 Response to surveys. Flow chart illustrating response to surveys All 40 participants were nationals of African countries and resided in the countries of Cameroon, Ethiopia, Mali, Ghana, Kenya, Nigeria, Tanzania, South Africa and Uganda. Most participants (75%) were male between the ages of 30–39 years old (Table 1 ). Fifty percent (n = 20) had a masters degree, 42% (n = 17) were either doctoral degree students (n = 3) or had a doctoral degree (n = 14). Almost all participants had an undergraduate degree in the social sciences and chose various social sciences disciplines for specialization during their postgraduate training. Table 1 Age and Sex Distribution (n = 40) Age group Male Female Percentage 20–29 1 0 2.5% 30–39 17 5 55.0% 40–49 9 4 32.5% 50–59 2 1 7.5% 60+ 1 0 2.5% Total 30 10 100.0% Of the 18 participants selected for a phone interview, 55% (n = 10) were between 30–39 years and 22% (n = 4) were female. Of those with postgraduate degrees, 50% (n = 9) had a masters degree, 44% (n = 8) either had a doctoral degree or where doctoral degree students while 5% (n = 1) had a bachelor's degree. When difficulties arose in ascertaining whether the professional discipline linked to the social sciences, participants were contacted for clarification. Eighty-five percent of the participants (34/40) received funding for postgraduate training from their national government, international organizations (such as the WHO or DFID), or other foreign governments. Sources of career development information included the Internet, friends and colleagues, local universities, conferences, journals, professional networks and local newspapers. Enabling factors Interest in malaria When asked if participants were interested in malaria during their academic training, 82% (33/40) of the participants said yes. Only 36% (12/33) of those interested included malaria as a research topic in their thesis or dissertation. Three participants (7.5%) who were not interested in malaria included malaria in their thesis. Post graduation, 35% (14/40) of the participants actively sought employment in malaria control. Three of the 14 (21%) who sought employment in malaria were turned down for jobs due to insufficient technical knowledge of malaria control, while six participants (15%) returned to previous health care positions, some of which were positions in malaria control. Although some participants were not currently working on malaria-related projects, nearly all participants, at some point in their career, had worked as a consultant in malaria control in addition to their full time positions. Participants who specialized in malaria for postgraduate degrees were often funded by a national or international organization, on condition that they would return to their jobs in malaria following the completion of studies. Eleven of the 40 participants said that the primary factor that prevented those with an interest in malaria from seeking further research and employment opportunities in malaria control was the perception that social scientists could not be employed in malaria control. As one participant remarked "Malaria is an endemic disease in my community, so I can't say I was never interested. I wanted to do a thesis on malaria, but I realized that social scientists could not get jobs in malaria so I had to look elsewhere." When followed up during the phone interviews in Phase II, it was clear that some participants turned down jobs in malaria due to competing employment in areas such as HIV/AIDS, while others declined malaria control contracts due to the competing demands of doctoral-level training. Another issue that was revealed in Phase I was the influence of the epidemiology of malaria on participants' interest in malaria. Eleven of the 18 participants during the phone interview explained that they developed an interest in malaria after learning about the complex nature of the disease, its high prevalence and the possibility of access to research funds. "For years I thought the field of malaria was boring and I was not interested in it. I only became interested in malaria when I realized that it was a complex disease with a major public health impact and it appeared that funding for research might be available." Factors influencing employment in malaria All 40 participants were asked to identify factors that attracted social scientists into general employment opportunities, as well as malaria-specific opportunities (Table 2 ). Responses in Phase II provided further clarification on the specific factors affecting career trajectories. Of the 18 participants included for an in-depth interview in Phase II, 11 indicated that a senior lecturer or mentor during the practical training experience had encouraged participants to work with them on their research project. Mentors helped to develop participants' research and proposal writing skills and identify funding and publication opportunities, and in some cases international contracts. These contracts often resulted in participants gaining local and international visibility within the larger malaria community, which led to subsequent employment opportunities. Table 2 Factors Important for Employment (n = 40) Malaria Employment Frequency General Employment Frequency Sufficient funds to complete job 11 Potential for career advancement a 26 Supportive environment that facilitates the translation of research findings into programmatic use 9 Competitive salary a 13 Ability and opportunity to contribute alternative solutions to malaria control from a social science perspective 6 Proximity to family a 12 Type of social science dimension to job 5 Social value of job b 5 Sufficient technical skills to complete jobs 5 Epidemiology of malaria 4 a Indicates factors that were identified as important for both general employment and malaria specific employment. b The social value of a job was defined as the positive impact that a given job made in the community, as well as the professional and community respect awarded to the position. Participants were more likely to seek out employment from organizations with proven track records of using research findings to improve the health of the community. Opportunistic events also shaped career development. For example, the successful completion of a project, fellowship or consultancy often produced a cascade effect by opening up other opportunities in malaria control, resulting in individual professional recognition by local and international collaborators as an expert in the field. Attending workshops sponsored by international organizations, such as the World Bank and WHO, enabled participants to get their name in the organizations' database for possible consultancies. Strong writing skills were identified as essential for job and training opportunities by most of the 18 participants in Phase II. Ten of the 18 participants (55%) in the phone interview noted that while it was initially difficult to identify and develop fundable proposals, receiving constructive feedback from rejected proposals helped improve their writing skills and led to the development of more successful proposals. "Most of the rejected proposals do not give reasons for rejection, which is unfortunate. I have been lucky to have one rejected proposal that was returned with explanations as to why it was rejected. I was told the issue we raised was very broad and vague and it made me realize that I had to be very clear in my framework. After that rejection, I was able to develop projects that had a clear framework and include a multidisciplinary team to strengthen other key areas and the next proposal we developed was accepted." When asked why they chose employment in malaria, one of the participants echoed many others by saying, "In Africa we take what we get. One rarely ends up in a profession of their choice. People take the job that is at hand and learn to deliver as best as they can." This was true for both those interested in malaria originally and those not interested. During the in-depth interviews, we also asked participants who did not initially express an interest in malaria but sought and gained malaria related employment, why they choose to stay in malaria? Of the 18 participants eight agreed with the following view: "I would probably stay in malaria because it is a major problem in my country, and I feel I have gained significant experience in malaria over the years. This has strengthened my confidence and ability to handle current and hopefully future opportunities, and maybe I am too old right now to start learning something new." Constraining factors Malaria employment challenges Participants were also asked to identify factors that constrained employment in malaria. All 40 participants were more likely to refer to employment challenges specific to malaria research, rather than malaria control, as national malaria control programmes employ very few social scientists. Ten of the 18 participants in Phase II noted that social science involvement in malaria control was a recent development in their countries, as biomedical scientists had previously dominated malaria control. Another phone interview participant remarked that "specifically focusing on malaria as the only area for job opportunities narrows ones fields for job opportunities. Although malaria is an endemic disease in my country, it is largely dominated by physicians and there are not sufficient job opportunities for social scientists in my country so it is impossible to focus only on malaria. You need something to fall back on besides malaria." Participants also noted that a paucity of research centres and limited funding for social science research also contributed to the lack of job opportunities. In situations where job opportunities existed for social scientists, they were generally limited to short-term rather than long-term career paths. Individual perceptions of malaria as a 'normal' everyday event limited participants' understanding of the impact of malaria and, thus, diminished their interest in malaria control. As one of the phone interviewed participants remarked, "At first, I did not realize the impact of malaria in my community. Everybody was talking about HIV/AIDS. I think it is because people have lived with malaria for so long that they treat it at home. It was only after doing some training at the hospital (as part of the national training requirement), that I realized how many people, especially children and mothers, die of malaria and I got interested in working in malaria control." Of the 40 participants in Phase I, 36 identified the lack of professional development opportunities as a constraining factor to employment in malaria. The lack of a supportive work environment with good communication and mutual respect was mentioned by those working primarily with biomedical scientists. "I prefer working on maternal and child health issues, as well as HIV/AIDS, because personnel in these areas are able to recognize the role of social science and request social science input. This makes me confident that my discipline is important and I can make a contribution." Insufficient technical skills in proposal writing and professional isolation from other social scientists hampered the development of collaborative relationships, which made it difficult to develop competitive proposals for funding and publication. As a participant observed, "Research is largely uncoordinated. We have no knowledge of who is doing what, there is no database of social scientists and the projects that they're working on from which we can develop collaborative projects. We neither have the infrastructure nor the access to current literature, which makes it difficult to develop manuscripts for publication." In order to meet the challenges of employment, eight of the 18 participants in Phase II stressed the importance of understanding all technical aspects of malaria control. One participant echoed the view of several others in their advice to junior social scientists, "try to learn not only about social science in malaria control, but all other aspects of malaria control as well, such as entomology, vector control and treatment. Having only a social science background without any understanding of the other areas of malaria control might make it difficult to effectively apply the social science aspect within malaria control. Besides you will always be working as part of a multidisciplinary team, therefore, you will need to understand their language in order to be able to effectively communicate the social science dimension to them." Participants also emphasized the importance of linking students to practical training opportunities in endemic areas for fieldwork experience and possible long-term opportunities. As one participant noted, "Encourage donors to develop grants for student researchers working with senior scientists, and identify conferences where students could present research findings." Integrating social science in malaria control Participants in Phase II were asked to describe strategies for improving the integration of social science in malaria control. Of the 18 participants interviewed, 13 (72%) stressed the need for social science advocacy as an important step in malaria research and control. Advocacy efforts were recommended for social scientists and non-social scientists alike, as well as policy makers in health and education and those who fund research. Five participants said their involvement in malaria projects was as a result of specific requests for social scientists in the call for proposals. All 18 participants emphasized the need to develop outreach efforts for junior social scientists in order to enhance their understanding of the potential contributions that social scientists could offer, the type of training needed, and the types of jobs they could perform in malaria control. Three of the 18 participants also suggested the revision of graduate and medical school curricula to include malaria and social science courses, but noted that support had to come from the national level for curricula change to take place. The establishment of a sustainable network of African social scientists working in malaria control was identified as a useful tool for integrating social sciences in malaria control by six of the 18 participants. Possible functions of the network were described as developing a mentoring programme for junior social scientists, a forum to disseminate current local social science research, information relating to employment and training and funding opportunities. To facilitate the application of social science research findings to national malaria control policies, one of the phone-interviewed participants explained how to include policy makers in the process of social science research. "Make the research process more participatory. Involve policy makers and consult with them so that eventually they are the ones who demand the research and in this way, social science research can influence policy. We developed working papers with key findings for distribution to various stakeholders. We published our findings on the Internet and conducted department seminars or short courses during which we discussed our research and presented the information in a very manageable manner. At least, in this way, we were able to get our findings into the hands of policy makers and hopefully begin a dialogue with them." Discussion Results of this study indicate that factors such as having an interest in malaria, a mentor during one's academic and professional career, strong writing skills, and technical experience in malaria made it possible for participants to take advantage of opportunistic events and job opportunities that led to employment in malaria control activities. As well, limited job opportunities, the lack of career development opportunities, and the lack of understanding about social scientists' role in malaria limited the involvement of social scientists in malaria control. This study revealed that many of the enabling and constraining factors for social scientists' involvement in malaria control are not unique to malaria control but are common to African researchers in general. An examination of the intrinsic and extrinsic factors regarding career trajectories of social scientists in this study revealed that the majority of participants did not specifically seek malaria as a career path but, rather, were looking for employment opportunities in general, some of which happened to be in malaria. It is unclear to what extent the career development process in many African countries contributes to the way employment is sought or if this is due to the relatively new role that social science plays in malaria control, which was noted by numerous participants. Career development is a process that ideally begins during one's formal academic training and continues throughout one's professional life. However, if career development is concentrated more towards academic training and less on postgraduate employment opportunities, it is no wonder that post graduation, students are forced to 'take whatever they can get,' as opposed to seeking opportunities that complement their interest and training. The lack of proper career counselling could also limit a student's understanding of what courses and practical experiences are most beneficial for social science and health careers, without which social scientists cannot be hired as part of a health intervention team [ 39 , 41 ]. More initiatives that provide research-training grants, career development fellowships and proposal development workshops, such as those currently established by the UNDP/World Bank/WHO/TDR, are needed to strengthen the social science capacity in endemic countries. However, creative strategies need to be identified to capture those who do not easily have access to such information and opportunities. In order to effectively design and implement malaria control interventions, it is necessary to have social scientists that are knowledgeable about the technical issues that underlie malaria control. This knowledge is acquired over time and needs to be supplemented with direct field experiences. However, given the economic constraints facing national malaria control programmes and Ministries of Health, this study shows that it might not be reasonable to expect that social scientists will be assigned solely to malaria control. Given the limited number of trained social scientists with practical experience and the variety of deadly diseases competing for attention in sub-Saharan Africa, there are few social scientists that are both willing and able to dedicate their expertise only to malaria control. Ministries of Health and Education should work with social scientists to establish employment opportunities in communicable diseases. Given the wide array of skills that social scientists have, it would be cost effective to employ social scientists to work broadly on issues common to communicable diseases, rather than solely on malaria. This "generic" approach would also support the programmatic shift from malaria as a vertical programme to its being integrated within primary health services or the wider arena of communicable diseases. Although this may not be ideal for building strong social science capacity specifically for malaria, this offers a pragmatic approach to utilizing scarce resources. Lessons learned from behavioural interventions in other diseases can also be applied to malaria control, thus strengthening social science contributions to malaria control. The acknowledgement by many participants of their need for a better understanding of both biomedical and technical aspects of malaria control is reinforced by data from other successful projects that are aimed at establishing collaborative multidisciplinary research teams. For example, the International Clinical Epidemiology Network (INCLEN) social science programme seeks to enhance clinicians' understanding of social science by exposure to relevant issues in social science design and measurement and evaluation issues, while teaching principles of clinical epidemiology and biostatistics to established social scientists. As a result, strong collaborative partnerships among individual clinical epidemiologists, social scientists, and biostatisticians have been developed to produce the interdisciplinary solutions required for priority health problems in society [ 42 ]. A key factor identified as important for employment in malaria was the ability to contribute social science solutions to malaria control and the integration of research findings into malaria control programmes and policies. Social scientists are more likely to work directly with members of the community therefore, it is important that strong relationships are developed with local communities and their leaders, as a trust building mechanism and a matter of social prestige for the researcher. This link to the community facilitates the application of research findings on a local level [ 39 , 42 ]. Limitations While findings from this study give us greater insight into factors that affect career trajectories of African social scientists, caution must be used when interpreting the results. The use of convenience sampling and snowball sampling techniques to identify eligible participants appeared to have excluded the younger more recently qualified social scientists who might not have been members of the various networks we used to identify eligible participants for this study. It is possible that some of the junior social scientists do not have much work experience in malaria and are not yet known by the more experienced social scientists that helped with referring other participants for the study. In addition, although telephone interviewing enabled us to reach social scientists from several countries, we were not able to reach those social scientists with no access to the Internet and telephone who might have had other unique challenges not captured in this study. Further research should examine younger or more recent social science graduates as well as those with no access to the Internet. Conclusion Malaria control is at a point where there is good evidence-based data to demonstrate that various approaches can make a difference in malaria-related morbidity and mortality. Prompt and timely treatments with an effective drug, intermittent preventive treatment for malaria in pregnancy, and use of insecticide-treated materials are examples of interventions that have been shown to be effective. However, the success of these approaches depends, to a great extent, on human behaviour. Epidemiologists and clinicians, who work in malaria control, traditionally, do not have the necessary training to incorporate behavioural and social science knowledge into the development, implementation, and evaluation of control interventions. It is time to broaden the perspective of malaria control, which can be best achieved through employing a multidisciplinary team that includes social scientists. To support such collaboration, attention needs to be given to understanding the factors that influence job satisfaction, recruitment and retention of the social scientists. At this point in time, social science capacity is severely limited in most African malarious areas due to insufficient funding, lack of career structure, loss of trained individuals to international postings, and a limited understanding of how social scientists can contribute to malaria control. In order to improve this situation, capacity development is needed as a first step. Joint collaborative efforts should be made to offer technical malaria knowledge to social scientists and social science methodology to epidemiologists and control personnel. Social scientists need forums and networks to exchange information, learn about existing research and educational opportunities and promote collaborative partnerships with national malaria control programmes. However, these mechanisms cannot be realized without adequate levels of funding. In order to attract skilled social scientists to Ministries of Health, a new line of thinking needs to emerge that situates social scientists more broadly within the ministry. Rather than focusing on how to integrate social scientists solely within malaria, the emphasis should shift to thinking creatively about how to best utilize social science expertise across an array of specific programmatic areas. Novel approaches within malaria control interventions are starting to emerge, such as integrating the distribution of insecticide-treated nets with measles immunizations. This same type of non-traditional thinking should be applied when developing career tracks for social scientists within Ministries of Health. However, incentives for employment must rival or exceed those offered in other areas. Pay scales must be equitable and the value of multidisciplinary perspectives must be appreciated and desired by national malaria control programmes and Ministries of Health in order for career structures for long-term employment of social scientists to be established. For contractual employment, the same considerations should be shown. Short-term salaries for malaria-related projects should be competitive with salaries offered for similar work being conducted with other diseases, such as HIV/AIDS. In the last five years, there has been a noticeable increase in the collaboration among social scientists and others engaged in malaria control. The interest in malaria by social scientists is clearly present. What are needed now are structures for training and employment that offer a professional career path over time. Authors' contributions HAW and CJ obtained funding for the project. All authors contributed to the study conception and design. PN coordinated fieldwork, data collection and analysis with ongoing supervision from HAW and CJ. PN wrote the first draft of the article with critical revisions from HAW and CJ. PN, HAW, CJ, IN, SD and FG all read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544396.xml
539341	Multiple Campylobacter Genomes Sequenced	null	In 1995, the first complete bacterial genome sequence was published. Now, nearly 200 bacterial genomes have been completed, and a new one hits the scientific press most weeks. This burgeoning industry is not just scientific “stamp collecting,” however. Having all these genome sequences may provide useful clues about why some bacteria cause human disease, how to control their spread, and how to treat the infections caused by them. By comparing genome sequences, scientists can learn much more about what makes a bacteria tick than they can learn from a single sequence. Derrick Fouts and his colleagues have taken this comparative approach with Campylobacter . Infection with a Campylobacter species is one of the most common causes of human bacterial gastroenteritis. In the US, 15 out of every 100,000 people are diagnosed with campylobacteriosis every year, and with many cases going unreported, up to 0.5% of the general population may unknowingly harbor Campylobacter in their gut annually. Diarrhea, cramps, abdominal pain, and fever develop within 2–5 days of picking up a pathogenic Campylobacter species, and in most people, the illness lasts for 7–10 days. But the infection can sometimes be fatal, and some individuals develop Guillain-Barré syndrome, in which the nerves that join the spinal cord and brain to the rest of the body are damaged, sometimes permanently. Campylobacteriosis is usually caused by C. jejuni , a spiral-shaped bacterium normally found in cattle, swine, and birds, where it causes no problems. But the illness can also be caused by C. coli (also found in cattle, swine, and birds), C. upsaliensis (found in cats and dogs), and C. lari (present in seabirds in particular). Disease-causing bacteria generally get into people via contaminated food, often undercooked or poorly handled poultry, although contact with contaminated water, livestock, or household pets can also cause disease. Genome sequencing and comparison of four species of Campylobacter In 2000, C. jejuni was the first food-borne pathogen to be completely sequenced, but we still know little about how Campylobacter species cause disease. In their search for clues, Derrick Fouts and coworkers have completely sequenced the genome of C. jejuni strain RM1221 (isolated from a chicken carcass) and compared it with the previously sequenced C. jejuni strain NCTC 11168 and with the unfinished sequences of C. coli strain RM2228 (a multi-drug-resistant chicken isolate), C. lari strain RM2100 (a clinical isolate), and C. upsaliensis strain RM3195 (taken from a patient with Guillain-Barré syndrome). The researchers describe numerous differences and similarities between these different Campylobacter strains and species. For example, there are major structural differences between the genomes caused by the insertion of new stretches of DNA. Some of these pieces of DNA may carry genes that improve bacterial virulence or fitness, so their presence could help to explain the different biological behaviors of these strains. There are also major variations in the genes responsible for synthesis of molecules that are important for the interaction of Campylobacter with the environment. Such differences could underlie the host specificity of the different species. Differences between the Campylobacter species in genes that are likely to be involved in aspects of bacterial virulence, such as adherence, motility, and toxin formation, are all detailed by Fouts et al., who also describe a new putative Campylobacter virulence locus. Further work is needed to relate these genomic differences to functional differences, but this detailed comparative genomic analysis provides the core blueprint for this important family of human pathogens. And in doing so, it lays the foundation for the development of new ways to monitor and control Campylobacter in the food chain and in human infection.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539341.xml
524377	Limited Durability of Viral Control following Treated Acute HIV Infection	Background Early treatment of acute HIV infection with highly active antiretroviral therapy, followed by supervised treatment interruption (STI), has been associated with at least transient control of viremia. However, the durability of such control remains unclear. Here we present longitudinal follow-up of a single-arm, open-label study assessing the impact of STI in the setting of acute HIV-1 infection. Methods and Findings Fourteen patients were treated during acute HIV-1 infection and subsequently subjected to an STI protocol that required retreatment if viral load exceeded 50,000 RNA copies/ml plasma or remained above 5,000 copies/ml for more than three consecutive weeks. Eleven of 14 (79%) patients were able to achieve viral loads of less than 5,000 RNA copies/ml for at least 90 d following one, two, or three interruptions of treatment. However, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most individuals. By an intention-to-treat analysis, eight (57%), six (43%), and three (21%) of 14 patients achieved a maximal period of control of 180, 360, and 720 d, respectively, despite augmentation of HIV-specific CD4+ and CD8+ T cell responses. The magnitude of HIV-1-specific cellular immune responses before treatment interruption did not predict duration of viremia control. The small sample size and lack of concurrent untreated controls preclude assessment of possible clinical benefit despite failure to control viremia by study criteria. Conclusions These data indicate that despite initial control of viremia, durable viral control to less than 5,000 RNA copies/ml plasma in patients following treated acute HIV-1 infection occurs infrequently. Determination of whether early treatment leads to overall clinical benefit will require a larger and randomized clinical trial. These data may be relevant to current efforts to develop an HIV-1 vaccine designed to retard disease progression rather than prevent infection since they indicate that durable maintenance of low-level viremia may be difficult to achieve.	Introduction The use of highly active antiretroviral therapy (HAART) can dramatically prolong the life of individuals infected by human immunodeficiency virus 1 (HIV-1) [ 1 ], but early hopes for virus eradication have not been realized [ 2 ]. The successful use of HAART is limited by drug-related toxicities, high costs, and drug resistance [ 3 ], factors which have led to the development of alternative therapeutic strategies, including the use of supervised, or structured, treatment interruption (STI). This approach, involving recurrent limited exposure to autologous virus, has not been successful in chronic infection [ 4 , 5 ], but has been shown to lead to at least transient containment of viremia after intervention in the acute phase of infection in humans and animals exposed to AIDS-associated retroviruses [ 6 , 7 , 8 , 9 ]. In the present study, we performed a detailed longitudinal assessment of the impact of early treatment followed by STIs in patients treated during acute or early HIV-1 infection. The main hypothesis of the study was that early treatment of acute HIV-1 infection followed by STI would lead to immune boosting and subsequent control of viremia without the need for drugs. The primary endpoint was the time to viral rebound above 50,000 copies/ml once or above 5,000 copies for three determinations separated by a week each. The early results of this trial were previously reported, showing that five of eight patients were able to achieve a plasma viral load of 500 copies/ml or less at a median of 6 mo off therapy [ 6 ]. The current study investigates the frequency and durability of control achieved with this intervention, with follow-up to a median of 5.3 y after infection, and with an increase in size of the cohort to 14 patients. Our results indicate that, although the majority of patients treated in the acute phase of infection go on to control HIV-1 to less than 5,000 RNA copies/ml plasma for at least 6 mo off therapy, the ability to contain viremia below this level over the long term is maintained in a minority of patients. Methods Objective The hypothesis of the study was that early treatment of acute HIV-1 infection would confer immunologic maturation and subsequent control of HIV-1 without the need for ongoing drug therapy. Alternatively, if a breakthrough of virus replication was observed, this would provide a boost in HIV-1-specific immunity after reinstitution of antiviral therapy. The primary endpoint was the time to viral rebound to more than 50,000 copies/ml or viral loads above 5,000 copies/ml for three determinations separated by a week each. The secondary objective was to correlate immunologic and virologic parameters with any observed effects including evolution of HIV-1-specific T helper and cytotoxic T lymphocyte responses. The original study protocol, including the patient consent form and the institutional review board approval, can be found in Protocols S1–S4 . Study Population Fourteen patients presenting with acute or early HIV-1 infection were enrolled in this study between July 1997 and January 2000 ( Table 1 ). Acute HIV-1 infection was defined by the presence of HIV-1 RNA in the plasma, a negative or weakly positive HIV-1 antibody by HIV-1/2 ELISA, and the detection of no more than three bands in an HIV-1 Western blot; early HIV-1 infection was defined by a positive ELISA and confirmation of early infection by either detuned negative ELISA or previously known negative ELISA. All participants in the study had symptoms compatible with the acute retroviral syndrome and were treated with HAART (one protease inhibitor and two nucleoside reverse transcriptase inhibitors) within a median of 19 d (range, 9–33) from the onset of symptoms. Study participants were recruited from the Massachusetts General Hospital, the Brigham and Women's Hospital, and the Fenway Community Heath Care Center in Boston. All individuals gave written informed consent to participate, and the study was approved by the respective institutional review boards and conducted in accordance with the human experimentation guidelines of the Massachusetts General Hospital. Table 1 Characteristics of the Study Participants a All patients were males. All individuals were White Americans except AC-45, who was Hispanic b ELISA and Western blot positive, but infection within 180 d from time therapy was initiated Ind, indetermined; NA, not applicable; neg, negative; pos, positive Six of the 14 individuals were investigated in an interim study [ 10 ]. These patients were AC-02 (AS2 in [ 10 ]), AC-05 (AS5), AC-14 (AS1), AC-15 (AS3), AC-25 (AS6), and AC-46 (AS4). STIs Entry criteria included treatment with HAART before or shortly after HIV-1 seroconversion, viral suppression on HAART to less than 400 RNA copies/ml for 2 mo, HIV-1 viral load at the time of entry into the study of less than 50 RNA copies/ml, and lack of significant mutations conferring drug resistance [ 11 , 12 ]. Lymphocyte proliferative response to recombinant HIV-1 p24 protein had to exceed a stimulation index of ten before treatment discontinuation, and net counts per minute had to be 800 or greater. All antiretroviral drugs were discontinued simultaneously. After a treatment interruption, patients were seen at least once a week for the first 24 wk, and then monthly, with a total of at least 30 visits for the first year after cessation of therapy. In the second year, visits continued monthly. Treatment was restarted if viral load remained above 5,000 RNA copies/ml plasma for greater than three consecutive weeks, or was in excess of 50,000 copies/ml on any single occasion. Human Leukocyte Antigen Typing High- and intermediate-resolution human leukocyte antigen class I typing was performed at a commercial laboratory (Dynal Biotech, Oxford, United Kingdom) by sequence-specific PCR as described [ 13 ]. Detection of GB Virus C RNA GB virus C (GBV-C) RNA was detected using a two-step nested PCR amplification reaction from whole plasma RNA. Briefly, GBV-C RNA was extracted from plasma using the Qiagen Viral RNA Mini Kit (Qiagen, Valencia, California, United States) according to the manufacturer's instructions. Extracted RNA was reverse transcribed using the Qiagen OneStep RT-PCR Kit and amplified by nested PCR; in both steps primers specific for the 5′ UTR of GBV-C were used, as described previously [ 14 ]. Chemokine Receptor Genotyping In order to analyze the chemokine receptor (CCR) 5Δ32 deletion polymorphism, the region spanning the 32-nt deletion was amplified by PCR, and the two alleles were separated by gel electrophoresis [ 15 ]. The CCR2–64I polymorphism was detected by PCR–restriction fragment length polymorphism analysis as described previously [ 16 ]. Synthetic HIV-1 Peptides We synthesized 410 synthetic peptides 15–20 amino acids long at the Massachusetts General Hospital Peptide Core Facility on an automated peptide synthesizer using Fmoc technology, as described [ 17 ]. Peptides overlapped by 10 amino acids and spanned the entire HIV-1 clade B 2001 consensus sequence. ELISPOT Assays ELISPOT assays were carried out as described previously [ 18 ]. Peripheral blood mononuclear cells (PBMCs) were incubated overnight at 50,000 to 100,000 cells/well in 96-well polyvinylidene plates that had been precoated with 0.5 μg/ml anti-human interferon-γ monoclonal antibody (Mabtech, Stockholm, Sweden). The final concentration of the peptide per well was 14 μg/ml. The numbers of spots per well were counted using an automated ELISPOT plate reader (AID EliSpot reader system, Autoimmune Diagnostika, Strassberg, Germany). A response was considered positive if there were more than 50 spot-forming cells (SFCs)/10 6 PBMCs and if the well had at least three times the mean number of SFCs in the three control wells. The dependence of responses on CD8+ T cells was determined by measuring the depletion of CD4+ T cells using the Minimacs cell depletion system (Miltenyi Biotec, Bergisch-Gladbach, Germany). When HIV-1-specific CD8+ T cell responses were detected against adjacent peptides, and therefore might represent targeting of the overlap region, responses to the weaker peptide were excluded for calculations of magnitude and breadth, as previously described [ 19 ]. Proliferation Assays Freshly isolated PBMCs (10 5 cells) were incubated with baculovirus-derived recombinant p24 protein (Protein Sciences, Meriden, Connecticut, United States) at 5 μg/ml for 6 d and then pulsed with 3 H thymidine at 1.0 μCi for 6 h before harvesting as previously described [ 20 ]. A stimulation index of five or greater was considered significant. Statistical Analysis Time to failure during the different treatment interruptions was assessed by Kaplan-Meier analysis. Patients who were still controlling viremia at the time of last visit, who failed because they restarted therapy without meeting the criteria of virologic failure, or who were lost in follow-up were included in the analyses, but the data were censored at the last evaluable time point. Equality of survival distributions for the first and second treatment discontinuations was evaluated using the Wilcoxon matched-pairs signed-ranks test. CD4+ T cell losses were calculated on regression lines based on least squares fit. A Cox proportional hazards regression model was used for the analysis of continuous variables such as days following onset of symptoms, CD4+ T cell count, HIV viral load, and time to rebound of viremia, as well as for the estimation of hazard ratios for the categorical variables of ELISA, Western Blot, coreceptor polymorphism, and GBV-C status. Statistical analyses of CD8+ and CD4+ T cell responses were based on a Student's t test, a multiparametric ANOVA test, a Wilcoxon matched-pairs signed-ranks test, or a Mann-Whitney U test, as indicated. p -values lower than 0.05 were considered to indicate statistical significance, and all reported p -values are two-sided. Statistical analysis and graphical presentation were performed using SPSS, SAS, and Prism software packages. Results Longitudinal Assessment of Control of Viremia following Treated Acute or Early Infection Fourteen patients identified at the time of acute or early infection ( Table 1 ; Figure 1 ) were entered into this protocol, and they were followed for a median of 5.3 y from the time of infection (range, 494–2,475 d). Patients underwent successive treatment interruptions after an initial treatment period of at least 8 mo (median, 508 d; range, 245–1,096 d) and were required to restart therapy when viral load exceeded 50,000 RNA copies/ml plasma on a single occasion, or 5,000 copies/ml for three consecutive weeks. For purposes of analysis, patients who dropped out of the study or who reinitiated therapy without meeting criteria were considered to have lost the ability to contain viremia. Figure 1 HIV-1 Viral Loads and CD4+ T Cell Counts in the 14 Study Participants Time zero corresponds to first institution of highly active antiretroviral therapy (HAART). Closed squares, HIV-1 plasma viral loads; open circles, CD4+ T cell counts; shaded areas, treatment with HAART according to protocol; diagonally shaded areas, patient restarted therapy without meeting criteria of virological failure; vertical dotted lines, virological failure without reinstitution of HAART. Patients are ordered by increasing number of supervised treatment interruptions. Using these criteria for reinitiation of therapy and to define failure, 11 of 14 patients (79%) were able to achieve virologic control to less than 5,000 RNA copies/ml plasma for at least 90 d after one, two, or three treatment interruptions ( Table 2 ). The period of longest containment was after one interruption for five patients, after two interruptions for eight patients, and after three interruptions for one patient ( Table 3 ). Table 2 Period of Viral Control Achieved Off Therapy Table 3 Time to Failure during the STIs a Numbers correspond to time until virological failure, unless otherwise specified Red indicates the longest time off therapy until failure or last follow-up b Last follow-up visit; patients still meeting criteria of virological control c Failure because patient restarted antiviral therapy without meeting criteria of virological failure d Virological failure due to HIV-1 superinfection Once control was achieved, the majority of the patients experienced a subsequent rise in viremia. The median time between cessation of therapy and rebound of viremia (having a viral load greater than 50 copies/ml) was 17 d (range, 7–169 d). Six of 14 patients (43%) achieved a period of control after stopping therapy for 1 y, but only three of 14 (21%) were able to control viremia off therapy at less than 5,000 RNA copies/ml plasma for more than 2 y. Duration of viremia control during successive treatment interruptions was highly variable, and there was no increase in the sustainability of viral containment during successive STI cycles. The three patients achieving control of viremia for more than 2 y did so during the first (AC-10), the second (AC-02), and the third (AC-14) treatment interruption, respectively ( Figure 2 A). A paired comparison (Wilcoxon matched-pairs signed-ranks test) showed no significant difference in the length of viremia control with subsequent treatment interruptions. Although patients experienced rebound viremia with discontinuation of therapy, none of the patients experienced recurrence of symptoms associated with acute HIV-1 infection. Figure 2 Evolution of Viral Load and CD4+ T Cell Counts during STI (A) Survival curves of time to virologic failure during the first three supervised treatment interruptions. Virologic failure was defined as having a viral load of greater than 5,000 copies RNA/ml plasma for 3 wk or greater than 50,000 copies once. Patients still achieving viral control at the last visit and individuals restarting therapy without meeting criteria or lost in follow-up are censored at the last evaluable time point. The horizontal axis represents the time off therapy since the beginning of the treatment interruption, the vertical axis corresponds to the number of patients maintaining control of viremia. The curves for first, second, and third STIs do not differ significantly from each other (log-rank test, p > 0.05). (B) Evolution of CD4+ T cell counts during the longest treatment interruption. Slopes of CD4+ T cell counts during the first year of the longest treatment interruption are shown for patients who experienced a cessation of therapy of at least 12 mo (all except AC13, AC25, and AC45), compared to the natural decline of CD4+ T cell counts in untreated patients of the MACS cohort with early chronic HIV-1 infection (CD4+ counts of >350 cells/mm 3 ). CD4+ T cell losses were calculated on a regression line based on least squares fit. The two groups differed significantly from each other (Mann-Whitney U test, p = 0.02). (C) CD4+ T cell count at intercept and CD4+ T cell slopes during the longest treatment interruption. The CD4+ T cell slopes of the same 11 patients shown in (B) and of untreated patients of the MACS cohort are represented according to the CD4+ T cell count at the intercept of the regression line based on least squares fit with the vertical axis (day 0 of treatment interruption). These data show that at least transient control of viremia to less than 5,000 RNA copies/ml plasma was achieved in the majority of study participants during at least one of the treatment interruptions, but that durable viral control in participants following treated acute infection occurred infrequently. Moreover, the data do not show a consistent pattern of augmentation of viral control with sequential treatment interruptions. Effect of Treatment Interruptions on CD4+ T Cell Counts Although viral load is a strong predictor of disease progression, CD4+ T cell loss is an additional, independent predictor [ 21 ]. Early treatment of acute HIV-1 infection led to normalization of CD4+ T cell counts in most patients (median, 753 cells/mm 3 ; range, 492–986), but the effect of treatment interruption was variable, even in those doing well, as defined by sustained low viral loads. Overall, 11 of 14 patients interrupted therapy for at least 12 mo, and these individuals were evaluated regarding the effect of treatment interruption on CD4+ T cell loss ( Figure 2 B and 2 C). The rate of change in CD4+ T cell counts during the first year of the longest period off treatment ranged from +157 to −438 cells/mm 3 /y (median, −192). Of the three patients who did not meet viral load criteria for restarting therapy for more than 2 y, one (AC-02) had an increasing CD4+ T cell count of 157 cells/mm 3 /y, one (AC-10) had a stable CD4+ T cell count (−9 cells/mm 3 /y) , and one (AC-14) experienced a decline of 344 cells/mm 3 /y. Comparison with data from the Multicenter AIDS Cohort Study (MACS) showed that the kinetics of CD4+ T cell loss was faster (Mann-Whitney U test , p = 0.02) than in untreated patients with early chronic HIV-1 infection (average loss of −67 cells/mm 3 /y in patients with a CD4+ T cell count of more than 350 cells/mm 3 at baseline). However, CD4+ T cell loss rate was in the same range as what has been described after treatment interruption in chronic HIV-1 infection [ 22 , 23 ]. Analysis of CD4+ T cell decline during the second year for the three individuals who controlled viremia for more than 2 y revealed similar trends in CD4+ T cell slopes, although they were less steep: AC-02, +88 cells/mm 3 /y; AC-10, +44 cells/mm 3 /y; and AC-14, −110 cells/mm 3 /y. When the first 3 mo off therapy were excluded in order to minimize the potential effects of recent treatment on CD4+ T cell number, the rate of change in CD4+ T cell counts during the first year off therapy no longer differed statistically from the MACS data (median, −207 cell/mm 3 /y; range, +119 to −699; Mann-Whitney U test, p = 0.07). A possible reason for steep CD4+ T cell slopes may be high CD4+ T cell counts at time of treatment interruption. Comparison with MACS data ( Figure 2 C) showed that several of the study participants still behaved as outliers when this factor was considered. These results indicate that periods of relative control of viremia were associated with declining CD4+ T cell counts in most patients. Correlation of Clinical and Genetic Markers with Duration of Viremia Control Although the study was small, we evaluated clinical and laboratory parameters to see if any was predictive of duration of viral control. Analyses included clinical and laboratory parameters at time of presentation with acute HIV-1 infection, genetic markers associated with different rates of disease progression, and the presence or absence of GBV-C coinfection. All patients presented with symptomatic acute infection. Time between onset of symptoms and institution of therapy did not affect duration of control following STI (Cox proportional hazards regression model, p > 0.05). The individuals who controlled viremia for a longer time either during the first STI or during any of the treatment interruptions were not different from those who experienced earlier breakthrough as measured by ELISA and Western blot status at initiation of HAART, coreceptor polymorphisms (CCR5delta32, CCR2 V64I), or the presence or absence of GBV-C coinfection (Cox proportional hazards model, p > 0.05 in all comparisons; data not shown). The only parameter that was predictive of prolonged viral control during the first treatment interruption was a low viremia at time of institution of therapy ( p = 0.01): there was a 2.8-fold increase in hazard per order of magnitude increase in viral load. This factor was no longer predictive when the period of longest control of viremia was considered. The time to rebound of viremia (>50 copies/ml or >400 copies/ml) did not correlate with the duration of viral control. Although 11 out of 14 individuals achieved at least transient control of viremia, and three experienced prolonged control, none of these patients possessed the HLA alleles B27 or B57 associated with better disease outcome [ 24 , 25 ]. Relationship of Magnitude and Breadth of HIV-1-Specific CD8+ T Cells to Duration of Viremia Control To assess the relationship between the clinical outcome and evolution of HIV-1-specific CD8+ T cells, we longitudinally analyzed the breadth and magnitude of CD8+ T cell responses using an interferon-γ ELISPOT and a panel of 410 overlapping peptides spanning the entire HIV-1 clade B consensus sequence. At the beginning of the first STI, HIV-1-specific CD8+ T cells were weak (median of 590 SFCs/10 6 PBMCs) ( Figure 3 A) and narrowly directed at a median of two epitopes ( Figure 3 B). CD8+ T cell responses increased significantly ( p < 0.05) during the first off-treatment period, reaching a median total magnitude of 2,725 SFCs/10 6 PBMCs and targeting a median of eight epitopes, and then were sustained when therapy was reintroduced. A further increase in the magnitude and breadth of HIV-1-specific CD8+ T cells was observed in the subsequent off-treatment periods, although these augmentations failed to reach statistical significance. The CD8+ T cell–mediated immune responses emerging during these consecutive cycles of treatment interruption were broadly directed, targeting all structural and most accessory and regulatory HIV-1 gene products (data not shown). However, the magnitude of HIV-1-specific CD8+ T cell responses at the beginning of the first ( r = 0.01, p = 0.76), second ( r = 0.16, p = 0.54), or third ( r = 0.1, p = 0.55) treatment interruptions was not predictive of the time the study participants were subsequently able to stay off therapy according to study criteria. Figure 3 Evolution of HIV-1-Specific CD4+ and CD8+ T Cell Responses during STI (A–D) Magnitude and breadth of increase of HIV-specific CD8+ T cells during supervised treatment interruptions. (A and B) Magnitude (A) and breadth (B) of HIV-specific CD8+ responses at the first day of treatment interruption (black bars) and at the last day off therapy (white bars). Data represent the mean and standard deviation. (C and D) Correlation between the increase of the magnitude (C) or breadth (D) of CD8+ T cell responses and the time off therapy during the first treatment interruption. (E and F) Evolution of CD4+ T helper cell responses during supervised treatment interruptions. (E) Magnitude of CD4 T helper cell responses at baseline and at the first day of treatment interruption (closed circles) and last day off therapy (open circles). Horizontal bars correspond to median values. An stimulation index greater than five was considered significant. (F) Correlation between the magnitude of p24-specific lymphocyte proliferative responses at the beginning of the first treatment interruption and the time patients were able to remain off therapy during the subsequent STI. The periods off treatment allowed for assessment of the relationship between exposure to virus and evolution of immune responses. There was a highly significant positive association between time until virologic failure during the first treatment interruption and change in the magnitude of HIV-1-specific CD8+ T cell responses ( r = 0.92, p < 0.001) ( Figure 3 C). Similarly, the longer a patient remained off therapy during the second and third interruptions, the greater the augmentation of the total magnitude of HIV-1-specific CD8+ T cell responses ( r = 0.83, p < 0.016; r = 0.74, p = 0.05, respectively). The increase in CD8+ T cell epitopes targeted during the first treatment interruption was also linearly correlated to the duration until virological failure ( r = 0.81, p < 0.001) ( Figure 3 D). However, no significant relationship was observed between the augmentation of epitopes targeted during the second and third treatment pauses and the time the study participants were able to remain off therapy in the respective treatment interruption. These data suggest that the duration of a treatment interruption, and therefore the duration of exposure to plasma virus, correlates positively with the magnitude and breadth of HIV-1-specific CD8+ T cell responses that emerge during off-therapy time periods. Yet, CD8+ T cell responses prior to treatment interruptions were not significantly predictive of the duration of time that patients are able to spontaneously control HIV-1 replication, as defined by the study criteria. Relationship of Magnitude of Lymphocyte Proliferative Responses to p24 Antigen to Duration of Viremia Control We next analyzed evolution of lymphoproliferative responses to recombinant HIV-1 p24 Gag protein in order to assess HIV-1-specific CD4+ T cell function. Most individuals had no detectable response at baseline prior to treatment, consistent with prior reports of patients with acute HIV-1 infection [ 20 ]. After initiation of therapy, all individuals generated HIV-1-specific lymphoproliferative responses ( Figure 3 E), which was a criterion for inclusion in the study. During treatment interruptions, there was a variable decline in magnitude, and comparisons between responses on the first day of treatment interruption and last day off therapy did not reach statistical significance (first STI, p = 0.72; second, p = 0.12; and third, p = 0.60, respectively). These HIV-1-specific CD4+ T cell responses also tended to rise with reinitiation of therapy, and some of them were very robust, with stimulation indices over 50 detected in several individuals ( Figure 3 E). Similar to CD8+ T cell responses, the magnitude of HIV-1-specific CD4+ T helper cell responses at the beginning of the first ( r = 0.05, p = 0.43) ( Figure 3 F), second ( r = 0.16, p = 0.54), or third ( r = 0.1, p = 0.55) treatment interruption was not statistically predictive of the time the study participants were subsequently able to stay off therapy according to study criteria. Discussion Although early treatment of acute HIV-1 infection followed by treatment interruptions may enhance control of viremia [ 6 , 8 ], the durability of this control remains unclear. Here we analyzed the long-term impact of initiation of antiviral therapy during acute HIV-1 infection followed by STIs in a cohort of 14 patients. Although initial control of viremia to less than 5,000 RNA copies/ml plasma was achieved in the majority of the individuals studied, a gradual increase in viremia and decline in CD4+ T cell counts was observed in most patients, even after a year or more of viral containment. Durable virologic control occurred infrequently, despite the presence of robust HIV-1-specific CD4+ and CD8+ T cell responses detected by standard assays. Moreover, even during periods of successful control of viremia, progressive loss of CD4+ T cells was frequently observed. These data indicate that although early treatment of acute and early infection is frequently associated with transient control of viremia after STI, ongoing low-level viral replication is associated with ultimate virologic breakthrough in most patients. The standard immunologic assays and virologic assessments in this cohort revealed considerable heterogeneity among the study participants, and did not show a consistent pattern in duration of viremia control during successive treatment interruptions. Eleven of 14 patients (79%) were able to maintain a viral load of less than 5,000 copies/ml for at least 90 d, but progressive loss of control ensued in the majority of patients and only three patients (21%) were able to maintain control for more than 2 y. These three patients did so during the first (AC-10), the second (AC-02), and the third (AC-14) STI. Clinical, genetic, and immunological parameters did not distinguish these three individuals from the other 11 patients, nor did they predict the duration of control following treatment interruption. Indeed, the longer a patient was off therapy, the stronger and more broadly directed the CD8+ T cell responses became, but these were still not sufficient to maintain prolonged control in most patients. Although three patients did not complete the study as initially intended (patient AC-45 withdrew from the study after viral breakthrough on the first STI, AC-13 restarted therapy despite a viral load of less than 5,000 copies/ml during both the first and second STIs and then withdrew, and AC-05 restarted therapy prematurely during the second STI but then failed to control during the third STI), the results are not substantially different if these three are censored rather than considered to have failed to control. Loss of viral control in this cohort occurred not only in the presence of strong CD8+ T cell responses, but in most cases also in the presence of virus-specific CD4+ T cell responses, although the CD4+ T cell responses often declined during periods of viremia. In addition, total CD4+ T cell numbers were also monitored and declined in most patients over time, including one of the three patients who were able to maintain low viral loads for at least 2 y. Mechanisms leading to rapid CD4+ T cell loss need to be further studied in future STI trials. Other parameters including chemokine receptor polymorphisms [ 26 ] and GBV-C coinfection [ 27 , 28 ] similarly failed to explain the different courses following treatment interruption. The only parameter found to be associated with longer control of viremia during the first treatment interruption was a lower viral load at time of institution of antiviral therapy. Given the multiplicity of comparisons made, the true significance of this finding is uncertain. The reasons for progressive loss of control despite augmentation of virus-specific CD4+ and CD8+ T cell responses remain to be defined. In one individual (AC-06), HIV-1 superinfection in the setting of strong and broadly directed HIV-specific cellular immune responses was associated with the loss of viral control, as previously reported [ 29 ]. No other cases of superinfection have been identified in these patients (data not shown). The immunologic studies performed failed to show an association between increases in viral load and loss of immune responses, but this may be due to the use of the current standard IFN-γ assays to quantify immune function. Numerous studies now indicate that IFN-γ production alone is not associated with viral load [ 19 , 30 , 31 ] but rather that functional characteristics of CD4+ and CD8+ T cells may be better associated with viral control [ 32 , 33 , 34 , 35 ]. Such studies will be important to pursue. In particular, even a low level of viremia correlates with a low or undetectable frequency of interleukin-2-producing HIV-1-specific memory CD4+ T cells endowed with proliferative capacity in vitro [ 36 , 37 , 38 , 39 ], thus abrogating CD4+ T cell help crucial to maintain efficacy of CD8+ T cell functions. In an interim study of a subset of six of the 14 patients presented here (patients AC-02, AC-05, AC-14, AC-15, AC-25, and AC-46), a fully differentiated effector phenotype of HIV-1-specific CD8+ T cells for selected epitopes was found to be associated with better control of viremia [ 10 ]. Other factors that may contribute include functional defects in antigen-specific cell-mediated immunity [ 35 , 37 , 40 , 41 , 42 ], and progressive immune escape [ 43 , 44 , 45 ]. HIV-1-specific humoral immunity can also affect viral control after treatment interruption [ 46 ], and viral factors including viral fitness [ 47 , 48 ] and infection with multiple viral variants [ 49 ] can influence viral set point and the rate of disease progression. Virus sequencing studies currently in progress in this cohort indicate that viral breakthrough is associated with sequence changes within and outside known CTL epitopes (data not shown). Full evaluation of the relationship between immune escape and viral breakthrough will require extensive additional analyses, including detailed analysis of responses to autologous virus [ 50 , 51 ]. Assessing the changes in CD4+ and CD8+ T cell functions over time as well as viral evolution under immune selection pressure will be important to evaluate immune correlates in this cohort. These data are important in light of other recent data on treatment interruption in both acute and chronic infection. In chronic HIV-1 infection, STI studies showed only marginal, if any, improvements of HIV-1 viremia control following a number of treatment interruptions cycles, despite at least transient increases in HIV-1-specific CD8+ and CD4+ T cell responses [ 4 , 5 , 52 , 53 , 54 , 55 ]. In the setting of infection with a multidrug-resistant virus, this strategy may even be deleterious [ 56 ]. Other studies of STI after treated acute HIV-1 infection have shown limited benefits [ 9 ], including recent trials such as the PrimSTOP trial [ 57 ] and the QUEST study [ 58 ]. However, little is known about the relationship between scheduling of HAART and treatment interruptions and the characteristics of viral rebound after therapy has been discontinued. Although durable control of viremia was not achieved, it is noteworthy that the majority of patients were able to achieve transient relative containment of viremia, providing rationale for future studies aimed at further enhancing immune control. Early treatment alone should still be considered an important therapeutic option. Therapeutic vaccinations administered after treated acute HIV-1 infection and before cessation of therapy have given disappointing results thus far [ 9 ], but the availability of new and more potent immunogens requires reassessment of this approach. Indeed, the ability to enhance CD4+ T helper cell responses in the chronic phase of infection has been demonstrated [ 59 ], but whether this will enhance CD8+ T cell function requires additional studies. Some promising results have been obtained using immunomodulatory drugs, including cyclosporine [ 60 ] and hydroxyurea [ 61 ], in combination with antiviral therapy, presumably because of the limitation of T cell activation. Administration of granulocyte-macrophage colony-stimulating factor blunted the viral rebound following interruption of HAART, and largely prevented a decrease of CD4+ T cell counts in an STI trial in chronic HIV-1 infection [ 62 ]. These additional therapeutic interventions deserve further investigation in future STI studies. Although the present study shows progressive viral breakthrough, it was not designed to address whether there might be a change in set point viremia achieved or overall clinical benefit through transient early treatment of acute HIV infection. The definition of failure chosen for this study was a viral load of greater than 5,000 RNA copies/ml plasma, which at the time the study was initiated corresponded to the level of viremia at which treatment was recommended. Larger randomized trials will be needed to determine the potential clinical and virologic benefit of approaches based on STIs. In studies of untreated infection, there is only a 5-fold difference in viremia separating the quartile with the slowest disease progression from the quartile with the most rapid progression [ 63 ], suggesting that small differences in steady-state viremia may influence clinical outcome. In the meantime, STI probably should be avoided outside the setting of controlled clinical trials. The data in this study may also be relevant to current efforts to develop a therapeutic AIDS vaccine designed to retard disease progression rather than prevent infection, since they suggest that durable maintenance of low-level viremia may be difficult to achieve. Supporting Information Protocol S1 Study Protocol (68 KB DOC). Click here for additional data file. Protocol S2 Protocol Amendment (47 KB PDF). Click here for additional data file. Protocol S3 Patient Consent Form (187 KB PDF). Click here for additional data file. Protocol S4 Institutional Review Board Approval (36 KB PDF). Click here for additional data file. Patient Summary Background Highly active antiretroviral therapy (HAART), which is used to treat patients with HIV, can have nasty side effects and is expensive. As a result, for the last five years scientists have been trying to determine if it is possible to give patients breaks from taking antiretroviral drugs, without patients' health suffering. It is likely that such a treatment strategy (called “supervised treatment interruption,” or STI) will not work in patients who have been infected with HIV for a long time. However, until now, the jury was out about whether STI could be of benefit to patients who had only recently been infected with HIV. Some research suggested that if newly infected patients had short “holidays” from taking HAART, it might help to boost their immune system—which in turn might help to keep HIV at bay. What Did the Researchers Do and Find? The researchers studied 14 patients who had recently been infected with HIV. Patients were treated until their viral load was below the limit of detection by using a very sensitive method. As they were then watched after treatment interruption, most of them were able to maintain a low, though detectable, viral load for some time. The researchers then stopped the treatment and carefully watched the patients over a period of up to five years. If the number of viruses in the patients' blood rose too high, the researchers gave the patients HAART again until the number of viruses fell again. In about half the patients, treatment needed to be restarted within a year because the viruses had started becoming more numerous in the blood. The doctors had hoped that it would take much longer for the number of viruses to reach this level. In other words, giving patients “drug holidays” did not help to keep the virus at bay for long periods of time. What Are the Limitations of the Study? It is relatively rare for doctors to be able to diagnose HIV shortly after an individual becomes infected with the virus. Many people have HIV for a long time before they see a doctor. Therefore, even if the STI strategy had worked, it would only have been relevant to a few patients. In addition, the study was small and preliminary, so we have to be careful about reading too much into the results. A limitation in the study is that it did not address whether there might be a long-term clinical benefit despite a gradual increase in viral load. What Does This Study Mean for Patients? It is important to remember that this was an experimental trial—patients with HIV should not stop taking antiretroviral drugs unless their doctor specifically tells them to do so. Although it looks as though STI may not work in the way everyone had hoped, these results may help scientists develop an HIV vaccine that is designed to keep the disease at bay. Whether early treatment of acute infection has an overall benefit in terms of time-to-development of AIDS or need for long-term treatment after drug discontinuation will need to be answered in larger clinical trials designed to answer these important questions. Resources on the Web. AIDSinfo: http://www.aidsinfo.nih.gov/ AIDSmap: http://www.aidsmap.com/ Medline Plus AIDS Information: http://www.nlm.nih.gov/medlineplus/aids.html	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524377.xml
539355	The DiAMOND trial protocol: a randomised controlled trial of two decision aids for mode of delivery among women with a previous caesarean section [ISRCTN84367722]	Background Caesarean section (CS) has become an increasingly common method of delivery worldwide, rising in the UK from 9% of deliveries in 1980 to over 21% 2001. This increase, and the question of whether CS should be available to women on request, has been the subject of considerable debate, and national reports and guidelines have specifically highlighted the importance of patient choice in the decision making process. For women who have already experienced CS, the UK National Institute of Clinical Excellence recommends that the decision should consider maternal preferences and priorities in addition to general discussion of the overall risks and benefits of CS. Decision aids for many different medical treatment and screening decisions have been developed and evaluated, but there is relatively little evidence for the use of decision aids for choice of mode of delivery among women with a previous CS. The aim of the study is to evaluate two interventions to assist decision making about mode of delivery among pregnant women with one previous CS. Methods/design Women with one previous CS are recruited to the trial during their booking visit at approximately 12–20 weeks' gestation in participating maternity units in Bristol, Weston and Dundee. Using central randomisation, women are allocated to one of three arms: information programme and website; decision analysis; usual care. Both interventions are computer-based, and are designed to provide women with detailed information about the potential outcomes for both mother and baby of planned vaginal delivery, planned CS and emergency CS. The decision analysis intervention additionally provides a recommended 'preferred option' based on maximised expected utility. There are two primary outcomes (decisional conflict and actual mode of delivery), and five secondary outcomes (anxiety, knowledge, perceptions of shared decision making; satisfaction with decision making process, proportion of women attempting vaginal delivery). Primary follow up for the questionnaire measures is at 36–37 weeks' gestation, and a total of 660 women will be recruited to the study. The primary intention-to-treat analyses will comprise three pair-wise comparisons between decision analysis, information and usual care groups, for each of the two primary outcomes. A qualitative study will investigate women's experiences of the decision making in more depth, and an economic evaluation from the perspective of the NHS will be conducted. Discussion Provision of information to women facing this decision appears variable. The DiAMOND study aims to inform best practice in this area by evaluating the effectiveness of two interventions designed to aid decision making.	Background Over the last 20 years caesarean section (CS) has become an increasingly common method of delivery. The CS rate in the United Kingdom rose from 9% of deliveries in 1980 to 21% in 2001 [ 1 ]. This increase, and the question of whether CS should be available to women on request, has been the subject of considerable debate [ 2 ]. The optimal mode of delivery for women who have experienced a previous CS is complicated by the difficulty in balancing the risks of repeat CS with those of vaginal birth after caesarean section (VBAC). An evaluation of caesarean delivery by the American College of Obstetricians and Gynecologists reported that first time mothers with term singleton cephalic pregnancies and women with a previous CS account for two thirds of all caesarean deliveries in the US [ 3 ]. The Changing Childbirth report has emphasised the importance of patient choice when decisions need to be made in relation to the management of pregnancy and childbirth [ 4 ]. However the views of women who have experienced CS and their preferences for future deliveries have received little attention to date. Obstetricians tend to focus on the risks of uterine rupture and emergency caesarean section in labour [ 5 ], which may influence the advice they give to women uncertain about future mode of delivery. Others have focused on the increased morbidity following CS [ 6 ] and on the negative impact of operative delivery on first postnatal contact between the mother and her baby [ 7 ]. In Chile, where there is a very high rate of CS in the private sector, only a minority of women reported that they had wanted this method of delivery [ 8 ]. In a Scottish study, more women who delivered by elective CS reported they were satisfied with their involvement in the decision making process compared with women who underwent emergency CS [ 9 ]. The advantages and disadvantages of planned CS versus planned vaginal delivery has also been debated in north America in response to a growing number of requests for elective CS [ 10 ]. In an attempt to ensure appropriateness of CS in the UK, a set of evidence-based guidelines on indications for CS have recently been published [ 11 ]. The guidelines, commissioned by the UK National Institute for Clinical Excellence, make a specific recommendation that for women with a previous CS, the decision should consider maternal preferences and priorities in addition to general discussion of the overall risks and benefits of CS. It is essential that the process of decision making about future mode of delivery is evaluated and enhanced to achieve the safest and most satisfactory outcome for both mother and baby. It has been proposed that the way in which clinical decisions are made lies on a continuum, from paternalistic (clinician decides) through partnership (clinician and patient share the decision) to informed (patient decides) [ 12 ]. Although proposed as the preferred approach of determining patients' treatments [ 13 ], some problems with the concepts, terminology and practice of shared medical decision making have recently been highlighted [ 14 ]. The appropriateness of the shared model may also depend on the clinical context as well as patients' and clinicians' preferences for involvement in decision making [ 15 ]. In addition, there is some evidence that patients and health professionals often have different treatment preferences, potentially making agreement on a treatment strategy more difficult [ 16 ]. Decision aids are designed to help people select between various treatment strategies by providing information on the options and outcomes relevant to a person's health. Decision aids for many clinical conditions have been developed [ 17 ], and evaluations of these decision aids have been the subject of systematic reviews [ 18 , 19 ]. A north American trial found no difference in terms of VBAC rate between written versus personal counselling interventions that actively promoted vaginal delivery [ 20 ]. An Australian trial of a paper-based decision aid for women who have previously experienced CS is currently underway [ 21 ]. The specific content of decision aids may vary, but in general they aim to present more than one strategy for clinical management, help people understand the probable outcomes of treatment choices and allow people to consider the personal value they place on benefits versus harms. Decision aids can take several formats, such as leaflets, interactive videodisks, individualised decision analysis, personal counselling sessions and audio workbooks. Interventions to assist patient decision making can improve knowledge about treatment options, make patients more realistic in their expectations, reduce decisional conflict and increase active involvement in decision making [ 18 ]. As an intervention to aid patient decision making, individualised decision analysis has so far received limited attention. By explicitly combining patients' values regarding treatment outcomes and individual probability information, decision analysis attempts to provide a rational framework to guide patient decision making. The use of individualised decision analysis has been debated [ 22 ], but empirical evidence demonstrates that it is feasible and acceptable and has value as an aid to patient decision making [ 23 ]. Aim The aim of this paper is to describe the protocol for a randomised controlled trial of two interventions to aid decision making about mode of delivery among pregnant women with one previous CS. The interventions being assessed are (1) Decision analysis, and (2) Information programme and website. Both decision aids will be compared with usual care given by the obstetric team. The interventions will be assessed in terms of decisional conflict, planned and actual mode of delivery, anxiety, knowledge, perception of shared decision making and satisfaction with the decision making process. Development and piloting of the interventions took place in 2003–2004, and the main phase of the study started in May 2004 and will continue until December 2006. Ethical approval for the study was obtained from the UK South West Multi-Centre Research Ethics Committee. Methods/design Recruitment and allocation of participants The sample will comprise pregnant women with one previous lower segment CS (all parities will be included, but the most recent delivery must have been CS), no current obstetric problems and delivery expected at ≥ 37 weeks. Women are being recruited to the study by research midwives during their initial booking visit at approximately 12–20 weeks' gestation. Recruitment takes place from maternity units in St Michael's and Southmead Hospitals in Bristol, Weston General Hospital, and Ninewells Hospital in Dundee. The current CS rates for these units range between 18 and 24% and are representative of rates for other units throughout England and Scotland. The women are informed that although both vaginal delivery and repeat CS carry their own benefits and risks, the best method of presenting this information in order to assist women in reaching a decision is not known. Women expressing an interest in participating in the trial at the booking visit are given an information sheet, a written consent form and a baseline questionnaire to take home. Following receipt of the baseline questionnaire and written informed consent to enter the trial, women are randomised to one of three arms as detailed below. Allocation is stratified by maternity unit and preferred mode of delivery and blocked (using random permuted blocks of sizes 6, 9, 12 and 15) to ensure reasonable balance between the trial groups through time. The randomisation sequence was generated by a member of the study team (AAM), and allocation of participants is performed by a staff member with no other involvement in the study. Interventions Both interventions are computer based. Women allocated to receive an intervention have an appointment with a researcher to allow the decision aid to be delivered using a laptop computer, usually in the woman's own home or workplace. (i) Information programme and website This intervention provides information about the outcomes associated with planned vaginal delivery, planned CS, and emergency CS. This includes descriptions of outcomes for both mother and baby, and the probabilities of these outcomes based on the best available evidence. Both the probabilities of having and not having the event are given, and all probabilities are presented in both numerical and pictorial format [ 24 ]. The programme easily allows women to choose the information that they view, and the information each women accesses is logged. At the end of the initial appointment with a researcher, women are given a password that allows them to access the information programme via the internet as often as they wish. Womens' use of the programme via the internet is also logged. (ii) Decision analysis There are generally four main steps involved in a decision analysis. The first is to draw up a decision tree that maps out the likely outcomes of the strategies in question [ 25 ]. These outcomes are then assigned utilities that represent how an individual values a particular outcome. A utility is a number between 0 and 1, often representing the outcomes 'death' and 'perfect health' respectively [ 26 ]. Probability information is then included in the tree to represent the chance of each outcome occurring [ 26 ]. Finally, strategies are compared by calculating the weighted sum of the utilities of all possible outcomes [ 27 ]. The recommended strategy is that with the highest expected utility value, or in other words, the one that gives an individual the best chance of achieving an outcome that is valued. The decision analysis intervention in the trial proceeds according to the principles described above. First, women are given information about the outcomes associated with planned vaginal delivery, planned CS, and emergency CS. This includes descriptions of outcomes for both mother and baby, but not the probabilities of these events. These are embedded in the decision tree which is not visible to users. Second, women are required to rate (assign a utility value between 0 and 1) each possible outcome using a visual analogue scale. Finally, the programme combines the elicited utilities and the probabilities of each outcome in a decision tree to produce a recommended 'preferred option' based on maximised expected utility. Each woman is given a computer printout of the outcome of the decision analysis. (iii) Usual care This comprises care normally given by the obstetric and midwifery team. Women allocated to decision analysis or information programme receive these interventions in addition to usual care. Women in both intervention arms are contacted by letter at 35 weeks' gestation. The purpose is to encourage discussion of the intervention with their obstetrician and/or midwife when they attend the clinic at 36–37 weeks to finalise their planned method of delivery. Participation in the study is recorded in the medical records of all women in the trial. Outcome measures There are two primary outcomes: (1) Decisional Conflict Scale (DCS) [ 28 , 29 ]. This is a 16 item questionnaire that measures degree of uncertainty about which course of action to take and the main modifiable factors contributing to uncertainty. Previous research indicates that effect sizes of about 0.35 to 0.5 standard deviations can discriminate between individuals who make a decision and those who delay or are unsure [ 28 ]. Assuming a standard deviation of 15 points [ 18 ], this is equivalent to differences of 5.25 to 7.5 points on the total DCS 100 point scale. (2) Actual mode of delivery (vaginal, elective CS, or emergency CS). Unlike a previous trial [ 20 ], we are not seeking to promote one method of delivery over another. However differences between the arms in the proportions of different modes of delivery may have important healthcare resource implications. There are five secondary outcomes: anxiety; [ 30 ] knowledge; perception of shared decision making; satisfaction with decision making process; proportion of women attempting vaginal delivery. Follow up The primary and secondary outcomes are assessed in all three groups at baseline, and approximately two weeks after randomisation. The questionnaire at two weeks will constitute a secondary follow up, and will enable sufficient time for delivery of the appropriate interventions. As part of usual care, women in the trial normally attend the clinic at around 36–37 weeks' gestation to finalise plans for their preferred method of delivery. Questionnaire outcomes are measured again after this visit, and this constitutes the primary follow up for this trial. Actual and attempted mode of delivery (cross-checked with hospital records) and satisfaction with choice are assessed by a further postal questionnaire at approximately six to eight weeks after giving birth. Justification of sample size As noted above, differences in excess of 0.35 standard deviations have been considered as important for the total DCS score, and differences of this magnitude are feasible for interventions of this kind [ 23 ]. With regard to mode of delivery, UK data indicate that about 33% of women with one previous CS are delivered vaginally [ 1 ]. The sample size calculation for a previous trial of written versus verbal counselling in north America presumed a vaginal delivery rate of 30% for a minimal intervention, and in the event observed that 51% of women achieved vaginal delivery for the trial groups overall [ 20 ]. A change from 30–33% to 51% corresponds to an odds ratio of about 2.1–2.4, and this would certainly be considered as clinically important. With two-sided 1% alpha, a total sample size of 600 provides 82–99% power to detect a standardised difference of 0.35–0.5 in total decisional conflict score between the groups, and 84–95% power to detect odds ratios of 2.1–2.4 in women achieving vaginal delivery. A pair-wise alpha of 1%, corrected for multiple comparisons using Tukey's procedure, maintains an overall study-wise alpha of 3.4%. In order to allow for pre-term deliveries, malpresentations, and losses to follow-up, we will therefore recruit 660 women to the trial. Statistical analysis Data analysis will proceed according to CONSORT guidelines for randomised controlled trials. The first stage of the analysis will be to use descriptive statistics to describe the group of individuals recruited to the trial in relation to those eligible, and to investigate comparability of the trial arms at baseline. The primary analyses will comprise three pair-wise intention-to-treat comparisons between decision analysis, information and usual care groups, for each of the two primary outcomes. These comparisons will use appropriate (that is, standard or logistic) multivariable regression models, adjusting for maternity unit, initial preference regarding mode of delivery, and value of the outcome variable at baseline. Full attention will be paid to the estimates and confidence intervals for these comparisons as well as the p-values, with the latter adjusted for multiple comparisons using Tukey's procedure. Secondary outcomes will then be analysed in the same way, using appropriate multivariable regression models depending on the nature of the outcomes. Other secondary analyses will involve investigation of the short-term effects of the interventions using data from the two week follow up, and the effects at 36–37' weeks gestation adjusted for both baseline and two week follow up. Pre-planned subgroup analyses employing appropriate interaction terms in the regression models will be used to ascertain any differential effects of the interventions on the two primary outcomes across the following categories of women: previous caesarean section occurring before or after labour; previous successful vaginal delivery; stated preferred mode of delivery. Since the trial is powered to detect overall differences between the groups rather than interactions of this kind, the results of these essentially exploratory analyses will be presented using confidence intervals as well as p-values, and interpreted with due caution. Finally, we will investigate the effect of differential use of the information intervention via the internet using descriptive statistics and appropriate comparisons with the other groups. Qualitative study Qualitative research methods will be used in order to explore aspects of the interventions and women's experiences of the decision making in more depth. Specifically, semi-structured interviews will be conducted with a sample of women from each of the intervention arms (Decision Analysis and Information), across the research sites in Bristol and Dundee. These interviews will explore: (1) Women's views and experiences of the intervention and its delivery – for example, the quality and relevance of the decision aid/information, what they felt about the risk information presented to them, and which particular aspects of each intervention were helpful or unhelpful. (2) Which factors women felt had most influenced their preferences regarding method of delivery. (3) Whether the women had prior preferences about method of delivery, whether/how these changed during their pregnancy and in the case of the decision analysis, what they felt about the method of delivery proposed by the intervention compared to any prior preferences. (4) Any other information sources women sought and used to help them make a decision about method of delivery (for example, information from health professionals, partner/family/friends, internet, media, books). (5) Women's views and feelings about their actual method of delivery compared with any prior preferences and the method of delivery suggested by the intervention (for decision analysis). A small number of interviews may also be conducted with women in the usual care arm of the trial to explore what support and advice was normally provided during pregnancy to those who did not receive an intervention. A sample of approximately 30 women across the two intervention arms will be interviewed in depth to ensure a thorough exploration of emergent themes and concepts. Within each arm, women will be purposefully chosen to include those with different parities/ages/socio-economic backgrounds, and where possible different methods of delivery/outcomes, following a maximum variation sampling strategy [ 31 ]. The interviews will take place a short time after the primary follow up, to avoid any influence of the interview on these measures. A subset of the women will be interviewed a second time six to eight weeks after birth in order that they can reflect upon their decision regarding preferred mode of delivery with the actual mode of delivery. In addition, a small number of interviews may take place closer in time to receipt of the intervention if this is deemed (a) feasible given recruitment and (b) worthwhile in terms of providing new information to that gleaned from qualitative interviews undertaken in the development phase of the trial. Interviews will be conducted in the womens' homes or other suitable setting chosen by them. A check-list of topics will be used to ensure that the primary issues are covered, whilst allowing flexibility for new issues to emerge from each interview. Interviews will be recorded on mini-disc, fully transcribed and anonymised to protect confidentiality. Transcripts will be studied in detail and a list of common themes and concepts drawn up. Data collection and analysis will run in parallel and the coding index added to or refined and coded material regrouped as new themes and categories emerge from subsequent interviews [ 32 ]. Further analysis will employ the constant comparison method of grounded theory in which the textual data is scrutinised for differences and similarities within themes keeping in mind the context in which mention of these themes arose in each interview [ 32 ]. In addition to the interviews with women, a small number of focus groups with health professionals (e.g. obstetricians, midwives) in each research site may be conducted near the end of the trial, resources and time permitting. Focus groups are often used in evaluations of new services/interventions and are useful for exploring group views, concerns and preferences (e.g. consensus or disagreement about an issue) [ 33 ]. They may be valuable for exploring professionals' views about the intervention and issues around implementation into routine practice. Economic evaluation The aim of the economic evaluation is to compare the costs and benefits of the two interventions with usual care. The analysis will be from an NHS perspective and will be based on the costs incurred during pregnancy, delivery and 6–8 weeks following delivery. Incremental cost effectiveness ratios will be formed comparing (i) the cost per point improvement on the Decisional Conflict Scale (DCS) and (ii) the cost per extra patient able to make a decision (represented by a 0.5 standard deviation change on the DCS). We will also compare the average cost of care per patient in each arm of the trial with patient satisfaction. Differences in the cost of care of women receiving the two interventions will be compared with usual care from the point at which patients are randomised. The analysis will include all resources under the control of the NHS that may differ as a result of the interventions and will include resources used by both mother and baby. The costs identified as being of relevance are: antenatal appointments in addition to routine appointments, including both primary and secondary care; mode of delivery and related hospital stay for mother and baby; follow up care for mother and baby, including primary and secondary care appointments, outpatient appointments, inpatient stays, A&E visits, and prescribed medication. Data on resource use will be collected principally by two questionnaires completed by the women. The first questionnaire, completed at 36–37 weeks' gestation, will provide information on all antenatal appointments, and the proportion of these that involved discussion about mode of delivery. Women will be asked how often mode of delivery was addressed at (i) routine appointments, and (ii) appointments initiated by them to discuss mode of delivery. A sample of 10 hand-held records from each arm at each main centre (St Michaels, Southmead and Ninewells, total 90) will be scrutinised to validate the information given in response to the questionnaire. The second questionnaire, completed six to eight weeks after delivery, will provide information on mode of delivery and all non-routine postnatal health service contacts and prescriptions for both mother and baby. The principle of opportunity cost will underlie the valuation of resource use. In many cases, market rates will act as a proxy for opportunity cost. National data sets such as the Unit Costs of Health and Social Care [ 34 ] and the British National Formulary [ 35 ] will be used to value primary care consultations and prescribed medication. Secondary care contacts will be coded according to Healthcare Resource Group (HRG) and valued using the Department of Health National Reference Costs [ 36 ]. Costs and outcomes will not be discounted, as the economic evaluation will be limited to a period of 12 months. Sensitivity analysis will be conducted in areas where there is uncertainty around assumptions about resource use measurement and/or valuation. Variation in resource use and the effectiveness of the intervention is not captured by a cost-effectiveness/utility ratio. We will use bootstrapping to address this, and construct a cost effectiveness acceptability curve. Discussion Women with an uncomplicated pregnancy and expected term delivery who have previously delivered by CS face a choice between repeat elective CS or attempted trial of labour. Guidelines in the UK emphasise the importance of involving women in the decision making process and taking account of maternal preferences and priorities, but the type and amount of information available to women facing this choice appears variable. The DiAMOND study is a randomised trial that aims to inform best practice in this area, by evaluating the effectiveness of two interventions to assist decision making in terms of decision quality and actual mode of delivery. Competing interests The author(s) declare that they have no competing interests. Authors' contributions Alan Montgomery, Deirdre Murphy, Ali Shaw and Sandra Hollinghurst drafted the manuscript, with input from the other members of the DiAMOND Study Group. The Decision Aids for Mode Of Next Delivery (DiAMOND) Study Group comprises the following members: Clare Emmett, Tom Fahey, Peter Gregor, Sandra Hollinghurst, Claire Jones, Beverley Lovering, Alan Montgomery, Irene Munro, Deirdre Murphy, Roshni Patel, Tim Peters, Ian Ricketts, Anne Schlegelmilch, Ali Shaw, Kav Vedhara, Kate Warren. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC539355.xml
535534	Recombinant activated factor VII (rFVIIa) as salvage treatment for intractable hemorrhage	Background Recently, there has been an increased use of recombinant activated factor VII (rFVIIa) to promote hemostasis in various hemorrhagic conditions. The objective of this study was to determine the outcome of patients treated with rFVIIa who had intractable bleeding associated with cardiac surgery (CSP) or as a result of other causes (OBP). Methods The medical records of 40 consecutive patients treated with rFVIIa were retrospectively reviewed for blood product use before and after treatment. In all patients, rFVIIa was given only after all other measures to stop bleeding had failed. The number of transfused units of red cells (R), platelets (P), fresh frozen plasma (F), and cryoprecipitate (C) were determined both before and after administration of rFVIIa, and the results compared. Mortality at 4 hours and 30 days was assessed. Patients dying within 4 hours of rFVIIa administration were not evaluable for response. Patient characteristics were also assessed as risk factors for mortality. Results Twelve of 24 CSP survived for more than 4 hours. These 12 patients required an average of 17 units (U) of R, 18 U of P, 18 U of F and 15 U of C pre-treatment compared to an average of 6 U, 10 U, 9 U and 4 U of R, P, F and C respectively, post-treatment. These differences were statistically significant. For the OBP, 11 of 16 survived more than four hours. These 11 patients required an average of 10 U of R, 11 U of P, 14 U of F and 10 U of C pretreatment compared to an average of 1 U, 2 U, 2 U and 0 U of R, P, F, and C respectively, post-treatment. With the exception of C, there was a statistically significant decrease in blood product use following treatment with rFVIIa. Of the survivors in each group, 6 of 12 CSP and 2 of 11 OBP died between 3 and 30 days post-treatment from causes other than bleeding. Mortality at 30 days for CSP and OBP survivors was 50% and 18% respectively, whereas overall 30 day mortality was 75% for CSP and 44% for OBP. Conclusions rFVIIa is effective in decreasing blood product use and promoting hemostasis in patients with intractable bleeding associated with cardiac surgery and a variety of other causes.	Introduction Recombinant factor VIIa (rFVIIa), originally developed for the treatment of acquired inhibitors associated with hemophilia [ 1 , 2 ], has been successfully used for bleeding due to acquired or congenital thrombocytopathy [ 3 , 4 ], extensive trauma and a variety of surgical procedures, including anecdotal reports of use in cardiac surgery patients [ 5 - 9 ]. It reverses the effect of warfarin in healthy volunteers [ 10 ], and corrects the prothrombin time in patients with hepatic failure [ 11 ]. Most critically ill patients with trauma suffer profound coagulopathy. Coagulation abnormalities in these patients have been attributed to multiple factors including disseminated intravascular coagulation (DIC), excessive fibrinolysis due to release of tissue plasminogen activator (tPA), dilutional coagulopathy from fluid replacement and massive blood product transfusion, dysfunctional platelets, and metabolic abnormalities including acidosis and hypothermia [ 12 - 18 ]. In addition, the use of cardiopulmonary bypass in patients undergoing cardiac surgical procedure may exacerbate the coagulopathy [ 19 ]. Although the mechanism of action of rFVIIa remains unclear, many investigators have suggested that it binds to the surface of activated platelets and directly activates Factor X, thus bypassing the early steps of the coagulation cascade. Activated Factor X (Xa) then combines with activated Factor V (Va) on the platelet surface, leading to rapid conversion of prothrombin to thrombin [ 20 , 21 ]. Hemostasis is promoted through high concentrations of thrombin generated near activated platelets at the site of vascular injury. Based on its mechanism of action, rFVIIa may be effective in controlling hemorrhage due to trauma, surgery and other causes. The present report is a retrospective review of 40 consecutive patients treated with rFVIIa (NovoSeven, Novo Nordisk, Denmark) for intractable hemorrhage. Twenty-four of these patients had bleeding associated with cardiac surgery, and the remaining 16 suffered with bleeding from other causes. The need for transfusion of blood products both before and after treatment and the mortality at 4 hour and 30 day were assessed. Patient characteristics were also assessed as risk factors for mortality. Methods Patients The study was approved by the Institutional Board Review of the Washington Hospital Center, Washington DC, USA. The medical records of 40 consecutive patients treated with rFVIIa between July 2001 and June 2003 at the Washington Hospital Center, a tertiary care teaching hospital and level 1 trauma center, were reviewed. The patients were divided into two groups: 24 patients who had undergone cardiac surgical procedures (CSP), and 16 patients who had bleeding from other causes (OBP). There were more male patients in both groups (19 males and 5 females in CSP, 10 males and 6 females in OBP). The median age in the CSP was higher at 65 years (range 26–85 years) compared to that of OBP (median age 42; range 23–82 years). The type of surgery in the CSP group included 15 coronary artery bypass grafts (CABG), 3 aortic arch repairs either elective or for dissection, 2 aorta rupture repair (one also underwent CABG), 3 valve replacements and 1 ventricular rupture. In the OBP group, 5 patients had suffered either stab or gunshot wounds, 2 had traumatic liver lacerations, 2 had undergone joint replacement, 3 had clotting factor abnormalities (acquired Factor VIII inhibitor, congenital Factor VII deficiency, acquired VWF deficiency), and one each had post partum hemorrhage, warfarin overdose, bowel resection, and trauma from a motor vehicle accident. Administration of rFVIIa Commercially available rFVIIa, (Novo-Seven, Novo Nordisk, Denmark) was used in all patients. There were no predetermined criteria for administration. In most patients, treatment was given after extensive blood product support had failed to control hemorrhage, and the decision to treat was made jointly by the surgeon, hematologist and blood bank pathologist. rFVIIa was administered as a bolus dose of 90 mcg/kg. The first two patients received a second dose of 90 mcg/kg 6 hours after the first. Endpoints and Assessment of Risk The total number of products given before and up to twenty-four hours after rFVIIa administration, or until death, whichever came first, was quantified. Patients who died within 4 hours of treatment were not evaluable because blood product support was suspended shortly after rFVIIa administration in these hemodynamically unstable patients deemed non-salvageable. Also, given the short half-life of rFVIIa, this time frame did not allow for an accurate determination of efficacy. The number of patients surviving at 4 hours and 30 days after the administration of rFVIIa was compared. Thirty days was chosen as an end point since this is the operative mortality cut off point, as defined by the Society of Thoracic Surgeons, for inpatient and out of hospital death. For patients surviving more than 4 hours, charts were reviewed for thromboembolic complications during hospitalization. In an attempt to determine risk factors for poor outcome, patients were assessed for age, gender, comorbid conditions, left ventricular ejection fraction (LVEF), use of cardiopulmonary bypass (CPB), hemoglobin, platelet count, arterial blood gas results and coagulation parameters including prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and D-Dimers. Laboratory parameters were not available for some patients. Statistical Considerations The Paired Student's t-Test was used to compare the number of blood product transfusions before and after treatment with rFVIIa, and, since the number of pair groups was less than 50, this result was confirmed with the Wilcoxon Matched-Pairs Signed-Ranks Test. Results Cardiac Surgery Patients (CSP) Outcome Twelve of the 24 patients survived for more than 4 hours after the administration of rFVIIa, and the results of their pre- and post-treatment blood product use are listed in Table 1 . From the start of surgery until the administration of rFVIIa, patients received on average 17 U of R (range 5–39), 18 U of P (range 6–37), 18 U of F (range 6–33), and 15 U of C (range 0–50). Post rFVIIa treatment, an average of 6 U of R (range 0–28), 10 U of P (range 0–19), 9 U of F (range 0–28) and 4 U of C (0–20) were transfused. The difference between the pre- and post-transfusion requirement was significantly lower for all blood products: R (p = 0.007), P (p = 0.038), F (p = 0.009) and C (p = 0.03). Eight of the 12 patients showed a rapid hemostatic response to rFVIIa, as measured by a decrease in R transfusion to an average of 2 U (range 0–6). The other 4 patients (Patient # 24, 27, 32 and 35) continued to require blood product support beyond 24 hours after rFVIIa administration. Table 1 Summary of rFVIIa treated cardiac surgery patients (CSP) who survived >4 hours P# Age sex Procedure Co-morbidities Response to rFVIIa Outcome CPB EF% Pre rFVIIa Post rFVIIa R P F C R P F C 1 75M CABG - >45 18 18 8 10 0 4 0 0 Survived 13 36M Aortic arch repair + >45 28 18 16 10 6 19 20 10 Died 14 64M Aortic dissection - >45 39 37 32 50 2 6 4 0 Survived 17 63M AVR/MVR repair - <25 7 18 16 15 1 12 6 0 Survived 18 70M CABG + >25 6 20 20 20 0 0 0 0 Survived 20 72M CABG - >25 21 12 10 0 0 0 0 0 Survived 24* 79M CABG + >25 10 12 12 10 5 4 4 0 Died 27* 64M CABG, IVAD + <25 30 30 33 20 28 16 28 20 Died 30 85M CABG, ruptured aorta + >45 13 12 6 10 3 6 8 0 Died 32* 83M CABG + >25 5 18 18 10 6 18 20 0 Died 33 78F Ruptured aorta + <25 14 18 20 20 3 12 6 0 Died 35* 54M Aortic arch repair + >45 9 6 18 10 15 24 12 20 Survived Average 17 18 18 15 6 10 9 4 P value .009 .038 .009 .03 P: Patient R: Red Blood Cell; P: Platelet; F: Fresh Frozen Plasma; C: Cryoprecipitate M: Male; F: Female CPB: Cardiopulmonary Bypass EF: Left Ventricular Ejection Fraction CABG: Coronary Artery Bypass Graft AVR: Aortic Valve Replacement MVR: Mitral Valve Replacement IVAD: Intraventricular Assisted Device * Patient continued to require blood transfusion past 24 hours post rFVIIa treatment. Six of the 12 patients who survived more than four hours were eventually discharged to home (Table 1 ). The remaining 6 patients (Patient # 13, 24, 27, 30, 32, and 33) died between 3 and 30 days after rFVIIa treatment from a variety of causes including multi-organ failure and infections. Amongst survivors beyond 4 hours, mortality at 30 days was 50% and overall mortality for the entire group was 75%. Of the 12 patients who died from multiorgan failure and hemodynamic instability within 4 hours of receiving rFVIIa, 11 died within 2 hours and 1 died within 4 hours (8 during surgery and 4 in the recovery room). Due to the limited time frame, it was not possible to determine if bleeding in these patients diminished after rFVIIa treatment. In the urgent setting in which these patients were treated, determining the amount and timing of blood loss and blood transfusion was difficult. Moreover, for patients deemed non-salvageable, blood product transfusion was suspended before response to rFVIIa could be determined. Risk factors and outcome in CSP Overall, 17 of 24 CSP were over the age of 60. Twenty underwent CPB, and 15 had a LVEF below 25%. Fourteen underwent mediastinal re-exploration for bleeding. Twenty-three patients had a significant coagulopathy defined as thrombocytopenia below 100,000/mm 3 , prolonged PT and PTT or both (data not given due to wide variations in the results). Seven of the 24 patients did not have a documented arterial blood gas (ABG) around the time of rFVIIa administration; however, among the other 17 patients, pH's of 7.0 in 1, 7.2 in 5, and 7. 4 in 11 patients were noted. With regards to rFVIIa administration, there was no correlation between pH, PT, PTT, thrombocytopenia, anemia and outcome. Of the 12 patients who died within 4 hours, all underwent CPB for over 30 minutes (time on CPB for 8 patients was more than 2.5 hours). All 12 had an LVEF of less than 25% and 7 had mediastinal re-exploration for bleeding. In contrast, only 3 of the 12 surviving patients had EF <25%, 8 had surgery with CPB and 7 underwent mediastinal re-exploration. The median age and coagulation parameters were not different between the two groups. Other Bleeding Patients (OBP) Outcome Eleven of the 16 patients with bleeding due to other causes survived for more than 4 hours after rFVIIa administration, and the results of their blood product use before and after rFVIIa are summarized in Table 2 . From the beginning of surgery or time of trauma until the administration of rFVIIa, patients received an average of 10 U of R (range 2–28), 11 U of P (range 0–36), 14 U F (range 0–34) and 10 U of C (range 0–70). Post administration of rFVIIa, an average of 1 U of R (range 0–6), 2 U of P (range 0–6), 2 U of F (0–10) and 0 U of C were transfused over 24 hours. The difference between the pre- and post-rFVIIa treatment blood product requirement was significantly lower for R (p = 0.004), P (p = 0.017), and F (p = 0.017), but not for C (p = 0.122). Nine of the 11 showed a rapid response to rFVIIa, as measured by a decrease in R transfusion to an average of <0.5 U (range 0–3). The other 2 patients (Patient # 2 and 15) continued to require blood product support beyond 24 hours post rFVIIa treatment. Table 2 Summary of rFVIIa treated other bleeding patients (OBP) who survived >4 hours P# Age Sex Procedure Co-morbidities Response to rFVIIa Outcome Pre rFVIIa Post rFVIIa R P F C R P F C 2* 48F Colectomy Colon cancer 4 6 24 70 2 0 2 0 Died 15* 26F Cystectomy, nephrectomy, pelvic fracture, termination of Pregnancy MVA, 20 weeks gestation 18 12 8 0 6 6 10 0 Survived 16 53M Left femur fixation Multiple myeloma 12 12 6 0 0 0 0 0 Survived 19 82M Shoulder replacement HTN, CAD, Renal failure 7 10 20 10 2 6 4 0 Survived 21 54F Liver laceration Trauma 23 17 34 20 3 6 10 0 Survived 22 24M Neck soft tissue trauma FVII deficiency 2 0 4 0 0 0 0 0 Survived 23 64F Liver laceration Dialysis 28 36 33 0 0 0 0 0 Died 28 30F Postpartum hemorrhage None 12 18 6 10 0 0 0 0 Survived 29 64M Gastrointestinal bleed FVIII inhibitor 2 0 0 0 0 0 0 0 Survived 38 60M Thyroidectomy VWF deficiency 4 12 12 4 0 0 0 0 Survived 40 40F Splenic subcapsular bleed Amyloidosis, coumadin overdose 4 0 8 0 0 0 0 0 Survived Average 10 11 14 10 1 2 2 0 P value .004 .017 .017 .122 P: Patient R: Red Blood Cell; P: Platelet; F: Fresh Frozen Plasma; C: Cryoprecipitate M: Male; F: Female EF: Left Ventricular Ejection Fraction VWF: von Willebrand Factor * Patient continued to require blood transfusion past 24 hours post rFVIIa treatment. Nine of the 11 surviving patients were discharged to home and 2 (Patient # 2 and 23) died between 3 and 30 days from a combination of infection, cardiac arrest and multiorgan failure. Amongst survivors beyond 4 hours, mortality at 30 days was 18% and overall mortality for the entire group was 44%. All five of the 16 patients who died within four hours of rFVIIa treatment had massive injuries either from gunshot or stab wounds. Upon arrival to the emergency room, these patients were hemodynamically unstable, with hemoglobin values ranging between 3–6 g/dL. rFVIIa was given as a last resort, but these patients were not evaluable since transfusions were suspended shortly after infusion of rFVIIa. Risk factors and outcome in OBP The median age of this group was 42 years. None underwent CPB, and two of the 7 patients tested had an EF <25%. Only 1 patient had a second exploratory laparotomy for bleeding. The patient suffering with a warfarin overdose had a splenic subcapsular bleed complicating systemic amyloidosis. At the time of rFVIIa administration, a wide variation in laboratory parameters was observed in these patients (data not shown). There was no difference in coagulation parameters between patients who died and those who survived. Eight of the 16 patients did not have an ABG documented. In the 5 patients who died within four hours, the pH was lower (6.9–7.0) than for those who survived (7.3–7.4). Thromboembolism after rFVIIa Only one of the 23 patients in both the CSP and OBP groups who survived more than 4 hours had a documented thromboembolic complication after receiving rFVIIa. Deep vein thrombosis (DVT) of the left subclavian vein was confirmed by a Doppler ultrasound 2 days after rFVIIa administration. The thrombus was related to a central line placed before rFVIIa treatment. No other clinical or laboratory evidence of thromboembolism was documented in the medical records of the other patients throughout their hospital stays. None of the other 17 patients in both the groups who died within 4 hours were evaluable for thromboembolic complications. Discussion This retrospective case series suggests that rFVIIa is effective in promoting hemostasis even when given as a last life-saving measure in poor prognosis patients with massive, transfusion-refractory hemorrhage. A statistically significant decrease in blood product transfusion was evident in 12 CSP and 11 OBP who survived more than 4 hours after rFVIIa infusion. Six of 12 CSP and 2 of 11 OBP died later between 3 and 30 days after rFVIIa infusion, but these deaths were due to causes other than hemorrhage. Despite the obvious limitations of a retrospective analysis, this is the largest series of such patients reported in the literature to date. Al Douri et al [ 8 ], described 5 patients who underwent open-heart surgery for valvular disease and experienced uncontrollable intraoperative or postoperative bleeding unresponsive to a blood product support protocol. The study was prospective, had well-defined criteria for "excessive bleeding" and excluded patients with a history of ischemic heart disease, stroke or venous thromboembolism. Hemostasis was achieved in all patients following a single dose of 30 ug/kg of rFVIIa, and there was no mortality from bleeding, although 1 patient died 3 days later of an unrelated cause. Overall, the 4-hour and 30 day mortality in the present series was high compared to that of Al Douri et al [ 8 ], but it is difficult to compare these two very different patient populations. Factors predictive of inferior outcome after cardiac surgery include age over 60, low LVEF, significant co-morbidity, the use of CPB and re-exploration after initial surgery [ 22 - 24 ]. The use of CPB increases the rate of fibrinolysis and induces an inflammatory response [ 19 , 25 ]. Moreover, prolonged time on CPB, defined as 2.5 hours or more, is associated with increased bleeding and need for re-exploration. Two-6% of patients undergoing CABG require re-exploration for bleeding and is associated with a mortality rate of up to 22% [ 19 , 26 , 27 ]. In this series, the CSP group had multiple poor prognostic factors. The median age was 65 years and in addition, all 12 of the CSP who died within 4 hours and 3 of 6 CSP who died later had preoperative LVEF of less than 25%. All of the 12 CSP who died within 4 hours in this series, required CPB and 8 of these patients were on CPB for more than 2.5 hours. More than half of our CSP (14 of 24) required re-exploration, which undoubtedly influenced the mortality rate. Both, the CSP and OBP groups had a significant decrease in blood product use after rFVIIa treatment but the short and long-term survival appeared to be worse in CSP probably due to multiple poor prognostic factors as discussed above. Poor outcome has also been independently associated with peri-operative transfusion of more than 7 units of R [ 28 ]. The CSP and OBP who survived received an average of 18 and 11 units of R in the peri-operative period, respectively. The R requirement of patients who died within 4 hours in both groups was probably higher but could not be reliably estimated. All 5 of the patients in the OBP group who died within 4 hours suffered from major blood vessel injury as a result of stabbing or gun shot wounds. These patients were unstable upon arrival to the emergency room with hemoglobins in the range of 3–6 g/dL. Excessive hemorrhage requiring massive transfusion can lead to hypothermia, DIC, excessive fibrinolysis, dilutional coagulopathy, and metabolic acidosis, which may further exacerbate bleeding and morbidity. [ 14 - 17 ] A reduction in the pH to 7.0 nearly abolishes rFVIIa activity as reported by Meng et al [ 18 ]. In this present series, there was no obvious correlation between the coagulopathy and mortality in both the CSP and OBP groups. Five of the OBP who died within 4 hours had a pH of 7 or less. However, acidosis did not appear to play an important role in CSP. The optimal dose and timing of administration of rFVIIa for these patients is unknown. The standard dose (90 mcg/kg) was first used in hemophiliacs and was based on the in vitro dose-dependent reduction in aPTT in plasma from these patients [ 1 , 29 ]. The 90 mcg/kg dose of rFVIIa corresponds to concentration of 1.25 mcg/ml FVIIa in plasma. Martinowitz et al [ 7 ], used a median dose of 120 mcg/kg (range 120–210 mcg/kg) to achieve hemostasis in 7 cases of trauma. In 2 patients, 120 mcg/kg was insufficient, and 2 additional doses of 60 mcg/kg were required to achieve hemostasis. Kenet et al. [ 30 ], have shown better efficacy with a 300 mcg/kg bolus dose followed by continuous infusion. The standard therapeutic dose of 90 mcg/kg of rFVIIa was used in this series of patients although a lower dose of 30 mcg/kg dose has also been shown to be effective [ 8 ]. However, in considering the data from the aforementioned studies, it would appear that higher initial doses, or additional doses, might improve outcome. It is also tempting to speculate that earlier treatment with rFVIIa may have prevented rapid clinical deterioration and complications associated with massive blood product transfusion in this series of patients. This question would best be addressed in controlled studies using standard dosing protocols and well defined criteria for intractable hemorrhage. Although rFVIIa is expensive, it would appear to be cost effective when compared with the combined cost of large amount of blood products. Previous clinical experience with rFVIIa supports a good safety profile in patients with hemophilia and trauma. Less than 1% of patients receiving rFVIIa had thrombosis and thrombosis related complications [ 21 , 31 ]. In our series, only one patient had a deep vein thrombosis of the left arm associated with an indwelling line and not considered to be due to rFVIIa. There were no other documented cases of thrombosis or microemboli. Conclusions This retrospective study suggests that rFVIIa can play a beneficial role as an adjunctive hemostatic agent in patients after cardiac surgery or extensive trauma who experience bleeding that cannot be controlled by conventional therapies. Prospective studies are necessary to determine optimal patient selection, and dose and timing of rFVIIa administration.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535534.xml
526275	i-Genome: A database to summarize oligonucleotide data in genomes	Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.	Background During the last decade, many genomes have been successfully and completely sequenced. Summarized information about the oligonucleotides in genomes provides biologists, who interests in the evolution and growth of genomes, to work in comparative genomics, oligonucleotide probe design, primer design and the analyses of genomic repetitive features. The computation of the occurrences and the frequency of all oligonucleotides in a complete genome is very elaborate and time-consuming, especially when the genome size is very large, such as the human and mouse genomes. A database that summarizes the occurrences and the frequencies of oligonucleotides in complete genomes can facilitate the biological and the statistical analyses of genomes. The contents of the database can be used in many biological applications, such as comparative genomics and evolution analyses [ 1 , 2 ], the prediction of regulatory sequences by detecting the over-represented oligonucleotides [ 3 - 8 ] and primer/probe design based on the uniqueness of oligonucleotides [ 9 ]. Table 1 shows the biological applications of the database entries. The entries in the database are divided into two types, namely, the occurrence positions of oligonucleotides and the frequencies of oligonucleotides. The oligonucleotide occurrences and the oligonucleotide frequencies in both the coding regions and the non-coding regions are summarized. For instance, these information can be used in statistical analyses to study the over-representation of the regulatory sequences in upstream promoter regions in genes. van Helden et al. systematically searched the promoter regions of potentially co-regulated genes for over-represented oligonucleotides which may be transcription factor binding sites [ 3 ]. They presented a simple and fast method for isolating DNA binding sites for transcription factors from families of co-regulated genes, illustrating their results using Saccharomyces cerevisiae. Although conceptually simple, the algorithm efficiently extracted the upstream regulatory sequences that had been previously been determined experimentally for most of the yeast regulatory families already analyzed. Other studies [ 4 - 8 , 10 - 12 ] on the prediction of gene regulatory sequences have been based on oligonucleotide analysis. Table 1 Applications and the relevant data in the database. Database entries Entry types Biological applications Oligonucleotide occurrences Positions 1. Oligonucleotide analysis for regulatory sequences 2. Oligonucleotide probe design 3. Primer design Oligonucleotide frequencies Counts 1. Oligonucleotide analysis for regulatory sequences 2. Evolutionary analysis 3. Oligonucleotide probe design 4. Primer design Gene coding regions Positions 1. Oligonucleotide analysis for regulatory sequences 2. Evolutionary analysis 3. Oligonucleotide probe design 4. Primer design Repetitive element frequencies (LINE, SINE, Alu, and so on) Counts Evolutionary analysis Repetitive element occurrences Positions Evolutionary analysis Tandem repeats Positions Prediction for genetic disease marker Hsieh et al. [ 1 ] investigated the oligonucleotide distributions of typical microbial genomes and found that the microbial genomes have the statistical characteristics of a much shorter DNA sequence. This peculiar property supports an evolutionary model in which a genome evolves by random mutation but grows primarily by random segmental duplication. Repetitive elements, including LINEs, SINEs, LTR and Alu, can be investigated in evolution analysis [ 2 ]. It has been estimated that at least 43% of the human genome is occupied by four major classes of interspersed repetitive elements – LINEs, SINEs, LTR elements and DNA transposons [ 2 ]. Their analysis has yielded some insights into the evolution of the human genome. The tandem repeats provided in our database can be used for forensic analysis and the study of genetic diseases [ 13 - 16 ]. Another application of the established database is to facilitate the design of primers to amplify specific regions of the genomic sequence. The basic concept is that the sequences of primers from 15 to 40 bps should be unique, unlike the repetitive oligonucleotides in our database. Additionally, our database maintains the repetitive oligonucleotides that facilitate the design of oligonucleotide probes to allow the selection of signature oligonucleotides when identifying different organisms using DNA arrays [ 9 ]. The user can query oligonucleotides whose lengths exceed a threshold, such as 15 bps, to determine whether the oligonucleotides are repetitive. The non-repetitive regions of the target sequences without repetitive oligonucleotides can be used as the signatures for the target genomes. Construction and content Data sources and contents The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The data sources including the complete genomes and the gene annotation information are obtained from GenBank [ 17 ]. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase [ 18 ]. The database supports a range of complete genomes including human, yeast, worm, and 128 microbial genomes. The Appendix lists the organisms supported in the database [see additional file 1 ]. The occurrences and the frequencies of oligonucleotides from one to 50 base pairs are generated and accumulated from each of the complete genome sequence. Inputting the sequence of the oligonucleotide returns the positions of the oligonucleotides. Additionally, both the occurrences and the frequencies of the repetitive elements such as LINEs, SINEs, Alu and LTR are provided by computationally scanning whole genome sequences. The tandem repeats are computationally detected by the tandem repeat finder [ 19 ]. The database also provides the gene annotation information. For instance, Table 2 presents the number of occurrences of repetitive oligonucleotides in yeast. The oligonucleotide "ACCCTA" occurs 2,724 times in the yeast genome, 822 times upstream of a gene (-600 ~-1 bp, +1 bp denotes the gene translational start postion) and 793 times in the coding regions. The counts of the occurrence of ecah oligonucleotide between one and 50 bps are present in the database. Table 2 Number of occurrences of the repetitive oligonucleotides in yeast genome Repetitive oligonucleotide Amount of occurrences Repetitive oligonucleotide Amount of occurrences Genome Up-streams Coding region Genome Up-streams Coding region ACCCTA 2,724 822 793 CAATCCA 1,895 655 343 ACCCTC 2,917 881 795 CGTCTCC 592 199 148 AGTACT 3,073 933 879 CGTCTGA 652 196 165 AGTAGA 6,673 1,970 1,798 ACAAACTA 594 179 183 AGTAGC 4,912 1,545 1,299 ACAAACTC 514 175 112 GATACC 4,829 1,638 1,005 CACAGAAAC 146 38 46 GATAGA 7,030 2,163 1,807 CACAGAAGA 164 57 39 TGGTAA 10,513 3,493 2,214 ACATATAAAAA 54 9 29 TGTAAA 11,364 3,439 3,418 ACATATAAAAC 139 34 56 AAGGGGA 1,172 299 421 ACATATAAAAG 36 7 22 AAGGGGC 626 142 256 ACTTATGTCATC 57 17 23 AGAGTGG 983 310 271 ACTTCTAGTATA 159 44 67 AGAGTTA 1,859 610 441 ACTTTTTTTTCT 32 5 21 CAATCAG 1,358 445 320 ACTTTTTTTTTC 50 6 33 Data Generation A software is implemented to index systematically a complete genome sequence into a suffix-array using a perfect match approach [ 20 ]. This index is only able to find the perfect match for any oligonucleotide. The user can thus use it to find the positions of a designated oligonucleotide in a genome sequence. For each genome, the occurrences of all oligonucleotides shorter than 50 bps can be efficiently searched for. The occurrence is the position of the oligonucleotide in genome. The frequency is the count of oligonucleotide occurrences in a region. The regions are the complete genome, the coding regions and the non-coding regions. Frequencies of oligonucleotides with different lengths are stored in different flat-files. For example, the two chromosomes of the Vibrio cholerae genome are processed separately to allow the computation of the occurrences of oligonucleotides in each chromosomal sequence. RepeatMasker [ 21 ] and the repetitive element database, Repbase [ 18 ], are used to search the instances of the repetitive elements in eukaryotic genomes. The tandem repeat finder (TRF) is used to find the tandem repeats in genomes [ 19 ]. The TRF and RepeatMasker can find the instances of repetitive elements with imperfect matches. The settings used here for each software is described in below. The Tandem Repeat Finder uses seven parameters. These are match score, mismatch score, indel score, probability of match and insertion, minimum score of alignment and the maximum of tandem repeat pattern size. The corresponding values used here are 2, 7, 7, 80, 10, 20 and 500. The values are the default suggestions found in Tandem Repeat Finder documentation. The transposable elements (TEs) are detected by RepeatMasker. TEs in each genome are identified using the complete dataset available from REPbase Updates [Please add the citation]. The sensitivity and the speed of RepeatMasker are set as the default values. Utility Table 3 presents the two output formats – flat-files and the web query interface with a filtering function. In the flat-file format, the fields of each oligonucleotide (in a chromosome) include the sequences, the number of occurrences in the chromosome, the number of occurrences in the coding regions and the number of occurrences in the non-coding regions. The user that requires a large amount of such data can download them in this format [ 1 ]. Table 3 Output styles of the database. Database entries Entry types Output formats Oligonucleotide occurrences Positions Web interface Oligonucleotide frequencies Counts Web interface and flat-file Gene coding regions Positions Web interface Repetitive element frequencies (LINE, SINE, Alu, and so on) Counts Web interface and flat-file Repetitive element occurrences Positions Web interface Tandem repeats Positions Web interface The web interface enables users to query the occurrences of an oligonucleotide in a genome and the number of occurrences in each chromosome. Figure 1 shows this approach. The occurrences of the repetitive elements and the tandem repeats in the established database can also be queried via the web interface, as in the example given in Fig 2 . Figure 3 depicts the flat-file format of oligonucleotide frequencies. The first row in the flat-file presents the basic information for the oligonucleotide frequencies and the fields are the chromosome sequence/NCBI accession number, the length of the chromosome sequence, the size of coding regions, the size of non-coding regions, the length of the oligonucleotides and the minimum number of copies of the oligonucleotide. The directories labeled C10 are the files that contain the counts of oligonucleotides with at least ten occurrences in genome. Each file name includes the sequence/NCBI accession number, the length of oligonucleotides and the minimum occurrences of oligonucleotides. For example, "NC 000913 L30 C10" is the oligonucleotides, which are 30 nucleotides in length and have at least 10 occurrences in the genome. Figure 1 Web query interface (1/2). Figure 2 Web query interface (2/2). Figure 3 Database entries in flat-file format. Figure 4 shows the web interface for the occurrences of a specific oligonucleotide. The user submits the query oligonucleotide and selects particular species; the positions of the oligonucleotide are then shown. The first line is the user submitted data. Following this information are the positions of oligonucleotides in forward strand. This is followed by the same information for the reverse strand. Figure 4 The occurrence positions of the oligonucleotide are found by Oligos Locator. Conclusions We have constructed the databases of both the oligonucleotide occurrence locations and their frequencies in the coding and the non-coding regions in complete genomes. The data in flat-file format can be downloaded directly for further analyses in several biological applications. The user may also use the web interface to query and access the database contents. The database also provides a filtering function for retrieving the information about oligonucleotides under search conditions specified by the users. Furthermore, the database provides the occurrences and the frequencies of other repetitive elements, such as LINE, SINE, Alu and tandem repeats in genomes. Availability and requirements The database is now available at Authors' contributions FML implements the software and refinements the system. HDH conceives of the study and drafted the manuscripts. YCC and JTH participates the design and coordination. All authors read and approved the final manuscripts. Supplementary Material Additional File 1 Appendix listing the organisms supported in the database. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526275.xml
545951	Cyclic nucleotide binding proteins in the Arabidopsis thaliana and Oryza sativa genomes	Background Cyclic nucleotides are ubiquitous intracellular messengers. Until recently, the roles of cyclic nucleotides in plant cells have proven difficult to uncover. With an understanding of the protein domains which can bind cyclic nucleotides (CNB and GAF domains) we scanned the completed genomes of the higher plants Arabidopsis thaliana (mustard weed) and Oryza sativa (rice) for the effectors of these signalling molecules. Results Our analysis found that several ion channels and a class of thioesterases constitute the possible cyclic nucleotide binding proteins in plants. Contrary to some reports, we found no biochemical or bioinformatic evidence for a plant cyclic nucleotide regulated protein kinase, suggesting that cyclic nucleotide functions in plants have evolved differently than in mammals. Conclusion This paper provides a molecular framework for the discussion of cyclic nucleotide function in plants, and resolves a longstanding debate about the presence of a cyclic nucleotide dependent kinase in plants.	Background The discovery of cyclic 3'5'-adenosine monophosphate (cAMP) by Earl Sutherland in the late 1950s was one of the most significant paradigm shifts in biochemistry [ 1 ]. This breakthrough ushered in the concept of second messengers: intracellular molecules which transmit signals in cells and are derived from an extracellular signal. In the past half century, cyclic nucleotides (both cAMP and cGMP) have been implicated in a vast array of biological phenomena in all kingdoms of life [ 2 ]. The ubiquitous presence of cyclic nucleotides may be due to several characteristics which make it an ideal second messenger. Cyclic nucleotides are derived in a energetically favourable reaction from common metabolites (ATP and GTP), and can be broken down into non-toxic products (inorganic phosphate and AMP/GMP). The synthesis and degradation of cyclic nucleotides are controlled by enzymes termed adenylate (or guanylate) cyclases and cyclic nucleotide phosphodiesterases, respectively [ 3 , 4 ]. In plants, cyclic nucleotides have endured a checkered research history fraught with complications and setbacks. Despite this, recent work has shown unequivocally that cyclic nucleotides are present in plant cells [ 5 , 6 ], and that they play key roles in the regulation of plant physiology [ 7 - 9 ]. Furthermore, the recent identification and cloning of adenylate and guanylate cyclases in plants [ 7 , 10 ] may eventually give clues as to what signals the synthesis and degradation of these molecules in plants. Cyclic nucleotides are able to bind to two distinct protein domains, CNB domains and GAF domains. CNB domains were first identified in the regulatory subunit of mammalian cAMP-dependent protein kinase (RI and RII). Since several CNB domain containing plant proteins have been shown to be directly modulated by cyclic nucleotides, this indicates that the CNB domain in plants is functionally similar to CNB domains in other organisms [ 11 - 15 ]. GAF domains were initially identified as conserved domains in light sensing molecules but are known as small molecule binding domains in cyclic nucleotide regulated phosphodiesterases, the Anabaena cyclic nucleotide stimulated adenylate cyclase and several other proteins [ 16 ]. GAF domains have been shown to bind both cAMP [ 17 ] and cGMP [ 18 , 19 ]. Recent crystal structures of the GAF domains of human PDE2 [pdb:1MC0] and the yeast protein YKG9 [pdb:1F5M] have shown that this domain is an alpha/beta two layer sandwich with no structural or sequence homology to the CNB domain [ 18 , 20 ]. Therefore, GAF and CNB domains have evolved independently to bind cyclic nucleotides. In order to further explore the roles of cyclic nucleotides in plants, we performed a bioinformatics based analysis of the completed Arabidopsis thaliana and Orzya sativa genomes [ 21 - 23 ] in order to elucidate the potential targets of cyclic nucleotides in plants. Results and discussion GAF domains Based on the PDE2 crystal structure 11 residues were proposed to be involved in cyclic nucleotide binding [ 18 ], but comparison to the cAMP binding GAF domain of the Anabaena adenylate cyclase shows that these residues may only be strictly conserved in mammals. Further complicating our analysis is the fact that GAF domains are known to bind other small molecules such as 2-oxoglutarate, formate and bilins [ 24 - 26 ]. GAF domains form a structural scaffold which can be utilized to bind several possible small molecules depending on the functional groups on that scaffold. Therefore, their role in cyclic nucleotide signalling must be verified by biochemical means rather than strictly by sequence analysis. Our analysis indicated that in plants there are two types of proteins which contain GAF domains. These are the phytochrome proteins and the ethylene receptor proteins. Phytochromes Phytochromes are light sensing signal transduction molecules which function to control several aspects of plant biology. Interestingly phytochromes were found to function upstream of cyclic nucleotides in their signal transduction pathways since their functions can be mimicked by cGMP and calcium in phytochrome knockout cells [ 27 - 29 ]. The light sensing portion of the phytochrome molecule is a covalently linked bilin molecule which is known to be bound by the GAF domain. Therefore it is unlikely that the GAF domain of the phytochrome is also able to bind cyclic nucleotides directly, although it is clear that cyclic nucleotides are somehow involved in this signalling pathway. Ethylene receptors Ethylene responses have been documented for nearly a century in plants. This gaseous hormone is involved in many aspects of plant physiology, including fruit ripening, organ development, germination, seedling growth, flowering and response to challenges such as pathogens and stress [ 30 ]. There are five putative ethylene receptor isoforms in both Arabidopsis and rice as determined by genome sequencing [ 31 ]. All known ethylene receptors contain a GAF domain in a cytoplasmic region amino-terminal to the kinase domain. It has been speculated that this domain may be involved in cyclic nucleotide signalling but examination of heterologously expressed, functional ETR1 [Swiss-Prot: P49333] showed no detectable cyclic nucleotide binding (G. E. Schaller, personal communication). There are other ethylene receptors which have GAF domains and which to our knowledge have not been tested for cNMP binding, however, to date there is no evidence of cNMP regulation of ethylene receptors. Currently the function and ligands of the GAF domain in ethylene receptors is unknown. CNB domains From the alignment of CNB domains in animals, bacteria and plants, it was apparent that there are some strong similarities, as well as some significant differences (Figure 1A ). In order to visualize whether plant CNB domains could fold in a similar manner to the other well characterized CNB domains, we generated an in silico model. We chose the plant protein which showed highest similarity to known crystal structures ( Arabidopsis thaliana CNTE1) and based our model on the solved crystal structures of RIα, RIIβ, HCN2, CAP and Epac2 (Figure 1B and see additional file 5 ). We then examined our model's overall topology as well as its cyclic nucleotide binding site. The basic fold of the domain is two anti-parallel beta sheets consisting of four strands forming a sandwich, ending with an alpha helix (the hinge region). Connecting these sheets are exposed loops, the most important of which is the phosphate binding cassette [ 32 ]. It is important to note that our structure models very well against all CNB domains with excellent conservation of all secondary structure and most loops. We calculated the backbone root mean square deviation for our model versus each of the templates as: RIIβ domain 1: 0.76 Å; RIIβ domain 2: 0.83 Å; Epac1 domain 1: 1.08 Å; RIα domain 1: 0.85 Å; RIα domain 2: 0.94 Å; HCN2: 0.82 Å and CAP: 1.12 Å. Our model agrees in general with a previously reported model for the Arabidopsis CNGC2 [ 33 ], although a detailed comparison between the two models was not performed. The use of a less distant target (atCNTE1) as well as several templates (seven compared to one) adds to the reliability of our structure. In all mammalian cAMP-binding structures solved, there is a key arginine residue (Arg 209 in RIα) which forms a salt bridge with the cNMP's phosphate group. This residue is absent in some CNB domains, despite evidence that at least some of these domains do in fact bind cyclic nucleotides. For example, this residue is absent in the Drosophila ether-a-go-go channel, which is known to be modulated by cyclic nucleotides [ 34 ]. Examination of our model shows that in the region near the phosphate, there are two residues which may functionally replace the arginine, Tyr 91 and Ser 92 (Figure 1B ). In bacterial CAP, hydrogen bonding is provided to the phosphate by the Arg 82 sidechain, Ser 83 amide nitrogen atom and sidechain hydroxyl group, as well as a water molecule. In some mammalian isoforms, the serine residue is changed to an alanine and therefore is only able to provide backbone hydrogen-bonding. In our plant atCNTE1 model, the serine residue is conserved, but the arginine residue is missing. Since there was no good template to model the phosphate binding cassette onto, our model only approximates the position of this loop, and will require verification by other structural studies. The hydroxyl and amide groups of Ser 92, as well as the hydroxyl group of Tyr 91 are all within proximity of the cNMP phosphate and could play a role in stabilizing the cyclic nucleotide (see blue residues in Figure 1 ). Examination of the region which contacts the base, indicates that our model is most similar to the structure of CAP in this region, so it is likely that the base moeity of a cNMP is bound in a syn orientation as in CAP. This is in agreement with a previously reported atCNGC2 model [ 33 ]. Further analysis of the binding site for the nucleotide indicates that it is likely cGMP which binds to atCNTE1. This conclusion is based upon the presence of three residues (Tyr 80, Ser 92 and Ser 109) which could potentially differentiate between cyclic nucleotides, each of which has a preference for cGMP (Figure 1 and [ 35 , 36 ]). Other conserved structural features of our model are the hydrophobic pocket forming residues Tyr 36, Val 42, Val 43, Tyr 53, Leu 55, Ala 60, Phe 82, Ala 93 and Val 95 and Leu 105 (see green residues in Figure 1 ) as well as several conserved glycine residues which are involved in turns between the beta strands (39, 58 and 83) and the helix capping Asp 109. This residue signals the end of the hinge region alpha helix and is present in most CNB domains examined. Phylogenetic analysis indicates that the plant CNB domains segregate into three subfamilies (Figures 2 and 3 ). The phylogenetic distribution of the CNB domain matches their domain context in that CNGC, shaker-type and CNTE proteins form separate groups. Furthermore, for each of the three protein classes, the CNB phylogeny matches the phylogeny of the full-length protein, implying that these proteins obtained the CNB domain prior to isoform duplication (Figure 3 , [ Additional file 1 ], [ 37 ]). Since all three branches have been detected in both Arabidopsis and Oryza, it is likely that the specific plant cyclic nucleotide responses developed prior to monocot-dicot divergence. We did not find CNB domains in any protein kinases, transcription factors or guanine nucleotide exchange factors in our analysis. Each of the three classes of CNB domain containing proteins will be discussed below. Cyclic nucleotide gated ion channels Plant CNGC ion channels were first identified in a screen for calmodulin binding partners in barley [ 38 ]. There are now known to be 20 CNGC proteins in Arabidopsis likely indicating a high level of channel redundancy [ 37 , 39 ]. We also detected 16 CNGC proteins in rice by examination of the TIGR Rice Genome Annotaion Resource [ 40 ]. Electrophysiological studies have shown the CNGC channels to be permeable to potassium, sodium and calcium [ 13 - 15 , 41 - 43 ]. Cyclic nucleotides have been shown to activate channel opening in all CNGC proteins examined thus far leading to an influx of cations into the cell [ 13 - 15 , 33 ]. Mutagenic screens have shown that mutations in atCNGC2 and atCNGC4 create faulty pathogenic reactions [ 13 , 44 ]. When taken together with data showing that cyclic nucleotides are necessary for pathogen responses and that calcium and potassium influxes are characteristic of early phases of plant pathogen responses [ 45 ], this seems to imply that cyclic nucleotides may play a role in controlling plant immune responses. Finally, work by Maathuis and Sanders [ 8 ] has shown that cyclic nucleotides can modulate sodium uptake in Arabidopsis plants, implying that there is a cyclic nucleotide controlled channel which plays a role in salinity tolerance. They showed that cyclic nucleotides are required for limiting sodium uptake in root protoplasts, but the exact molecule (cAMP or cGMP) responsible for this effect has not been pinpointed. Shaker-type potassium channels Plant potassium channels fall into two classes, the KCO channels and the shaker-type channels [ 37 ]. In addition to the 9 shaker-type channels described in Arabidopsis thaliana , we have found 10 channels in Oryza sativa . A variety of mutational studies have implicated the shaker-type channels in several key processes involving the movement of potassium including: from the soil (AKT1, KAT3), long distance transport (AKT2), transport into growing pollen tube (AKT6), secretion into xylem sap (SKOR) and transport during guard cell opening either into the cell (KAT1, KAT2) or out of the cell (GORK) [ 37 ]. Shaker-type channels are voltage dependent outward (GORK, SKOR) or inward (KAT and AKT) rectifying channels. Analysis of heterologously expressed channels have shown that cyclic nucleotides function to adjust the activation potential of these channels [ 11 , 12 ]. Since cyclic nucleotides have already been implicated in some of the processes controlled by shaker-type channels [ 7 - 9 , 46 ], it is reasonable to believe that cyclic nucleotides are physiological regulators of shaker-type potassium channels. Cyclic nucleotide regulated thioesterases Initially we detected a short CNB containing protein which was only slightly larger than the domain itself. Sequencing of the EST provided by the Arabidopsis Biological Resource Center [ 47 ] showed the protein was actually mis-annotated by the automated gene-finding algorithm. Further analysis indicated that there are two isoforms of this protein in Arabidopsis and one in rice. Each protein contains an amino-terminal CNB domain and a carboxy-terminal acyl-CoA thioesterase domain. Searches of other partially sequenced plant genomes and EST databases indicated that these proteins are present in several plant species, but not in any other division of life and thus represents a novel plant-specific cyclic nucleotide target. Comparison of these protein sequences indicates a high level of conservation, including residues conserved for both catalysis and cyclic nucleotide binding domain structure. Arabidopsis CNTE1 had previously been partially characterized as a thioesterase and shown to have activity versus both 16:0-CoA and 18:1-CoA when over-expressed and partially purified from E. coli [ 48 ]. Fatty acid synthesis requires the use of acyl-CoA's as building blocks for incorporation into lipids. It is therefore possible, that these thioesterases function as scavengers which remove "irregular" fatty acids from the pool of available building blocks [ 48 ]. Furthermore, the thioesterases could divert fatty acids away from biosynthetic pathways and β-oxidation during germination or during stressful conditions. In most cases when a small molecule binding domain is connected to a catalytic domain on the same polypeptide, the catalytic domain is regulated by the small molecule [ 49 ]. The conservation of this protein across planta indicates that the CNB domain likely has a role in controlling the thioesterase activity of this enzyme, but it is unknown at this time exactly what role cyclic nucleotides play in this process. In order to address this we cloned and tried to express the atCNTE1 protein in E. coli , however after extensive trials we were unable to express and purify soluble protein. A cyclic nucleotide dependent protein kinase in plants? We found no PKG or PKA regulatory subunit homologs in the Arabidopsis genome. There has been a long standing controversy in the plant field as to the existence of a plant cyclic nucleotide dependent kinase [ 50 - 52 ]. As PKA is the major cAMP target in mammalian cells we chose a biochemical approach to further explore the possibility that a PKA-like enzyme may be present in Arabidopsis. We performed protein kinase assays with extracts of Arabidopsis thaliana using the PKA substrate Kemptide. Kemptide is a peptide which has a motif which was confirmed as the optimal substrate for PKA [ 53 , 54 ] and is routinely used in mammalian PKA assays. As Figure 4A shows, there was no detectable increase in kinase activity in the plant cell extracts when cAMP or cGMP are added. Fractionation of extracts, as well as testing a range of cyclic nucleotide concentrations also did not allow us to detect any differences in kinase activity with addition of cyclic nucleotides (data not shown). For comparison, adipocyte extracts (a cAMP responsive mammalian tissue) were assayed as well, illustrating the large increase in protein kinase activity in these cells when cAMP is added. Furthermore, blotting of A. thaliana extracts with polyclonal antibodies raised against mammalian PKA subunits (both the catalytic and the RIIα subunit) reveals that no structurally similar proteins are present in this extract (Figure 4B ). Blotting with a monoclonal antibody to the RIIβ subunit gave similar results (not shown). Although there is a weak band present in the Arabidopsis extract which cross-reacted with the catalytic subunit polyclonal antibody, it is likely unrelated to cyclic nucleotide signalling. Protein kinase catalytic domains are very highly conserved [ 55 , 56 ] and therefore a minor amount of cross-reactivity is not unexpected. Further adding to the validity of the western blotting experiment is the observation that several studies have shown that true PKA-like enzymes in non-mammalian eukaryotes do cross react with antibodies raised against the regulatory subunits of mammalian PKA [ 57 , 58 ], whereas this is not detected in our plant extracts. The lack of evidence for kinase activity could be attributed to substrate specificity, differences in binding affinity or expression levels in plant extracts relative to mammalian extracts, so our experimental approach does not exhaustively rule out the possibility of a cNMP dependent kinase activity. However, we feel that these data in concert with the genomic and blotting data strongly suggest that there is no cNMP dependent kinase in plants. Finally, our data imply that if such a protein exists, it would bear little or no sequence, structural or biochemical similarity to the classically studied mammalian enzyme. Conclusions As the understanding of cyclic nucleotide signalling in a variety of systems has progressed, it has been increasingly difficult to describe a general role for cyclic nucleotides in biology. They control 'well-fed' gene transcription in bacteria, and modulate signal transduction and ion currents in mammals, resulting in a large number of possible physiological responses. This analysis is potentially limited in that it only analyses cNMP domains which have already been previously identified and characterized in other systems. However, conservation of CNB and GAF domains as the only known cyclic nucleotide binding domains present over a wide cross-section of life indicates that these domains are likely to control most, if not all cyclic nucleotide responses. It is possible however, that plants have evolved entirely novel domains which can be modulated by these second messengers. It will be interesting to compare this in silico analysis with future biochemical data regarding the direct effectors of cyclic nucleotide signalling in plants. It is interesting that no homologous proteins in the CNGC, shaker-type or type II acyl-CoA thioesterase families have been found which lack CNB domains. This implies that cyclic nucleotide binding is indispensable to their cellular role. Although it would have been interesting if this analysis revealed more novel classes of plant cyclic nucleotide binding proteins, the fact that (with the exception of CNTE) all cyclic nucleotide binding proteins had been previously identified indicates that the previously attained biochemical data agrees with our bioinformatic evidence. The identification of no transcription factors or protein signal transduction molecules with CNB domains implies that cyclic nucleotides may be unable to directly modify the proteome of plant cells. This is in stark contrast to bacterial, yeast and mammalian systems. The only common domain context of CNB domains in animals and plants is the CNGC channels, however, even these channels appear to have evolved independently [ 39 , 59 ]. Therefore it is clear that the roles of cyclic nucleotides in prokaryotic and eukaryotic, as well as plant and animal systems differ and that evolutionarily distant branches of life have evolved different mechanisms by which these molecules are utilized. It is worth pointing out that the ubiquitous presence of cyclic nucleotides in all forms of life may indicate that although the means by which this particular biochemical tool is used differ, it is still an indispensable component of biology's toolbox. Methods Bioinformatics In order to identify the proteins which contain CNB or GAF domains, we initially used the Simple Modular Architecture Research Tool (SMART at smart.embl.heidelberg.de; [ 60 - 62 ]) to scan all predicted Arabidopsis proteins for CNB and GAF domains in the EMBL, TIGR or NCBI databases. Once redundancies were removed, a list of proteins was generated [see additional file 2 ]. In order to ensure broad coverage of possible variants, we also examined the Interpro collection of protein sequence analysis algorithms, all of which use slightly different methods [ 63 ]. As an additional method, the predicted proteins of the Arabidopsis genome were searched using the BLAST algorithm [ 64 ]. As search bait, we used several known cyclic nucleotide binding domains including those from GAF domains (human PDE2A [Swiss-Prot: O00408] and Anabaena cyaB1 [Trembl: P94181]) as well as CNB domains (human CGK2 [Swiss-Prot: Q13237], human RIIβ [Swiss-Prot: P31323], human Epac2 [Swiss-Prot: Q8WZA2], human rod CNGC [Swiss-Prot: P29973] and E. coli CAP [pir: E86000]). This yielded no new inclusions to our list of proteins, but did confirm each of our previous entries. For examination of the Oryza sativa spp . Japonica genome we performed BLAST searches using the aforementioned baits, as well as each of the Arabidopsis proteins. This search was performed using the Blast utility of the TIGR rice database [ 40 ]. The criterion for inclusion was that the CNB or GAF domain had to match the consensus motif with an E-value of less than 0.5 over the entire domain as determined by SMART. For newly identified proteins from the Orzya sativa , we named them so that they agreed best with the nomenclature of Maser et al . [ 37 ] [see additional files 1 , 2 , 3 ]. Sequence alignments were performed using the ClustalX [ 65 ] or T-COFFEE algorithms [ 66 ]and then inspected visually. Neighbor-joining trees were generated by ClustalX or PHYLIP [ 67 ], then were visualized with TreeView [ 68 ]. Trees generated using a variety of analysis methods (parsimony, distance and maximum likelihood) yielded similar results to the neighbor-joining trees. Sequencing of atCNTE1 One protein, which appeared to contain only a cyclic nucleotide binding domain and no other motifs was found in the Arabidopsis database. We obtained the clone corresponding to this putative gene from the Arabidopsis Biological Resource Center and sequenced it. Sequencing was performed at the University of Calgary Core Sequencing Facility. We determined that the gene prediction algorithm which scanned the genome improperly predicted the intron/exon structure of this gene. The new gene, which we named cyclic nucleotide regulated thioesterase 1 (atCNTE1) was deposited in the NCBI database [GenBank: AY874170]. A subsequent BLAST search using this gene found another isoform of this gene in Arabidopsis (atCNTE2) and one isoform in Rice (osCNTE1) which we also included in our analysis. Theoretical model of atCNTE1 We generated a model of the atCNTE1 cyclic nucleotide binding domain (residues 28–117) based on an alignment of atCNTE1 with the CNB domains of RIIβ [pdb: 1CX4] [ 69 ], RIα [pdb: 1RGS] [ 70 ], CAP [pdb: 1CGP] [ 71 ], HCN2 [pdb: 1Q3E] [ 72 ] and Epac1 [pdb: 1O7F] [ 32 ]. This was submitted to the SWISS-MODEL server via the DeepView program [ 73 ]. The alignment was iteratively refined to allow for best agreement of sequence and structural similarity. Cyclic nucleotide dependent protein kinase assays Unless otherwise indicated all chemicals were purchased from Sigma-Aldrich. Assays were performed on extracts of Arabidopsis cells grown in suspension culture [ 74 ] or isolated male Wistar rat adipocytes from epididymal fat pads [ 75 ]. Both cell types were homogenized in 50 mM Tris pH 7.5, 5% (v/v) glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine and 0.1% (v/v) 2-mercaptoethanol. Adipocytes were lysed by 10 strokes of a dounce homogenizer while plant cells were lysed by two passages through a french press cell at 15 000 psi. The extracts were clarified by centrifugation for 15 min at 4000 RPM in a SS34 rotor at 4°C. These extracts were assayed for kinase activity using 32 P-γ-ATP (Amersham-Pharmacia), 30 μM Kemptide substrate, 50 mM HEPES pH 7.4, 1 mM dithiothreitol and 10 μM cyclic nucleotide as specified. Reactions were allowed to occur for 10 minutes at 30°C and assays were terminated by spotting onto squares of P81 paper followed by extensive washing with 75 mM phosphoric acid [ 76 ]. Assays were performed in duplicate on three separate preparations with error bars indicating the standard error between preparations (n = 3). Protein concentration was determined by the method of Bradford with bovine serum albumin (ICN Biomedicals) as a standard [ 77 ]. Western blotting of extracts Bovine heart PKA catalytic subunit and RII were purified to homogeneity [ 78 , 79 ]. Purified protein was injected into rabbits and serum was obtained according to standard methods [ 80 ]. Extracts of adipose and plant cells were prepared as described above. Samples were boiled into SDS-PAGE buffer and separated on a 10% denaturing gel [ 81 ]. The proteins were then transferred to nitrocellulose for 2 h at 100V and blocked overnight with 5% (w/v) skim milk powder. Blots were probed with antibodies for 1 h and visualized by enhanced chemiluminesence. The PKA catalytic subunit antibody was affinity purified according to [ 82 ] and used at 0.5 μg/mL while RII was used as crude immune serum at a 5000X dilution. For the RII western blots, both a polyclonal and a monoclonal antibody (anti-RIIβ BD Transduction Laboratories) gave identical results. List of abbreviations CAP catabolite activator protein cAMP 3'5'-cyclic adenosine monophosphate CGK2 cGMP dependent protein kinase 2 cGMP 3'5'-cyclic guanosine monophosphate cNMP 3'5'-cyclic nucleotide (cAMP or cGMP) CNB cyclic nucleotide binding CNGC cyclic nucleotide gated channel CNTE cyclic nucleotide dependent thioesterase Epac exchange protein directly activated by cAMP GAF domain found in c G MP-phosphodiesterases, a denylyl cyclases and F hlA GORK guard cell outward rectifying potassium channel PKA protein kinase A SKOR stellar potassium outward rectifying channel SMART simple modular architecture research tool Authors' contributions DB performed the bioinformatic analysis, modeling and biochemical studies and drafted the manuscript. MEF participated in the modeling. GBG conceived of the study and participated in its design and co-ordination. All authors have read and approved the final manuscript. Supplementary Material Additional File 5 Co-ordinate file of model generated for Figure 1b . Click here for file Additional File 1 Phylogenetic analysis of CNGC and shaker-type channels from Arabidopsis thaliana and Oryza sativa . Un-rooted neighbor-joining trees were constructed for (A) shaker-type and (B) CNGC channels using full-length sequences. For the shaker-type channels, numbers at nodes indicate number of trees out of 100 in which the node occurred. These are omitted in (B) for clarity. Scale indicates number of differences per residue. Trees were generated using ClustalX [ 65 ] and visualized with TreeView [ 68 ]. Click here for file Additional File 4 Relevant Sequence Alignments Protein sequence alignments upon which phylogenetic analyses in Figure 2 , and additional file 2 are based. Click here for file Additional File 2 Cyclic nucleotide binding proteins in Arabidopsis thaliana and Oryza sativa . Proteins containing CNB domains including species, name, aliases, accession number, other domains present (AR indicates ankyrin repeat, CaM indicates calmodulin binding domain and TE indicates type II acyl CoA thioesterase domain), sequence length, residues encompassing the CNB domain and E-value for the CNB domain as determined by SMART [ 62 ]. All accession numbers are from NCBI except osCNGC5b, osCNGC5c and osCNGC18 which were obtained from the MIPS Oryza sativa database . Click here for file Additional File 3 Protein sequences of all sequences analysed in this manuscript. Sequences are named according to details in the text and are in FASTA format. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545951.xml
526261	Personalized versus non-personalized computerized decision support system to increase therapeutic quality control of oral anticoagulant therapy: an alternating time series analysis	Background The quality control of oral anticoagulant therapy (OAT) during the initiation and maintenance treatment is generally poor. Physicians' ordering of OAT (especially fluindione and warfarin ) can be improved by dose adjustment algorithms, taking into account the results of International Normalized Ratio (INR). Reminders at the point of care, computerized or not, have been demonstrated to be effective in changing physicians prescription behavior. However, few studies have addressed the benefit of personalized reminders versus non personalized reminders, whereas the personalized reminders require more development to access patient record data and integrate with the computerized physician order entry system. The Hospital Information System of George Pompidou European Hospital integrates an electronic medical record, lab test and drugs order entry system. This system allows to evaluate such reminders and to consider their implementation for routine use as well as the continuous evaluation of their impact on medical practice quality indicators. The objective of this study is to evaluate the impact of two types of reminders on overtreatment by oral anticoagulant: a simple reminder of text formatted dose adjustment table and a personalized recommendation for oral anticoagulant dose and next date of INR control, adapted to patient data. Both types of reminders appear to the physician at the moment of drug ordering. Methods The study is an alternating time series experiment with three 6 months periods, each one including every 2 months according to a Latin square scheme: a control period without any reminder, a period with the simple non personalized reminder, a period with personalized reminder. All patients hospitalized in departments using the computerized physician order entry system and ordered fluindione or warfarin , will be included in the study between November 2004 and May 2006. Main outcome will be the proportion of overcoagulation, as expressed by the proportion of observation time with INR over 4.5, assuming INR change linearly. Secondary outcome is the incidence of major haemorrhagic events. Data will be collected thanks to Hospital Information Systems databases. Data will be analyzed taking into account patient and physician clustering effect.	Background Iatrogenic effects of oral anticoagulants therapy According to a study carried out by French pharmacovigilance centres, haemorrhage subsequent to oral anticoagulant treatment (OAT) is the most common drug-related side effect resulting in hospitalisation in public hospitals in France (13% of such admissions, and 0.41% of the 3,137 admissions analysed). On the basis of these findings, the AFSSAPS (French Agency for Health Product Safety) has made the prevention of iatrogenic effects related to OAT one of its priorities. Many of these events are consequences of interactions between different drugs, resulting in inappropriate doses [ 1 - 3 ]. Implementation of a system of support when prescriptions are made out is likely to improve prescription practices and to decrease the frequency of side effects. It should be possible to integrate a support tool into the drug prescription system, by using nomograms to adjust OAT doses. Decision-making tools and appropriate practice The efficiency of reminders issued at the time of prescription has been demonstrated by a various studies [ 4 - 8 ]. These reminders can come in a paper, telephone [ 9 ] or computer form. Hunt et al . [ 10 ] reviewed all studies (randomised and quasi-randomised) evaluating the effect of computer-based clinical decision support systems on medical practice and treatment outcome. They showed that these systems are effective for drug prescription purposes and for the implementation of various medical strategies [ 10 ]. We have demonstrated the efficiency of reminders in the French situation, both in the form of "paper" reminders [ 11 ] and in the form of computer-generated reminders [ 12 ]. Computer-generated reminders appear the most promising given the development of computer programs including prescription aids for hospital usage. Several types of reminders can be issued at the time of prescription [ 13 ]: • simple, general information concerning the recommendations that should be taken into account (non-specific or non-personalised reminders), • "check list": includes questions or a precise list of practices that the doctor must tick to show that it has been done, • reminders including clinical data concerning a specific patient that must be taken into account for a given procedure (personalised reminders). The advantage of personalised reminders over non-personalised reminders has not been demonstrated in the literature. However, the production of personalised reminders necessitates better integration of existing information and is thus more expensive to develop. It is important to determine whether this personalised tool results in a better quality of care than non-personalised tools. Several randomised clinical trials have tried to evaluate decision support systems for the prescription of OAT, but failed to draw any conclusions about their efficiency for several reasons: heterogeneity and complexity of the systems evaluated, experimental designs difficult to apply and not necessarily adapted, and too few patients included [ 14 - 17 ]. Reminders issued at the time of prescription have been shown to be effective by experimental studies, but the difficulties of maintaining the effectiveness of interventions designed to improve clinical practices remains a major problem. We evaluated the effect of an active decision support system for the prescription of low molecular weight heparin as prophylaxis for venal thrombosis in an orthopaedic surgery department. In this study, the system was and was not used during alternate periods. It showed that such programs affect practices without affecting learning [ 12 ]. Other authors have looked at the problem of the routine use of computerised systems [ 18 , 19 ]. The long-term use of a computer program to modify medical practices necessitates a hospital information system that ties in prescriptions and the results of medical tests, and the continuous collection of indicators making it possible to evaluate use and effect. Georges Pompidou European Hospital (GPEH) Organisation of the information system at GPEH The hospital information system currently collates prescriptions and results of biological tests and imaging procedures. Eight hundred computers, both laptops and fixed posts, are used to in care procedures (in care departments and medical offices). The Dx-Care ® program is at the centre of care delivery. It is used by doctors and nurses: • to prescribe laboratory examinations and imaging tests for a patient, • to visualise the results of laboratory tests, • to establish and to consult nursing schedules, • to archive a structured observation, • to prescribe drugs. Dx-Care ® is integrated with other applications to allow circulation of information between departments, laboratories and the pharmacy. Prescriptions for laboratory tests are transmitted to the Netlab ® program which manages such tests (this program is used by all biochemistry, haemostasis and immunology laboratories, etc.) The laboratories return the results using this same program, which retransmits them to Dx-Care ® . Furthermore, prescriptions of drugs are transmitted to the Phedra program, which is used by the pharmacy to manage prescriptions. The prescription is validated by the pharmacy and this validation is then transferred to Dx-Care ® . The lab test prescription facility has been available in the hospital information system since 2000 and is used by all departments of the hospital. The drug prescription facility has been implemented later on, since January 2003, and its use is still increasing (currently by half of the hospital departments). The hospital information system thus allows to install decision support systems that are activated whenever a prescription is issued and routinely to collect evaluation criteria of prescription practices. If possible and validated, the use of the hospital information system to evaluate care procedures will make it possible to collect data regularly, and routinely to assess methods for the improvement of care practices. Organisation of quality assurance and system for recording undesirable events With the aim of quality of care and preventing risks, the hospital is developing a system, based on the Intranet network, of declaration of undesirable events. This system must be able to record all undesirable events and incidents linked to the use of health products (drugs, medical equipment, blood products, etc.) and care (e.g. falls, lost files, bedsores, long waiting times) as well as those due to the patient environment (building, security, malevolence, etc.) and the job of health care professionals (accidental exposure to blood, chemicals, radiation, etc.). When a health care professional decides to report an undesirable event, he or she must complete a dedicated form available on the Intranet with all relevant information. This incident form includes an item entitled "complications associated with anti-coagulants". When the doctor clicks on this item, a form specific to haemorrhagic accidents following anti-coagulant treatment appears (see form in appendix). Prevention of thrombosis at GPEH Among departments which already started to use the computerised drug order entry system, several (cardiology and vascular medicine departments) are heavy "consumers" of anti-thrombosis drugs (see Table 1 ). In 2001, a working group representative of all the hospital departments described a group of procedures. This was done by critically reading articles published in the literature. The procedures concerned the preventive and curative indications of anticoagulants (OAT and heparins) in arterial thrombosis and venous thrombosis, and the way in which patients receiving anticoagulants are monitored and handled in cases of overdose. The procedure concerning curative OAT included a nomogram for adjusting doses of fluindione and warfarin based on previous doses and the INR (see Tables 2 and 3 ). These nomograms are currently available on the hospital's intranet, although they are not widely used. Their inclusion in a computerised prescription aid could increase their impact. Study aims Main aims 1. To evaluate the effect on the frequency of overanticoagulation of the implementation in the computerised physician order entry system of two types of tool to adjust OAT doses (one personalised and one non-personalised). 2. To assess any advantages of using the personalised tool rather than the non-personalised tool. Secondary aims 1. To evaluate the frequency of haemorrhagic accidents in the context of the study. 2. To evaluate the feasibility of long-term implementation of the intervention. Methods Experimental design The study is an alternate time series experiment which consists of three successive six-month periods (timed to change with the changing of medical residents even though they will not be the only prescribers). Each phase will consist of: • a two-month period without active support during which evaluation criteria will be collected (period A), • a two-month period with non-personalised active support (period B), • a two-month period with personalised active support (period C). To limit the impact of a learning effect on appropriate OAT management practice within the department over time (possible for medical residents), the order of these three periods was determined by using a Latin square plan (see Figure). This experimental design can be considered valid for an impact study in this context whereas a randomised controlled design is difficult to apply with just one hospital [ 20 ]. Participants Patients This study will include all the patients who are prescribed OAT for any indication and are hospitalised in clinical care departments where physicians are using the hospital information system to prescribe drugs. The following table shows the number of INR examinations prescribed by these departments in a three-month period. It provides an estimation of the approximate proportion of overdoses among the INR that exceeded 2, which is supposed to be found in patients treated with OAT in these units. Physicians All doctors authorised to prescribe drugs in the participating departments will be included in the study: residents and fellows, registered and non registered university hospital doctors. Each six-month period will coincide with an internship semester. Intervention To prescribe a drug using Dx-Care ® , the doctor selects the required drug from an exhaustive list. This opens up a dialogue box in which the doctor types the dose, the frequency of intake and the mode of administration. From this window, it is possible to add a text comment or to consult particular protocols that have been defined by the departments. It is planned to integrate two types of decision support systems into the computerised prescription program: 1) non-personalised active system: when the drug is selected a window automatically opens giving the prescribing the nomogram for the adjustment of OAT doses in the form of a table (see Tables 2 and 3 ). 2) personalised active system: when the drug is selected a window automatically opens suggesting a dose recommended according to the nomogram (taking into account the doses previously received by the patient and the patient's INR), together with a date for next INR control and an explication. Definition of endpoints Overanticoagulation Proportion of patient observation time with INR results > 4.5, assuming linear change of INRs. Major haemorrhagic accidents Intra-cranial haemorrhage or spontaneous haemorrhage necessitating surgery or a transfusion or decreasing haemoglobin concentration by more than 2 g/dl. Assessment of evaluation criteria OAT overdose The Netlab ® application allows biological laboratories to receive prescriptions and to return results. All of the INR results can be extracted from the Netlab ® database accompanied by information making it possible to identify the patient, the treatment and dose received, the prescribing doctor, the hospitalisation unit, the date the test was prescribed. Data about overdoses can therefore be collected systematically by regular database searches. Furthermore, the storage of the information in a computerised tool will make it possible to determine previous doses and INR results each time a drug is prescribed. Haemorrhagic accidents When a health care professional decides to declare an undesirable event, he or she fills in a specific, pre-formatted form available on the Intranet. This form includes a list of events that must be declared at the GPEH. The declaration form includes an item entitled "complication of haemorrhagic accidents". When the doctor clicks on this item, a specific form for the declaration of a haemorrhagic accident associated with anti-coagulant treatment appears (see form in appendix). Determination of sample size The determination of the number of participants necessary requires the definition of the statistical unit of interest, information about the incidence of the evaluation criteria in the study population and a hypothesis about the efficiency of the intervention. Statistical unit In this study, the main aims are to guide each prescription and to reduce the number of anti-coagulant overdoses: the simplest statistical unit to study is therefore the INR result. This unit will be used to calculate the sample size. This choice is not, however, perfect and the efficacy results will be presented using other indicators of the quality control of anti-coagulant treatments: Given the low incidence of major haemorrhagic accidents (not currently measured at the GPEH but probably below 1%), it is not possible in this study to estimate the number of subjects necessary to demonstrate an effect of intervention on the "haemorrhagic accident" endpoint. Recording haemorrhagic accidents will give the frequency of such accidents, which will then be used for realistic estimates of power and sample size if further studies are carried out. In previous studies evaluating the efficacy of tools to aid the prescription of OAT, the unit considered was not always the same, taking into account the number of INR per patient and the time between INR measurements to greater or lesser extents. The most recent studies considered the number of patient-days according to the method described by Rosendaal [ 15 , 16 , 21 ]. This method can also be used to calculate the rate of haemorrhagic events as a function of the number of patient-days for a given range of INR values. We may also carry out an analysis for each prescribing doctor given that the intervention targets doctors directly. This will involve adjusting the effect of the intervention to the fact that intra-physician variability is a priori lower than inter-physician variability. Number of INR measurements and predicted frequency of overdoses During a six-month period (January to June 2004), 4 920 INRs were requested by the six departments which already routinely use the computerized physician order entry system. The frequency of overtreatment can approximately be estimated from the percentage of INR > 4.5 among INR >2. Among the 2620 INR > 2, 330 (12%) were higher than 4.5 (see Table 4 ). This frequency has been stable during these six months but differed considerably between departments (10% to 23%). Hypothesis about the efficacy of the intervention The number of INR tests necessary for a six-month period, with an α risk of 5% and a power of 80%, for the comparison of two percentages by classical methods (untreated group half the size of the treated group), for a basal incidence of the judgement criterion of 12% are and for the following hypotheses on relative reduction of the risk (RRR) of overdose, are: • RRR 30%: 2500 • RRR 40%: 1300 • RRR 50%: 800 • RRR 60%: 500 Carrying out approximately 5000 tests over six months will make it possible to detect an intervention effect of less than 30% in this period. The experimental design includes three six-month periods and should thus ensure adequate power. Statistical analyses Statistical analyses will be performed with the STATA statistical software (release 8, STATA corp, College station, Tex, USA) Standard statistical tests will be used to compare the baseline characteristics of the departments and patients. The main analysis concerns the effect of the intervention on the number of dangerously high INRs. The analysis will be carried out using a mixed effect analysis of variance model, in which the effect linked to the period will be considered fixed and that linked to the prescription tool will be considered random [ 22 ]. Rosendaal's method will be used to analyse the number of patient-days with INR over the target [ 21 ]. Regulatory aspects According to French policy, this study was exempt from medical ethics committee approval. The anti-coagulants being evaluated are prescribed as recommended by clinical studies validated within the GPEH. These recommendations are available on the hospital's Intranet and are thus accessible to all doctors. They conform to standard practices. Neither the patients nor the doctors will be randomised. The interventions are simply different means of giving valid information to physicians. Using funding from the PHRC (Hospital Clinical Research Program), we carried out two research studies related to this project. In the first (PHRC 95), an intervention aimed at modifying the way in which emergency department doctors handle ankle injuries, the study design was a randomised controlled study and the randomisation unit was the hospital [ 11 ]. In the second (PHRC 98), a computer-based decision-support system for the prevention of venous thromboses in orthopaedics, the experimental design was identical to that of our present study [ 12 ]. In both cases, the Ile de France branch of the Clinical Research Delegation considered that the project was exempt from medical ethics committee approval. List of abbreviations OAT: Oral Anticoagulant Therapy INR: International Normalized Ratio GPEH: Georges Pompidou European Hospital PHRC: Hospital Clinical Research Program Authors' contributions IC and PD, conceived, wrote the protocol and prepared the manuscript. GC is the statistical expert and performed the power calculations GC and ABR revised the protocol and the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC526261.xml
545936	Cough quality in children: a comparison of subjective vs. bronchoscopic findings	Background Cough is the most common symptom presenting to doctors. The quality of cough (productive or wet vs dry) is used clinically as well as in epidemiology and clinical research. There is however no data on the validity of cough quality descriptors. The study aims were to compare (1) cough quality (wet/dry and brassy/non-brassy) to bronchoscopic findings of secretions and tracheomalacia respectively and, (2) parent's vs clinician's evaluation of the cough quality (wet/dry). Methods Cough quality of children (without a known underlying respiratory disease) undergoing elective bronchoscopy was independently evaluated by clinicians and parents. A 'blinded' clinician scored the secretions seen at bronchoscopy on pre-determined criteria and graded (1 to 6). Kappa (K) statistics was used for agreement, and inter-rater and intra-rater agreement examined on digitally recorded cough. A receiver operating characteristic (ROC) curve was used to determine if cough quality related to amount of airway secretions present at bronchoscopy. Results Median age of the 106 children (62 boys, 44 girls) enrolled was 2.6 years (IQR 5.7). Parent's assessment of cough quality (wet/dry) agreed with clinicians' (K = 0.75, 95%CI 0.58–0.93). When compared to bronchoscopy (bronchoscopic secretion grade 4), clinicians' cough assessment had the highest sensitivity (0.75) and specificity (0.79) and were marginally better than parent(s). The area under the ROC curve was 0.85 (95%CI 0.77–0.92). Intra-observer (K = 1.0) and inter-clinician agreement for wet/dry cough (K = 0.88, 95%CI 0.82–0.94) was very good. Weighted K for inter-rater agreement for bronchoscopic secretion grades was 0.95 (95%CI 0.87–1). Sensitivity and specificity for brassy cough (for tracheomalacia) were 0.57 and 0.81 respectively. K for both intra and inter-observer clinician agreement for brassy cough was 0.79 (95%CI 0.73–0.86). Conclusions Dry and wet cough in children, as determined by clinicians and parents has good clinical validity. Clinicians should however be cognisant that children with dry cough may have minimal to mild airway secretions. Brassy cough determined by respiratory physicians is highly specific for tracheomalacia.	Background Cough is the most common symptom presenting to medical practitioners in Australia, the UK and USA [ 1 - 3 ]. Cough quality, specifically dry versus we t[ 4 ] or productive cough, is often used in epidemiological [ 5 - 7 ] and clinical research [ 8 , 9 ]. Clinically, physicians also often differentiate between dry and wet cough [ 10 - 12 ] but there are no studies that have evaluated if these are reproducible descriptors. In adults, productive cough is usually obvious but children however often swallow their sputum and hence a 'wet cough' is used inter-changeably with 'productive cough' to describe cough quality in young children who are unable to expectorate [ 10 , 13 ]. It is known that nocturnal cough is unreliably reported in both children [ 14 ] and adults [ 15 ] but there is no data on cough quality. Wet and dry cough are determined subjectively as there are no 'gold standards'. To date there are no human studies that have identified the objective relationship of the cough descriptors to mucus secretory states. The sound of a cough is due to vibration of larger airways and laryngeal structures during turbulent flow in expiration [ 16 , 17 ]. It is not known which generation of the airways is involved when the human ear identifies a wet cough and currently there are no validated human models that allow measurement of increased airway mucus. Mucus hypersecretory states in human diseases can occur from a variety of mechanisms which include; hypersecretion of stored mucin, hypertrophy or hyperplasia of goblet cells and/or increased synthesis from over-expression of mucin genes [ 18 ]. In disease states, it is not known which mechanism or site of production is the most important but in smokers with chronic bronchitis, a common cause of productive cough in adults, the larger bronchi (bronchi of diameter >4 mm ie segmental bronchi and above) [ 19 ] are the site of greatest inflammation [ 18 ]. Flexible bronchoscopy allows an in-vivo visual assessment of larger airways usually to the 3 rd (lobar bronchi) or 4 th generation (segmental bronchi) in young children. The study aims were to compare (1) cough quality (wet vs dry and brassy vs non-brassy) with bronchoscopic findings of secretions and tracheomalacia respectively and, (2) parent(s) vs clinician's evaluation of the cough quality (wet and dry). We hypothesised that clinical assessment of cough is good compared to bronchoscopic findings and that a wet cough is related to presence of airway secretions. Methods Children electively admitted for bronchoscopy without a known underlying respiratory diagnosis were seen by a member of the research team 0.5–3 hours prior to bronchoscopy. The clinician's assessment of cough quality (wet or dry) was recorded on a standardised sheet (based on the cough present on the day of the bronchoscopy), before the parent(s) independently evaluated the current (the morning of, or last 12 hours) cough quality (wet or dry) of their child. For clinician's assessment of wet/dry cough, when no spontaneous cough was heard or if child was too young to elicit a cough, cough quality (wet or dry) was deemed 'non-assessable'. Clinicians also rated cough as 'brassy' or 'non-brassy' based on coughs heard anytime before bronchoscopy. For assessment of reliability of cough quality (wet/dry and brassy/non-brassy), 21 cooperative children had their coughs digitally recorded (Acer Pocket PC n11, Taiwan) using music compact disc quality format (44.1 kHz, 16 bit) on the morning of their bronchoscopy. These stored cough sounds were later replayed (using headphones 30–10,000 Hz, Lanier, Japan) from a computer and re-scored in a blinded manner (blinded to the child's name and cough quality assigned earlier) for wet/dry and brassy/non-brassy qualities. Written consent was obtained from a parent and the study approved by the hospital's ethics committee on human research. Bronchoscopy and quantification of secretions seen during bronchoscopy Flexible bronchoscopy was performed under general anaesthesia as previously described [ 20 - 22 ]. Briefly, anaesthesia was induced with sevoflurane in 100% oxygen administered through a Jackson Rees T piece circuit, the vocal cords and upper trachea then sprayed (4 mg/kg lignocaine via a Cass needle). Atropine was given intravenously to most children aged <12 months. In all children a video flexible bronchoscope (BF 3C160, Olympus, Tokyo, Japan) entered the circuit via the port of a swivel right angle connector attached to a facemask. Images were projected onto a monitor (Sony Trinitron, Tokyo, Japan). A respiratory consultant (ABC or IBM) blinded to the child's history and cough quality scored the bronchoscopy sheet quantifying the amount of secretions at the time of the bronchoscopy in real time. When no scorer was available, the session was videotaped and played back. A secretion quantification card (figure 1 ) was visible to the scorer at all times. Secretions were quantified according to amount of mucus in the airways in relation to lumen size (fig 1 ) and scored from the trachea to the level of lobar bronchi (total of 9; trachea, right main stem, right upper lobe, right middle lobe, right lower lobe, left main stem, left upper lobe, left lingula, left lower lobe). When segmental bronchi were seen, the worst segment (ie segment with most secretions) was scored. These scores were used to obtain a final grade of bronchoscopic secretions (BS) from grades 1 to 6; Figure 1 Bronchoscopic secretion quantification card. BS Grade 1 = Nil secretions BS Grade 2 = Near dry = Bubbles only in < half total number of bronchi involved BS Grade 3 = Minimal = Bubbles found in > half total number of bronchi involved or Secretion type-I in < half total number of bronchi involved BS Grade 4 = Mild = Secretion type-I, > half total number of bronchi involved or Secretion type-II, < half total number of bronchi involved BS Grade 5 = Mod = Secretion type-II, > half total number of bronchi involved or Secretion type-III, < half total number of bronchi involved BS Grade 6 = Large = Secretion type-III, > half total number of bronchi involved Inter-rater reliability of BS grading was assessed by replaying the videotapes of the recorded bronchoscopy of 20 children, with the 2 nd assessor blinded to the child's condition. BAL was obtained from the macroscopically most abnormal lobe; when changes were generalised, BAL was obtained from the right middle lobe. Cell count was performed on the cell suspension, cytocentrifuge slides were prepared and stained (modified Wright's stain) for cell differential profile. All cellular examinations were performed by cytologists blinded to the children's medical history. Statistics Data were not normally distributed and thus non parametric analyses were used; medians and inter-quartile range (IQR) were used for all descriptive data and Kruskal Wallis for comparisons between groups. Cohen's kappa (K) with 95%CI was utilised for inter and intra-observer reliability and graded from 'poor' (K<0.2) to 'very good' (K = 0.81–1.0)[ 23 ]. For calculation of sensitivity and specificity, negative and positive predictive values (NPV, PPV); cough quality was assigned to dry when a history of cough was absent and bronchoscopy findings at two cut offs (grades 3 and 4) of BS grades were taken as the 'gold standard' eg for cut-off at BS grade 3, BS grades 1–2 were defined as no secretions and BS grades = 3 defined as secretions present. To determine if cough quality (wet/dry) was predictive of amount of secretions found during bronchoscopy, a receiver operating characteristic (ROC) curve was generated [ 24 ] where cough quality wet/dry was considered the true positive/negative and the bronchoscopic secretion scoring (1 to 6) as the ordinal rating scale. Two tailed p value of < 0.05 was considered significant. SPSS ver 11.1 was utilised for most statistical calculation. Results Median age of the 106 children (62 boys, 44 girls) enrolled was 2.6 years (IQR 5.7). Indications for bronchoscopy were chronic cough (n = 44, 41.5%), wheeze (n = 21, 19.8%), stridor (n = 16, 15.4%), investigation of persistent radiological changes (n = 14, 13.5%), recurrent pneumonia (n = 6, 5.8%), suspicion of aspiration lung disease (n = 3, 2.9%), BAL and suspected foreign body (n = 1 each, 2%). In four children, BS grades were not obtained (session was inadvertently not recorded and 'blinded' clinician not present at bronchoscopy). Scores of BS were done in real time in all but 9 children. In 30 children, cough was non-assessable. Agreement between clinicians and parents assessment of cough quality (wet/dry) was good (K = 0.75, 95%CI 0.58, 0.93). For cough quality of 'wet/dry', cough assessed by clinicians had the highest specificity, sensitivity, NPV, PPV and positive likelihood ratio for both BS cut-offs (tables 1 and 2 ). Parent(s) assessment were less precise but only marginally so. The area under the fitted ROC curve (figure 2 ) was 0.85 95%CI 0.77, 0.92. The specificity, NPV and likelihood ratio for brassy cough assessed against gold standard bronchoscopic finding of tracheomalacia was good (table 1 ) but less than that for cough quality of wet/dry. Table 1 Assessment of cough quality vs bronchoscopic findings with BS cut off at grade 3* Assessment type (clinical vs bronchoscopic findings) Sensitivity Specificity NPV PPV Positive LR Clinician 1.00 0.55 1 0.64 2.21 Cough quality (wet/dry) assessed by clinician (n = 96) Parent(s) 0.95 0.54 0.93 0.61 2.06 Cough quality (wet/dry) assessed by parents (n = 92) Combined (n = 100) 0.98 0.54 0.97 0.62 2.10 Tracheomalacia (n = 81)# 0.57 0.81 0.84 0.52 3.12 Cough quality (wet/dry) assessed by clinicians combined with parents. When cough was non-assessable by clinician and child has current cough, parental assessment of the cough (wet or dry) was taken. If child has no history of current cough, cough was assigned 'dry'. LR = likelihood ratio. Specificity, sensitivity of dry and wet cough was assessed against bronchoscopic findings as the gold standard where BS grades ≥ 3 were considered abnormal (secretions present) and ≤ 2 considered normal (no secretions). #That for tracheomalacia was assessed using clinicians record of presence/absence of brassy cough with bronchoscopic findings of tracheomalacia.[21] Table 2 Assessment of cough quality vs bronchoscopic findings with BS cut off at grade 4 Assessment type (clinical vs bronchoscopic findings) Sensitivity Specificity NPV PPV Positive LR Clinician 0.79 0.75 0.82 0.72 3.22 Cough quality (wet or dry) assessed by clinician (n = 96) Parent(s) 0.78 0.71 0.80 0.67 2.69 Cough quality (wet or dry) assessed by parents (n = 92) Combined * (n = 100) 0.77 0.73 0.80 0.69 2.88 Cough quality (wet/dry) assessed by clinicians combined with parents. When cough was non-assessable by clinician and child has current cough, parental assessment of the cough (wet or dry) was taken. If child has no history of current cough, cough was assigned 'dry'. LR = likelihood ratio. Specificity, sensitivity of dry and wet cough was assessed against bronchoscopic findings as the gold standard where BS grades ≥ 4 were considered abnormal (secretions present) and ≤ 3 considered normal (no secretions). Figure 2 ROC curve with 95%CI relating cough quality (wet/dry) to bronchoscopic secretion (BS) grades from 1–6. There was little difference in sensitivity and specificity between children grouped by indication for bronchoscopy (cough or other indications). Values were marginally better in older children (tables 4 and 5 in supplementary data additional file 2 ). Area under the fitted ROC curve was similar for both age groups (aged ≤ 2 years = 0.811, 95%CI 0.79, 0.84; age >2 = 0.84, 95%CI 0.74, 0.95). Agreement for clinicians vs parents cough quality (dry/wet) was better in children aged ≤ 2 years (K = 0.85, 95%CI 0.57, 1.0; n = 42 but 18 non-assessable) than that for those age >2 years (K = 0.70, 95%CI 0.49, 0.92; n = 64, but 12 non-assessable) (see additional file 1 ). Using recorded coughs, kappa scores were 'very good' for both intra-observer and inter-clinician agreement for wet and dry cough (K = 1.0 and 0.88 [95%CI 0.82–0.94] respectively). There was only one disagreement in wet and dry cough between clinicians and in this child the cough was mildly wet (BS grade of 3). Kappa scores for intra-observer and inter-observer clinician agreement for brassy cough was good, K in both was 0.79, 95%CI 0.73, 0.86. Inter-rater agreement for BS grades was 'very good' (weighted K = 0.95, 95%CI 0.87–1). Cellularity for total cell count, percentages of neutrophils and macrophages were significantly different between children grouped by BS grade cut-offs of 3 and 4 as well as wet/dry cough (table 3 ). Table 3 Cellular differential profile in BALs Median TCC (IQR) % M IQR) % N (IQR) % Lym (IQR) % Eos (IQR) BS cutoff at grade 3 ≤2 (n = 31) 195 (290) 82.0 (15.8) 5.0 (7) 13.5 (15.8) 0 (0) ≥3 (n = 70) 334.0 (425) 66.0 (45) 12.0 (38) 11.0 (16.0) 0 (0) p value^ 0.038 0.001 0.006 0.605 0.758 BS cutoff at grade 4 ≤3 (n = 52) 176 (257) 81.0 (17.0) 6.0 (8.0) 13.0 (16.0) 0 (0) ≥4 (n = 49) 368 (574) 51.5 (59.8) 20.0 (47.0) 11.0 (15.0) 0 (5) p value^ 0.0001 0.0001 0.0001 0.445 0.613 Cough quality Wet (n = 45) 365 (522) 51.5 (49.8) 25.0 (43) 13.0 (16) 0 0 Dry (n = 25) 176 (315) 80.5 (24.8)) 5.5 (13.0) 1.8 (16.0) 0 (0) No history (n = 28) 80 (310) 15 (16.5) 1 (7.5) 1 (11.5) 0 (0) 310 16.5 7.5 11.5 0 p value^ 0.017 0.0001 0.001 0.242 0.769 ^p value = examined using Kruskal Wallis test. *assessed by clinician TCC = total cell count; N = neutrophils, M = macrophages, L = lymphocytes, Eos = eosinophils, Discussion We have shown that clinical assessment of cough quality of wet/dry cough generally relates to bronchoscopic secretions determined using a standardised scoring system (BS grades). When cough is wet, secretions were always present; when cough was dry secretions if present, were usually minimal or mild. Clinicians were marginally better than parents at assessing wet/dry cough and agreement between the 2 groups was good. When clinicians detected presence of a brassy cough, tracheomalacia was usually present. Inter-rater clinician agreement for cough qualities of dry/wet and brassy/non brassy was good. Accuracy and reliability of symptoms are important in clinical and research settings. Cane and colleagues [ 25 , 26 ]. found that parental reports of wheeze and stridor are often not accurately reported in a clinic setting. There is no data on the validity of cough quality in spite its use in management and diagnostic guidelines [ 11 , 27 , 28 ] and cough being the most common symptom seen by general practitioners [ 1 - 3 ]. The level of agreement recommended for symptoms and signs to be used in clinical prediction rules is kappa value of ≥ 0.6 [ 29 ]. The kappa values we obtained in this study well exceeded 0.6. Specifically, intra and inter-clinician evaluation was very good and parental reporting of cough quality (wet/dry) also related well to clinicians' evaluation. When compared to bronchoscopic findings, this study showed that a wet cough is always associated with BS grades of 3 or more. Dry cough is less valid; the presence of dry cough does not necessary indicate absence of secretions. However BS grades are less in dry cough as shown in the ROC curve. The generation of cough sounds and some factors that influence cough sounds have been examined in the laboratory [ 16 , 30 ]. Using cough sound analysis (spectrogram and time-expanded waveform), productive and non-productive cough can be differentiated in the laboratory [ 30 ]. However to date there is no data on its clinical reliability and its relationship to quantification of airway secretions. In humans, it is not known how much mucus is required and where it has to be located for the human ear to detect presence of a moist cough. It is likely that mucus in the large airways is required for detectable difference in cough quality as the sound of cough is generated from vibration of larger airways and laryngeal structures during turbulent flow in expiration [ 16 , 17 ]. Laminar airflow, which occurs in smaller airways, is inaudible [ 31 ]. In an animal model, Korpas and colleagues showed that a certain amount of mucus is required to alter cough sound; 0.5 ml of mucus instilled into the trachea of cats altered cough sound, too little mucin had no effect on cough quality whilst too much mucin impaired breathing [ 32 ]. Our study findings support this and it is not surprising that when the cough is dry, BS grades were less. The rheological properties of airway mucus also influence cough sound [ 17 ]. It is not known how airway secretions in the more peripheral airways influences the sound of cough. One possible limiting factor of our study is the choice of cut offs for BS grades in determining presence or absence of significant secretions. We chose to use a cut off of 3 as a minor amount of bubbles in the airways can be present from trickling of lignocaine into the airways or spillage from the upper airways. BS cut-off at grade 4 resulted in improved specificity but decreased sensitivity. Children grouped by both BS cut-offs (3 and 4) had significantly different airway cellular profile. The clinical significance of minimal BS grades and appropriate cut-offs can only be determined in a prospective follow-up study which is not an aim of this study. This study did determine that our BS scoring method was easy to use (most done in real time) and had very good inter-rater agreement. The clinical outcomes of wet and dry cough were not the aims of this study and thus cannot be determined here. To relate clinical outcomes to cough descriptors would ideally require a randomised controlled trial with dry and wet cough as entry criteria. A follow-up cohort study with strict clinical diagnostic categories would be useful and we have shown in a preliminary study that dry cough was significantly more likely to naturally resolve than wet cough [ 33 ]. In addition to the limitation of quantifying airway secretions using a bronchoscopic method, this study is also limited by a number of factors. Firstly, clinical repeatability or agreement of cough sounds was assessed by doctors in a tertiary setting. Whether or not these findings can be extrapolated to the secondary and primary setting can only be speculated. Hay and colleagues showed that inter-observer agreement for clinical signs of fever, tachypnoea and chest signs were poor to fair (kappa of 0.12–0.39) in the primary care setting but these signs are known to have good agreement in secondary care settings [ 34 ]. However as parents were almost as good as clinicians in our study and are 'untrained' compared to medical practitioners, we would expect that this data can be extrapolated to most primary and secondary settings. Secondly, anaesthesia and atropine could possibly influence mucus quantity and properties. However this influence, if any, is likely to be small as both bronchoscopists (ABC, IBM) are experienced (our recorded average total theatre time is relatively short at 22 mins) [ 22 ], and atropine is given just immediately prior to commencement of bronchoscopy. Determining the validity of cough quality in children is important not only because of the commonality of the clinical problem of cough but also its use in guidelines and research studies [ 11 , 27 , 28 ]. A particularly important finding is the presence of small amounts of secretions in children with dry cough which may have implications in the management of suppurative lung disease; a dry cough may represent early disease process where only a small amount of mucous is present. Conclusion We conclude that the description of a cough as wet or dry cough as determined by clinicians and parents has good clinical validity as it has good agreement with, and relates to, quantification of airway secretions. However as minimal amount of secretions may be present in children with dry cough, clinicians should be cognisant that a dry cough may eventually become wet if airway secretions increase. Thus it should not be assumed that airway secretions are absent in children with chronic dry cough and cough quality in these children should be reviewed. We also conclude that the brassy cough determined by respiratory physicians is highly specific for presence of tracheomalacia. List of Abbreviations BAL Bronchoalveolar lavage BS Bronchoscopic secretion K Kappa NPV Negative predictive value PPV Positive predictive value ROC receiver operating characteristic Authors' contributions AC conceived the idea, designed the study, performed the data analysis and drafted the manuscript. JG participated in data acquisition and coordination of project. ME participated in electronic acquisition of data and software for sound recordings. JF and NC designed the microbiology and cytological components respectively and both helped draft the manuscript. IBM helped in formulation of overall study design, data acquisition and drafting of the manuscript. All authors read and approved the manuscript. Supplementary Material Additional File 1 Figure 3: ROC curve ROC curve with 95%CI relating cough quality (wet/dry) to bronchoscopic secretion (BS) grades from 1–6 in children grouped according into age (a) ≤ 2 years and (b) > 2 years. Click here for file Additional File 2 Table 4: Assessment of cough quality vs bronchoscopic findings in children grouped by indication for bronchoscopy 4a: Assessment of cough quality vs bronchoscopic findings in children whose indication for bronchoscopy was cough 4b: Assessment of cough quality vs bronchoscopic findings in children whose indication for bronchoscopy was others (ie not cough) Table 5: Assessment of cough quality vs bronchoscopic findings in children grouped by age 5a: Assessment of cough quality vs bronchoscopic findings in children aged ≤ 2 years 5b: Assessment of cough quality vs bronchoscopic findings in children aged > 2 years Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545936.xml
509282	Increased contractile responses to 5-hydroxytryptamine and Angiotensin II in high fat diet fed rat thoracic aorta	Background Feeding normal rats with high dietary levels of saturated fat leads to pathological conditions, which are quite similar to syndrome X in humans. These conditions such as hypertriglyceridemia, hypercholesterolemia, obesity, and hyperglycemia might induce hypertension through various mechanisms. Metabolic syndrome and the resulting NIDDM represent a major clinical challenge because implementation of treatment strategies is difficult. Vascular abnormalities probably contribute to the etiology of many diabetic complications including nephropathy, neuropathy, retinopathy, and cardiomyopathy. It has been shown that in Streptozotocin induced diabetic animals there is an increase in maximal responses to 5-Hydroxytryptamine and Angiotensin II. The purpose of this study was to evaluate High fat diet fed rats for the development of hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and hyperglycemia and to assess their vascular responses to 5-Hydroxytryptamine and Angiotensin II. Methods Male Sprague Dawley rats were used for this study and were divided into two equal groups. One of the groups was fed with normal pellet diet and they served as the control group, whereas the other group was on a high fat diet for 4 weeks. Body weight, plasma triglycerides, plasma cholesterol, and plasma glucose were measured every week. Intraperitoneal glucose tolerance test was performed after 4 weeks of feeding. At the end of fourth week of high fat diet feeding, thoracic aortae were removed, and cut into helical strips for vascular reactivity studies. Dose-response curves of 5-Hydroxytryptamine and Angiotensin II were obtained. Results There was no significant difference in pD 2 , with 5-Hydroxytryptamine and Angiotensin II in both groups but E max was increased. Conclusions These results suggest that hypertension in high fat diet rats is associated with increased in vitro vascular reactivity to 5-HT and Ang II.	Background Syndrome X comprises a plethora of conditions such as obesity, dyslipidemia, impaired glucose tolerance, insulin resistance and hypertension [ 1 ]. It places stress on multiple organ systems and plays a significant role in the development of other related cardiovascular disorders. Western style diet, which contains high levels of fat, is also considered to be one of the main factors in the development of obesity and insulin resistance. The most common reason for the development of hyperinsulinemia (decreased hepatic insulin clearance and/or increased insulin secretion) from insulin resistance is obesity. Excess fat deposits in the white adipose tissue affects insulin mediated glucose metabolism in non adipose tissues, causes disordered insulin response and increases lipid deposition [ 2 ]. However, pathophysiologies of vascular complications in syndrome X have not been fully understood. 5-hydroxytryptamine (5-HT) is shown to be related to pathogenesis of vasculopathy in diabetes. Various studies in humans and rabbits have reported increased plasma 5-HT levels and enhanced contraction to 5-HT in diabetes mellitus [ 3 - 5 ]. Another vasopressor peptide, Angiotensin II (Ang II) is involved in cardiovascular complications of diabetes mellitus. Many have reported that the vascular Renin angiotensin system is one of the key systems in the etiology of vascular alterations in early stages of diabetes. It is already established that 5-HT and Ang II responses are altered in the aortic rings of streptozotocin (STZ) induced diabetic rats [ 6 , 7 ] and high fructose diet fed rats [ 8 ]. However, very little information is available in the literature regarding the vascular contractile responses to vasopressor agents such as 5-HT and Ang II in HFD fed rats. The aim of this study was to elucidate the contractile responses to 5-HT and Ang II in HFD fed rat thoracic aorta which will provide an avenue for further exploratory studies. We have selected HFD fed rat model for our study because it is a useful model of the putative effects of excess fat intake in humans and it represents the major sub type of diabetes mellitus, non insulin dependent diabetes mellitus (NIDDM). Results Biochemical measurements HFD fed rats showed significant increase in body weight as compared to NPD fed rats (Table 2 ). In addition, the rats fed HFD showed significant elevation in the basal plasma glucose, triglyceride, total cholesterol and insulin levels at the end of four weeks of dietary manipulation as compared to NPD fed control groups (Table 2 ). The HFD fed rats exhibited significant elevation in basal fasting glucose and showed significant impairment in glucose tolerance to exogenously administered glucose (Fig 1 ). Estimation of AUC values indicated 28.4 % increase in plasma glucose of HFD fed when compared to NPD fed rats. Table 2 Various parameters of NPD and HFD fed rats Parameter NPD fed HFD fed Body weight (gm) 259.2 ± 5.3 315.0 ± 6.5* plasma glucose (mg/dl) 87.5 ± 2.5 111.0 ± 0.9* Plasma triglycerides (mg/dl) 41.3 ± 1.7 89.7 ± 7.2* Plasma cholesterol (mg/dl) 48.9 ± 3.7 87.5 ± 2.6* Plasma Insulin (ng/ml) 1.90 ± 0.15 3.05 ± 0.21* pD 2 of 5-HT 5.74 ± 0.09 6.00 ± 0.06 E max of 5-HT 30.0 ± 1.8 49.0 ± 2.0** pD 2 of Ang II 7.69 ± 0.08 7.31 ± 0.18 E max of Ang II 25.0 ± 3.4 34.0 ± 3.0* Each point is represented as mean ± SEM p < 0.05, p < 0.01 and *p < 0.001 Vs NPD fed group Figure 1 Effect of HFD on intra peritoneal glucose tolerance test (IPGTT) in rats, as compared to NPD fed group. All values are expressed as mean ± SEM (n = 8) p < 0.05, p < 0.01, p < 0.001 Vs NPD fed group Vascular Responses Cumulative concentration response curves of 5-HT and Ang II for both HFD fed rat thoracic aortae showed an increase in E max (Fig 2 and 3 ) with out any change in pD 2 values when compared to NPD fed rats (Table 2 ). There was no significant change in both E max and pD 2 values with KCl in both groups (data not shown). The order of potency of agonists in both groups was Ang II>5-HT>KCl. Figure 2 Cumulative concentration response curve to 5-HT in helically cut aortic strip preparations obtained from NPD fed and HFD fed rats. Each point is represented as mean ± SEM (n = 5) p < 0.05, *p < 0.01 Vs NPD fed group Figure 3 Cumulative concentration response curve to Ang II in helically cut aortic strip preparations obtained from NPD fed and HFD fed rats. Each point is represented as mean ± SEM (n = 5) p < 0.05, **p < 0.01 Vs NPD fed group Discussion Obesity is a major risk factor for several metabolic diseases, frequently clustering to form the metabolic syndrome or syndrome X [ 9 ]. Obese people have increased incidence of NIDDM with high percentage of mortality and morbidity [ 10 ]. Western style diet, which is abundant with calorically dense and saturated fatty foods, is considered to be the main factor in the development of obesity and insulin resistance. Our studies have shown that HFD causes increase in bodyweight when compared to NPD after four weeks of dietary manipulation in rats. Hyperglycemia is observed in insulin resistance where glucose utilization is reduced. We saw significant elevations in blood glucose levels. Intraperitoneal glucose tolerance tests confirm severe glucose intolerance. Oversupply of dietary lipids causes insulin resistance in rats [ 11 ]. Randle glucose fatty acid cycle suggested that the body prefers excess lipid stores to glucose for metabolic oxidation in insulin resistance [ 12 ]. Our HFD model also exhibited high plasma triglyceride levels. Hence studies on our experimental model in compliance with Randle et al findings suggested that HFD feeding causes insulin resistance [ 13 ]. Insulin resistance with compensatory hyperinsulinemia is a prominent feature of metabolic syndrome. The most common reason for the development of hyperinsulinemia in insulin resistance is obesity. It stands as one of the major cardiovascular risk factors in patients with obesity. The present study on HFD rats demonstrated higher plasma insulin levels than control values. This marked hyperinsulinemia could be due to a combination of increased β-cell mass and decreased insulin clearance, as well as failure of insulin to suppress hepatic gluconeogenesis [ 14 ]. Elevated cholesterol is also observed in insulin resistant individuals. For this reason we measured plasma cholesterol levels which were found to be more than normal values. Previous studies have reported a down regulation of LDL receptors and associated decrease in LDL clearance, increased total cholesterol levels [ 15 ]. According to National Cholesterol Education Program's Adult Treatment Panel III (Third report) easily measured clinical findings for syndrome X includes increased abdominal circumference, elevated triglycerides, low high-density lipoprotein-cholesterol, and elevated fasting blood glucose and/or elevated blood pressure. Three of these five are required for diagnosis. Our study demonstrated three of the clinical parameters indicating conditions of syndrome X in HFD fed rats [ 16 ]. Insulin resistance along with other conditions of syndrome X might induce hypertension by a host of mechanisms involving insulin itself, increased sodium reabsorption and/or enhanced intra cellular concentration of free calcium in vascular smooth muscle [ 17 ]. Although the etiology of vascular disorders in metabolic syndrome has not completely been revealed, it is suggested that alterations in the reactivity of blood vessels to neurotransmitters and circulating hormones are responsible for the functional abnormalities of blood vessels. In order to elaborate the pathways that connect syndrome X to hypertension we have studied the contractile responses to 5-HT and Ang II in both HFD and NPD fed rat thoracic aortae. Previously we have demonstrated increased contractile responses with synthetic alpha adrenoceptor agonist, phenylephrine, in HFD fed rat thoracic aorta [ 18 ]. This enabled us to explore the role of these endogenous mediators in the same animal model. The present vascular studies demonstrated that the magnitude of responses to 5-HT and Ang II was significantly enhanced in HFD fed animals without change in pD 2 value. Endothelial denudation obviates any related mechanisms such as impairment of NO release, increased destruction of EDRF and substrate availability for the production of EDRF. Hence, the probable reasons for these enhanced 5-HT and Ang II responses may be due to receptor mediated or non-receptor mediated pathways. The role of non-receptor mediated contraction can be ruled out for there was no change in contractile response to KCl. Vascular studies have also shown functional evidence that hypertension developed in HFD fed rats may be associated with enhanced vasoreactivity to various vasoconstrictor agents. Further the enhanced responses to 5-HT in HFD fed rats could be due to increased PKC as previously reported with STZ and alloxan (ALL) induced diabetic animals [ 19 ]. Increased contractions to 5-HT can also be related to 5-HT 2A upregulation as observed in spontaneously hypertensive rats or due to serotonin acting through alpha adrenoceptors [ 20 ]. Increased Ang II responses in HFD fed rats are may be due to upregulation of Ang II receptors as observed in hyperinsulinemia or via amplified secondary messenger systems [ 21 ]. A proposed scheme of events is given in Fig 4 . Figure 4 Proposed events underlying syndrome X in HFD rats. HFD leads to condition, in rats, similar to syndrome X. This includes obesity, insulin resistance, hyperglycemia and dyslipidemia which all are interrelated. These events are the initial steps in the cascade towards hypertension. This could be mediated via increased contractile responses to various endogenous mediators such as 5-HT and Ang II. In summary, the present study has shown that HFD feeding in rats produces conditions similar to syndrome X. Increased vasocontractile responses observed in the model are not only mediated via alpha adrenoceptors but also due to 5-HT and Ang II (see Figure 4 ). More robust studies on the secondary messenger systems of 5-HT and Ang II will provide valuable insights into the mechanisms underlying increased vascular contractility in insulin resistance. Materials and Methods Tissue Preparation Male Sprague-Dawley rats (central animal facility, National Institute of Pharmaceutical Education and Research (NIPER), India), 160–200 g, were kept in controlled environmental conditions with room temperature 22 ± 2°C, humidity 55 ± 5% and 12-h light/dark cycles. All the animals had free access to food and water. The rats were divided into two dietary groups and fed with standard rat normal pellet diet (NPD) (3.8 kcal/g, carbohydrate 67%, protein 21%, fat 12% kcal) and HFD (5.3 kcal/g, carbohydrate 17%, protein 25%, fat 58% kcal). Composition of HFD is described in Table 1 . Table 1 Composition of HFD (g) Powdered pellet diet 364 Lard 310 Casein 250 Cholesterol 10 DL-Methionine 3 Yee-sac powder 1 Vitamin and mineral mix powder 60 Sodium chloride 2 Biochemical measurements Blood samples from the retro orbital plexus of anaesthetized rats (Pentobarbitone 45 mg/kg, i.p.,) were collected into the heparinized tubes and immediately centrifuged at 5000 rpm for the separation of plasma. Plasma was stored at -20°C until assayed. The plasma was used for the estimation of glucose (Qualigens, Mumbai, India), triglycerides, and cholesterol (Chema diagnostica, Jesi, Italy) by commercial kits. Plasma insulin was determined by radioimmuno assay using rat insulin as standard (Linco research, St. Charles, MO, USA) Intra peritoneal glucose tolerance test (IPGTT) Glucose tolerance tests were carried out after four weeks of feeding of both NPD and HFD. After an overnight fast, blood samples were collected from the retro orbital plexus. Glucose levels were measured at time zero (0 min) and glucose was injected into the rats (2 g/Kg/4 ml, i.p.,). Additional blood samples were taken at 15, 30, 60 and 120 min. following the glucose load. Plasma glucose levels were measured by the glucose oxidase reaction (GOD/POD) using commercial kit (Qualigens, Mumbai, India). Area under the curve (AUC) was calculated for both NPD and HFD fed rats. Vascular Studies After 4 weeks of feeding, rats were sacrificed by cervical dislocation. The section of the aorta from between the aortic arch and the diaphragm was removed from the euthanized rats and placed in oxygenated, modified Krebs-Henseleit solution (KHS; in mM: 118 NaCl, 4.7 KCl, 25 NaHCO 3 , 2.6 CaCl 2 2H 2 O 1.2 NaH 2 PO 4 , 1.2 MgCl 2 6H 2 O, 5.5 glucose). With the help of a steel rod, aortic endothelium was deliberately denuded. The aorta was cut into helical strips 3 mm wide, 20 mm long and then placed in a well-oxygenated (95% O 2 -5% CO 2 ) bath of 10-ml KHS with one end connected to a tissue holder and the other to an isotonic transducer (Bio Devices, Ambala, India). The tissue was equilibrated for 60 min under a resting tension of 1.0 g. At the beginning of each experiment, aortic strips were primed with depolarizing concentration (90 mM) potassium chloride (KCl). After the equilibration period, contractile responses to various concentrations of 5-HT (10 nM-30 μM) and Ang II (1 nM-300 nM) were recorded. Data analysis Contraction responses are expressed as percentage. For each contractile agent, both the maximal contraction (E max ) and the concentration necessary to produce 50% of its maximal response (EC 50 ) were determined. The EC 50 values were converted to the negative logarithms and expressed as pD 2 . Results were shown as mean ± SEM; n refers to the number of animals from which vessels were taken. Agonist potencies and maximal effects were compared by student's t test by using statistical software (GraphPad Prism 3.01). Values were considered significantly different at p < 0.05. Drugs and solutions The following drugs were used in this study: 5-HT (RBI, USA), Ang II (Bachem, Switzerland), All drugs were dissolved in KHS. Drugs were added to the organ chambers in volumes not greater than 0.2 ml. List of abbreviations HFD – High fat diet NPD – Normal pellet diet NIDDM – Non insulin dependent diabetes mellitus STZ – Streptozotocin 5-HT – 5-Hydroxytryptamine Ang II – Angiotensin II KHS – Krebs-Henseleit solution GOD/POD – Glucose oxidase/peroxidase AUC – area under the curve E max – Maximal response EC 50 – Concentration required producing 50% of maximal response pD 2 – -log EC 50 Authors' contributions SG carried out the all experimentations. PR has conceived the study and participated in its design and coordination. Authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC509282.xml
522749	Comparison of glucose tolerance in renal transplant recipients and hemodialysis patients	Background Impaired glucose tolerance is a risk factor for atherosclerosis in hemodialysis patients and renal transplant recipients. Methods To check the relationship of impaired glucose tolerance with the other atherosclerotic risk factors, fasting blood sugar and the standard two hour glucose tolerance test, serum tryglyceride, serum cholesterol, cyclosporine through level (in renal tranpslant recipients) and hemoglobin A1C were measured in 55 stable renal transplant recipients, 55 hemodialysis patients and 55 healthy controls with similar demographic characteristics. Patients with diabetes mellitus and propranolol consumers were excluded. The mean age and female to male ratio were 39 +/- 7 years and 23/22, respectively. Results Four of the renal transplant recipients and twelve of the hemodialysis patients had impaired glucose tolerance. Significant linear correlation was observed with body mass index and IGT only in hemodialysis patients (r = 0.4, p = 0.05). Glucose tolerance also had a significant correlation with triglyceride levels (217.2 +/- 55 mg/dl in hemodialysis patients vs. 214.3 +/- 13 mg/dl in renal transplant recipients and 100.2 +/- 18 mg/dl in control groups, p = 0.001). The glucose tolerance had significant relationship with higher serum cholesterol levels only in the renal transplant recipients (269.7 +/- 54 in renal transplant recipients vs. 199.2 +/- 36.6 mg/dl in hemodialysis and 190.5 +/- 34 mg/dl in control groups, p = 0.0001). In the renal transplant recipients, a linear correlation was observed with glucose tolerance and both the serum cyclosporine level (r = 0.9, p = 0.001) and the hemoglobin A1C concentration (6.2 +/- 0.9 g/dl). The later correlation was also observed in the hemodialysis patients (6.4 +/- 0.7 g/dl; r = 67, p = 0.001). Conclusions We conclude that although fasting blood sugar is normal in non-diabetic renal transplant and hemodialysis patients, impaired glucose tolerance could be associated with the other atherosclerotic risk factors.	Background Mortality and morbidity due to cardiovascular diseases are frequent in patients with diabetes mellitus and high prevalence of diabetes and cardiovascular disease, also, are observed in patients with end-stage renal disease treated by renal replacement therapy, either renal transplantation (RT) and dialysis [ 1 ]. Although uremia is typically associated with impaired glucose metabolism via multiple mechanisms [ 2 - 4 ], hemodialysis improves, although not completely, the uremic induced glucose impairment [ 5 - 7 ]. Impaired glucose metabolism is also a common and an important problem after RT. By improvement of immunosuppression after RT, the incidence of post transplant diabetes (PTDM) has been decreased from 41% to 2.5% [ 8 , 9 ]. Although we routinely screen and treat only full-blown diabetes at the post transplant periods, an overlooked aspect is the impaired glucose tolerance, which may be a risk factor to induce atherosclerosis. Impaired glucose tolerance de novo , may be a risk factor of post-transplantation mortality and morbidity [ 10 ]. Although increased levels of glycosylated hemoglobin (HbA1C) and lipid concentrations have been shown in hemodialysis patients [ 11 ] and renal transplant recipients [ 12 ] with diabetes, their impairment is not clear in the both groups with impaired glucose tolerance without apparent diabetes mellitus. In this study we investigated glucose tolerance and lipid profiles in non-diabetic hemodialysis and renal transplant patients. Methods We selected fifty five RT recipients with more than one year of good renal allograft function (serum Cr < 1.5 mg/dl), under conventional triple therapy composed of cyclosporine A (CsA), azathiopurine and prednisolone. Their allograft sources were living donors. Fifty five stable HD patients and another fifty five healthy controls (C), were also enrolled in this study. The mean age (39 ± 7 years), sex (F/M ratio was 33/22), body mass index (BMI) 24.7 ± 1.28 kg/m 2 ) were similar in the three groups (see table 1 ). Patients with diabetes mellitus and propranolol consumers were excluded. Table 1 Demographical, biochemical, hematological and therapeutical factors in hemodialysis patients and renal transplant recipients. HD RT Control Age (years) 48 ± 3 46 ± 4 47 ± 4 Male/female ratio 33/22 33/22 33/22 BMI (Kg/m 2 ) 24.6 ± 1.4 23.8 ± 1.2 23.6 ± 1.3 Cholesterol (mg/dl) 199.2 ± 36.6 269.7 ± 54 190.5 ± 34 Triglycerides (mg/dl) 217.2 ± 55 214.3 ± 13 100.2 ± 18 HgA1C (g/dl) 6.42 ± 0.7 6.2 ± 0.9 5.7 ± 0.7 Hb level 10.9 ± 0.8 12.4 ± 1.1 13.3 ± 0.8 Therapy with vitamin D3 0.5 μg/day (number) 25 -- -- Impaired glucose tolerance (number) 12* 4** -- Significant correlation with BMI, serum triglycerides and HgA1C * Significant correlation with serum cholesterol, CysA concentration and HgA1C The levels of serum triglyceride, cholesterol (measured by enzymatic spectrophotometry)[ 13 ], CsA (measured by ELISA in whole blood, only in renal transplant recipients) and glycosylated haemoglobin concentration (Hb A1c) (measured by column chromatography) were measured after 10 hours fasting (in the hemodialysis group, in the early morning before hemodialysis). Fasting blood sugar and the standard 2 hours glucose tolerance test (after ingestion of 75 g of glucose) were detected in the three groups by spectrophotometry. Statistical analysis was performed by Kuruskal wallis, U-Mann Whitney, multiple comparison and regression correlation coefficient tests, using SPSS 10.05. Results On the basis of WHO classification [ 14 ], four of our (7.5%) renal transplant recipients and twelve (22%) of the hemodialysis patients had impaired glucose tolerance, i.e. the 2 hour of glucose tolerance test was between 140 and 200 mg/dl. It was more obvious at the end of the second hour of GTT. Although BMI was roughly similar in the three groups (Table 1 ), a significant linear correlation was observed between BMI and impaired glucose tolerance only in HD patients (r = 0.4, p = 0.05) (fig 1 ), but not in the RT recipients. The glucose tolerance (especially at the first hour) in the HD patients had a significant linear correlation with the level of serum triglycerides (r = 0.87, p = 0.001) (Fig 2 ). Serum triglyceride concentration was 217.2 ± 55 mg/dl in HD vs. 214.3 ± 13 mg/dl in RT and 100.2 ± 18 mg/dl in C groups, (p = 0.001). On the other hand the four RT recipients with IGTT (i.e. 100% of RT recipients with IGTT) had the higher serum cholesterol levels (308.4 ± 24.4 mg/dl)) compared with the remaining RT recipients with normal GTT (248.7 ± 55.6 mg/dl) with p = 0.031 (table 2 ). The mean of serum cholesterol was 269.7 ± 54 mg/dl in RT vs. 199.2 ± 36.6 mg/dl in HD and190.5 ± 34 mg/dl in C groups (p = 0.0001). A linear correlation was observed between impaired GTT and both of the serum Cyclosporine level (r = 0.9, p = 0.001) and HbA1c in RT recipients (Fig 3 ). The mean of HbA1c was 6.2 ± 0.6 gr/dl in the RT recipients with normal GTT vs. 4.34 ± 0.26 g/dl in the RT recipients with IGGT (p < 0.001, table 2 ). The later correlation was also observed in HD patients, in whom the mean of HbA1C level was 6.4 ± 0.7 gr/dl in the group (r = 67, p = 0.001). In contrast of a close relationship of IGTT and higher HbA1c, the gender, age, times after transplantation and BMI did not impact on IGTT in RT recipients. Although in logistic regression analysis higher serum level of cyclosporine was correlated with increased GTT impairment, we could not evaluate the implication of corticosteroids on this test, because all of the 55 RT recipients were received prednisolone at a doses of 5 to 10 mg/day. Figure 1 GTT has a linear relationship with BMI in hemodialysis patients. Impairment of GTT is more significant in the hemodialysis patients with higher BMI. Gtt2 = glucose tolerance test at the second hours of 75 gr oral glucose. bmih = body mass index in hemodialysis patients Figure 2 The glucose tolerance in the HD patients had a significant linear correlation with the level of serum triglycerides. gttd1= glucose tolerance test in dialysis patients, tgh= serum concentration of triglyceride in hemodialysis patients. Figure 3 Cyclosporine level and HbA1c have correlations with the IGTT in RT recipients ▲ = Serum Cyclosporine level ○ = HbA1c concentration Table 2 Impaired glucose tests in HD and RT recipients have higher values of serum triglyceride, serum cholesterol and cyclosporine concentration than patients with normal glucose tolerance tests. no. of cases Serum Triglyceride(mg/dl) Serum Cholesterol(mg/dl) HbA1c (gr/l) Cyclosporine (mg/dl) RT recipients with IGTT 4 231.4 ± 150 308.4 ± 24.4 7.34 ± 0.26 320.4 ± 36.6 RT Recipients With normal GTT 51 201 ± 75 248.7 ± 55.6 6.2 ± 0.6 295.1 ± 29 P = 0.59 P = 0.02 P = 0.001 P = 0.2 HD patients with IGTT 12 272.1 ± 41.3 201.1 ± 39 7 ± 1 HD patients with normal GTT 43 195.9 ± 45 198.5 ± 36.8 5.9 ± 0.7 P = 0.001 P = 0.87 P = 0.007 Controls 55 100.2 ± 18 190.5 ± 34 5.7 ± 0.7 Discussion Impaired glucose tolerance occurs in about 50% of patients with chronic renal failure (CRF) patients. It is due to multiple factors, which the two most important of them being insulin resistance at target organs and impaired insulin secretion from the pancreas [ 15 ]. Insulin sensitivity would be reduced by up to 60% in non-diabetic patients with CRF before dialysis [ 16 ]. Marked improvement in insulin sensitivity and consequently glucose tolerance has been reported in non-diabetic patients after 10 weeks of HD, although they are not completely returned to normal [ 15 ]. Thereby, impaired glucose tolerance during HD is secondary to non-effective removable toxins by HD compared with peritoneal dialysis. In the latter more effective removal of middle molecule toxins causes better glucose tolerance, although glucose rich dialyzet solution is used [ 16 ]. The other causes of impaired glucose tolerance in HD patients may be secondary to metabolic disturbances, such as anemia [ 17 ], malnutrition [ 18 ] and vitamin D3 deficiency [ 19 ]. Although all of our HD patients had normochromic-normocytic anemia, the severity was not proportionate with impaired glucose tolerance (The data has not been shown). The patients were well nourished and were under treatment with daily oral vitamin D3 (Rocaltrol), 0.5 micrograms per day. So malnutrition and vitamin D3 deficiency could not to contribute to impaired glucose tolerance in our HD patients. Impaired glucose tolerance was also observed in 7.5% of our RT recipients. All of the presumed risk factors for post transplant diabetes mellitus such as old age [ 18 ], family history of any known diabetes mellitus in their first relatives[ 21 ], cadaveric allografts [ 22 ] and obesity did not exist in the patients. Previously Boudreaux et al. [ 23 ] reported that those patients who weighed more than 70 kg had a higher incidence of post transplant diabetes mellitus (PTDM). A relative risk of 1.4 for developing PTDM for every 10 kg increase in body weight more than 60 kg has been shown [ 12 ]. Although in our study obese patients (BMI > 30 kg/m2) were not included in the both groups, a correlation was observed between impaired glucose tolerance and higher BMI in our HD patients. In RT recipients, the major risk factor for impaired glucose tolerance was immunosuppressive therapy. Through using higher doses of CsA and corticosteroids, PTDM was previously more common, but the complication has been decreased to 2–5% in FK506-based immunosuppressive protocols [ 24 , 25 ]. Although this relatively uncommon complication is a major cause of post-transplant mortality and morbidity, even minor glucose intolerance is associated with an increased long-term risk for cardiovascular disease [ 26 ]. The importance of impaired glucose tolerance should not be underestimated in these patients with high risk of atherosclerosis. Hyperlipidemia, another risk factor for atherosclerosis, on one hand accompanies the impaired glucose tolerance observed in the HD and RT patients and on the other hand increases the risk of atherosclerosis induced by impaired glucose tolerance. As reported previously, a tendency to higher pre-transplantation serum triglyceride concentration was associated with post-transplantation impaired glucose tolerance [ 27 ]. Hypertriglyceridemia is common complication in dialysis patients. In non-transplant populations it is regarded (along with low HDL cholesterol levels) as a prominent feature of insulin resistance syndrome, and also is a cardiovascular risk factor in organ transplant recipients [ 28 ]. Our study confirmed the relationship between impaired glucose tolerance and triglyceride levels in HD patients, and between impaired glucose tolerance and cholesterol levels in RT recipients. The latter was also accompanied by a higher level of HgA1C. Commonly used tests of HgA1C may be unreliable in patients with end-stage renal disease because of the presence of anemia, shortened red blood cell survival, and assay interferences from uremia. But HgA1C in the range of 6% to 7%, as was found in our study, estimates glycemic control within the range of patients without severe renal impairment [ 1 ]. So in the range of mild to moderate increased HgA1C in HD and uremic patients, it would be a reliable marker of impaired glucose tolerance. Conclusions There was increased HgA1C and impaired glucose tolerance in HD and RT patients. This was accompanied by hyperlipidemia in HD patients (with hypertriglyceridemia) and RT recipients (with hypercholesterolemia). The impact upon the progression of atherosclerosis needs more study in haemodialysis and renal transplant populations at a long term follow up. Competing interests None declared. Authors' contributions HA reviewed the literatures and wrote the manuscript and also helped to do statistical analysis, AN performed GTT and the other biochemical markers, MN participated as coordinator between laboratory and clinic, HTK selected the patients and collected data Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC522749.xml
535547	Using GIS to establish a public library consumer health collection	Background Learning the exact demographic characteristics of a neighborhood in which a public library serves, assists the collection development librarian in building an appropriate collection. Gathering that demographic information can be a lengthy process, and then formatting the information for the neighborhood in question becomes arduous. As society ages and the methods for health care evolve, people may take charge of their own health. With this prospectus, public libraries should consider creating a consumer health collection to assist the public in their health care needs. Using neighborhood demographic information can inform the collection development librarians as to the dominant age groups, sex, and races within the neighborhood. With this information, appropriate consumer health materials may be assembled in the public library. Methods In order to visualize the demographics of a neighborhood, the computer program ArcView GIS (geographic information systems) was used to create maps for specified areas. The neighborhood data was taken from the U.S. Census Department's annual census and library addresses were accumulated through a free database. After downloading the census block information from the data was manipulated with ArcView GIS and queried to produce maps displaying the requested neighborhood demographics to view in respect to libraries. Results ArcView GIS produced maps displaying public libraries and requested demographics. After viewing the maps the collection development librarian can see exactly what populations are served by the library and adjust the library's collection accordingly. Conclusions ArcView GIS can be used to produce maps displaying the communities that libraries serve, spot boundaries, be it "man-made or natural," that exist prohibiting customer service, and assist collection development librarians in justifying their purchases for a dedicated consumer health collection or resources in general.	Background Libraries have the objective to build collections that support the communities they serve. To build a viable collection the collection development librarian, outreach/marketing librarian, and others, must determine exactly what populations reside in the neighborhood, in respect to race, spoken language, educational level, and age groups. One method collection development librarians use to gather demographic information is to physically visit the communities and integrate into the neighborhoods. They may attend events within the community in order to analyze the attendees or walk around the neighborhood to get a feel for the community. Another method for collection development is to perform an informal survey with people that visit the library and learn about their preferences and/or what they like to read or look for on the Internet. Using census information can be a third way to gather community/neighborhood information. The US Census Bureau takes a census of the United States every 10 years and publishes the results on the Internet. Taking the Census information and transforming it into a graphical format provides an objective view of the communities surrounding a library. One way to visualize the census data is through the use of GIS, Geographic Information Systems. GIS are databases arranged by spatial coordinates that, when programmed, can produce maps [ 1 ]. The US Geological Survey office defines GIS as "a computer system capable of capturing, storing, analyzing, and displaying geographically referenced information; that is, data identified according to location [ 2 ]." Many disciplines have used GIS and many large academic libraries even support GIS by establishing a GIS department and employing a librarian to specialize in GIS information. In the field of library science articles have been published describing the establishment of GIS departments in libraries and what GIS librarians do [ 3 - 7 ], but little has been published describing how a library has used GIS. Governments use GIS to visualize land use planning, tax appraisal, utility and infrastructure planning and more [ 8 ]. Businesses have used GIS for real estate analysis, marketing, and demographic analysis [ 8 ]. Based on this approach, a research project was developed to test the use of GIS in collection development for a large metropolitan public library system. Building a collection for a library has become a sophisticated art. Johnson states that "collection building consists of four steps: identifying the relevant items, assessing the item to decide if it is appropriate for the collection and evaluating its quality, deciding to purchase, and preparing an order [ 9 ]." Each step in the process of collection development produces a layer of difficulties. In order to get to these steps a collection development librarian must be aware of their audiences. To name a few necessary pieces of information, the librarian must know the predominate age of their clients, their reading genre, their reading level, their culture, and their preferred language [ 9 ]. After one has gathered community information then the decision of what to purchase for use by the community, can be initiated and carried out [ 10 ]. This paper reports the steps taken to use GIS in assisting a large metropolitan public library system in visualizing neighborhoods that surround branch libraries in order to make an educated decision on whether a dedicated consumer health collection should be established to support the community. The objective of the project was to determine if GIS could be used to improve collection development. Methods After comparing large library systems in major metropolitan areas, (Chicago, Los Angeles, and New York City) Chicago was selected as a convenient sample to be mapped with GIS because of its size, number of public libraries, their geographic distribution within the urban area, and ease of access to data. Compared to other large cities, the New York Public Library system has 86 libraries and Los Angeles has 67 public libraries [ 11 ]. The city of Chicago has 77 public libraries with one central library. With the selection of the city of Chicago, the next step was to create a map of the metropolitan area. ESRI ArcGIS v9.0 geographic information software (GIS) [ 12 ] was selected as the GIS application. The software permits data manipulation and mapping capabilities. There are four components of ArcGIS that work together to give a high level of functionality to the program: ArcReader, ArcView, ArcEditor and ArcInfo. By inserting data files into ArcEditor the datum may convert into a graphical representation. Once a base map has been created with geographic boundaries, locations may be established to show distances within the map. In order to create the city map of Chicago, data files were obtained from the U.S. Census Bureau website. The Census Bureau produces TIGER (Topologically Integrated Geographic Encoding and Referencing) Files available for free to the public [ 13 ]. These files contain geographic structures, such as streets, highways, and addresses, for tabulation and dissemination. To set the boundary lines of metropolitan Chicago, Cook County (the county in which the city of Chicago resides) was selected as the source for the data file [ 14 ]. This TIGER file contained the street map for the city of Chicago. The next step required mapping distances between neighborhoods and neighborhood demographics. In order to achieve this, files were obtained from the U.S. Census Bureau website [ 15 ] which distributes the 2000 Census statistics in TIGER File format. Downloading the files for Census blocks in Cook County provided the necessary data to build a visual representation of neighborhoods. The final piece of information needed to build a map showing neighborhoods in respect to public library locations, was to map the public libraries within Chicago. Because the base map of Cook County was a street map, addresses of public libraries were entered into ArvView to mark all the public libraries within the city of Chicago. The state of Illinois provides a database available on the Internet that permits searching types of libraries and locations of libraries, . The output of this search was imported into Excel, cleaned, and converted into a dBase file. The dBase file was imported into ArcView to coordinate the library addresses to the street addresses of the base map. This enabled the software to place an image for each public library in the city map of Chicago. With the city of Chicago and its public libraries now in a GIS application, the location of libraries was displayed in respect to neighborhoods and distances from library to library (See Figure 1 ). Because the Census data was used to build the map, ArcView can be queried to show demographics of the city. This allows useful data manipulation in relation to points of interest, in this case, public libraries. Thus, ArcView was queried to show the distribution of all men and women within the city of Chicago as well as the breakdown of their age groups. The software application produced a graphic which used color schemes with a map legend. If a librarian wanted to compare the number of men in their 40s to women in their 40s, ArcView can map those fields with a well-structured query. ArcGIS was also capable of creating maps that display census ethnic groups. For example, Figure 2 shows a high percentage of African Americans in the southern neighborhoods of the city as compared to the northern part of the city. With maps such as these, a collection development librarian can graphically see the demographic breakdown of neighborhoods and build collections according to its clientele [ 16 ]. Figure 1 Location of public libraries in the city of Chicago Figure 2 Density of African American population in southern part of Chicago Results Fifteen maps were created showing a breakdown of each demographic with the city's public libraries. Each map provided information regarding gender, age, and race of the clientele surrounding the libraries. These maps used a combination of color schemes to graphically represent differences between groups. The maps displayed different age groups divided by ranges of age (30–39, 40–49, 50–64, 65 and up). The age ranges presented in the maps varied by query and census information. The groups analyzed were: women, men, white women (group age 40–49), African American women (group age 40–49), Asian (men/women, Latinos (men/women). The color schemes used represent the number of people that live within the census block as to the specified GIS query. In Figure 2 , the numbers, 0–992 in the legend are the African Americans residing within the census block. Hence the red blocks have a large population (403–992) of African Americans as compared to the dark green areas. Figure 3 represents a query for African American women in their 40s. The results produced a map showing that 26–59 African American women in their 40s live within the shaded red blocks. Figure 3 African American women in their 40s in proximity to public libraries In this project ArcGIS was used to build maps of neighborhoods in the city of Chicago, display public libraries and spatially represent distances from libraries to populations. The US Census demographic information was incorporated into ArcGIS and queried to produce 15 different maps. Queries were built to display neighborhood demographics through colors showing the percentages of the population(s). Each query produced a map for a specific age or race. Once the map was produced the public libraries were added to the map and analysis was made regarding collection needs according to the neighborhood population surrounding the library. To decide if a public library should establish a consumer health collection, a map showing the breakdown of women in their 40s was built. As in Figure 4 , the map shows a very high percentage of women 40–49 close to the Uptown Branch Library. The collection development librarian can now assess the libraries existing collection for health related materials. If there are very few resources then the collection development librarian should begin to purchase consumer health materials. If the library has a strong consumer health collection already then building maps to show the racial breakdown of the neighborhood could lead the collection development librarian to purchase culturally sensitive health materials. Figure 4 High density neighborhoods with all women in their 40s in proximity to public libraries Discussion By creating maps showing the demographic breakdown of the city, a public library can build, adjust, or verify its collection in respect to the neighborhoods it supports [ 17 ]. It is easier to define the characteristics of the library's service area clientele by making visual representations (maps) of neighborhoods surrounding a library. The neighborhood information may then be used to justify additional resource purchases, a change in collection policy and even a change in library services to fit the culture of the neighborhood. By using GIS, libraries will be capable of finding specific populations within the library's neighborhood to support or serve thus taking some of the guesswork out of collection development [ 17 , 18 ]. Collections may be tailored specifically to the neighborhood by using the subjective data provided with GIS. Many studies have been conducted analyzing what people search for on the Internet and who is searching. Studies have found that women in their 40s search for and utilize health information from the Internet more than men [ 19 - 24 ]. Using this information to assist in collection development, a librarian would need to know how large the population of women in their 40s is in the neighborhood the library serves. Using the map created by ArcGIS showing the distribution of women in their 40s in respect to library locations can assist the collection development librarian in purchasing appropriate materials. Using the maps showing the race of women in their 40s would also assist the librarian in purchasing appropriate materials. For instance, if most women are African American then the collection development librarian would purchase materials supporting race-related health needs such as Sickle Cell Anemia, Coronary Heart Disease, Diabetes and others (See Figure 2 and 3 ). Recommendations Using ArcGIS can assist in defining the exact make-up of the population that a library serves. By turning the census information into a graphic the library is provided a chance to see how far (physical distance) their services may reach. Library systems that have branch libraries may have one collection development librarian who buys for each library. By using ArcGIS the central library can analyze the communities surrounding each library to purchase materials appropriately without having to spend time in each library learning the environment. Purchase decisions may now be justified with hard data from ArcGIS. For example, if ArcGIS shows that the largest population surrounding a library speaks a minority language, materials should be purchased in the dominant language to best meet the needs of the community. Limitations There is a fairly high learning curve to use ESRI Arcview GIS. There are five programs in ESRI Arcview and each program has separate features that must be used within the specific program to be imported into the primary map. Learning what each program does and then how to incorporate the data into the map requires hours of reading. Acquiring the necessary data to build a map requires background study of the geographic area in order to assure that the map is configured correctly. Upon attaining all necessary data for the map, one must build queries to manipulate the data according to the information need, and then configure the map accordingly. While GIS presents a new technique to use in performing collection development, there was not a way to test the differences from a collection built using GIS information and the current methods of collection development. In addition, research has discovered that knowing the service area radius of the library doesn't mean that people within the service area will actually use or belong to the neighborhood library [ 17 , 25 ]. When using GIS one must take into account geographic barriers to library access such as major highways, and railways. People are often reluctant to cross these barriers and may elect to visit another library further away with no geographic obstructions [ 17 , 25 , 26 ]. These barriers must be represented in the GIS map to fully display the areas libraries serve. The TIGER files contain some geographic structures but most will have to be hand coded. Thus, all neighborhoods should be examined in order to assure the marking of geographic barriers. While GIS can take the census information and demonstrate the population surrounding a library, the census does not describe the specific users of the library [ 25 ]. To build a map showing a library's service area, patron addresses (information now protected by the Patriot Act) would need to be coded into GIS. Using patron zip codes of residence encompasses too much or too little area and there is no correlation between US Postal Service ZIP Codes and US Census Bureau Geography [ 27 ]. Thus, mapping or defining areas through zip codes is extremely difficult. Without patron address information, there can be no clear realization of exactly how far people will travel to a library, and if people cross-neighborhoods to use a certain library [ 18 , 25 ]. Conclusions Budget constraints will add a need for justification of purchases for a library's collection. Using GIS may provide one method to justify additions to collections. As health care changes and individuals take more control of their health, libraries need to have materials that provide valid information in an age and language appropriate format and be culturally sensitive. Branch libraries respond to the demands of the neighborhood [ 25 ] and knowing what those demands are will make an effective library [ 17 ]. GIS can assist collection development librarians located in a central library buy for branch libraries, by saving them time, and answering demographic questions about the population a library serves. Knowing details about its communities will make a more functional library within a neighborhood. To verify the use of GIS for collection development, an analysis of a library's collection should be conducted and compared with information provided from a GIS application to see if the collection satisfies the library's neighborhood. List of Abbreviations GIS = geographical information systems	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535547.xml
534103	Lipoprotein lipase in hemodialysis patients: indications that low molecular weight heparin depletes functional stores, despite low plasma levels of the enzyme	Background Lipoprotein lipase (LPL) has a central role in the catabolism of triglyceride-rich lipoproteins. The enzyme is anchored to the vascular endothelium through interaction with heparan sulphate proteoglycans and is displaced from this interaction by heparin. When heparin is infused, there is a peak of LPL activity accompanied by a reduction in triglycerides (TG) during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increase towards and beyond baseline. This suggests that tissue stores of LPL become depleted. It has been argued that low molecular weight (LMW) heparins cause less disturbance of the LPL system than conventional heparin does. Methods We have followed LPL activity and TG during a dialysis-session with a LMW heparin (dalteparin) using the same patients and regime as in a previous study with conventional heparin, i.e. a primed infusion. Results The shape of the curve for LPL activity resembled that during the earlier dialyses with conventional heparin, but the values were lower during dialysis with dalteparin. The area under the curve for LPL activity during the peak period (0–180 minutes) was only 27% and for the plateau period (180–240 minutes) it was only 36% of that observed with conventional heparin (p < 0.01). These remarkably low plasma LPL activities prompted us to re-analyze LPL activity and to measure LPL mass in frozen samples from our earlier studies. There was excellent correlation between the new and old values which rules out the possibility of assay variations as a confounding factor. TG increased from 2.14 mmol/L before, to 2.59 mmol/L after the dialysis (p < 0.01). From 30 minutes on, the TG values were significantly higher after dalteparin compared to conventional heparin (p < 0.05). Conclusion These results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does.	Background Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) in circulating lipoproteins [ 1 , 2 ]. This is a necessary first step in catabolism of the TG-rich lipoproteins as evidenced by the massive hypertriglyceridemia in patients with genetic deficiency of the enzyme [ 3 ]. Fine-tuned regulation of LPL activity is an important aspect of energy homeostasis [ 4 ]. Patients on chronic hemodialysis (HD) often have reduced LPL activity and derangements of lipoprotein profiles [ 5 - 7 ]. During HD, conventional heparin is commonly used as anticoagulant and this releases LPL from its binding sites at the vascular endothelium into the circulation. It has been suggested that repeated heparinisation may induce release and subsequent degradation of LPL that exceeds the rate of enzyme synthesis and thereby causes a depletion of LPL stores [ 8 - 11 ]. In a previous study we followed the LPL activity and the TG changes during a dialysis-session using conventional heparin as anticoagulant [ 12 ]. There was a peak of LPL activity accompanied by a reduction in TG during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increased towards and beyond the original baseline. When compared to a group of healthy control subjects, the peak LPL activity was only about 50 % in the HD-patients while the plateau activities were comparable. Our interpretation was that the functional pool of LPL, represented by the initial peak, was impaired in HD-patients, while the production of lipase molecules, reflected by the plateau, was only marginally reduced. In recent years, conventional heparin has increasingly been replaced by various low molecular weight (LMW) heparins. A major argument is the ease of administration [ 13 , 14 ]. A single injection of a LMW heparin can often replace a primed infusion of conventional heparin. The increase in plasma LPL activity is less pronounced after LMW compared to conventional heparin [ 15 ], and it has been suggested that this causes less disturbance of lipoprotein metabolism [ 10 ] although this conclusion has been questioned [ 16 ]. Direct studies of the lipase-heparin interaction have shown that a heparin decasaccharide is enough to fill the heparin-binding grove on the lipase molecule [ 17 , 18 ]. Decasaccharides fall in the middle or lower size range in preparations of LMW heparins [ 19 ]. Several lines of evidence indicate that also in biological systems, decasaccharides are sufficiently long to exert full effect on LPL. Chevreuil et al . found that on a weight basis, decasaccharides released more LPL from perfused rat hearts than conventional heparin did [ 20 ]. Several groups have reported that LMW heparins or decasaccharides release LPL from tissues in vitro or from cultured cells as efficiently as or even more efficiently than conventional heparin does. It is therefore unlikely that the lower plasma LPL activities after LMW heparin are due to less release of the lipase. More likely, LMW retards clearance of the lipase by the liver less efficiently than conventional heparin does. Two studies have directly demonstrated such a difference in liver perfusion experiments [ 20 , 21 ]. In a recent study we infused a LMW heparin (dalteparin) for eight hours to healthy volunteers to explore the influence on LPL activity and TG response [ 22 ]. The peak LPL activity was only about 30%, and the subsequent plateau activity only about 40%, compared to the activities observed during a similar infusion with conventional heparin. A bolus of conventional heparin given when the LPL activity had levelled off to a plateau brought out about the same amount of activity irrespective of if the subjects had been infused with dalteparin or conventional heparin. We concluded that dalteparin and conventional heparin had reduced the peripheral stores of LPL to a similar extent and that the difference in plasma levels of LPL activity was due to a more rapid hepatic clearance of the LPL-dalteparin complex. There was a tendency towards a more pronounced increase in TG after dalteparin compared to conventional heparin, indicating that lipoprotein metabolism might be more, rather than less, disturbed by the use of LMW heparin. As HD-patients are increasingly subjected to repeated treatment with LMW heparin during dialysis we have now followed LPL activity and TG during a dialysis-session with dalteparin using the same regime as in our previous study with conventional heparin, i.e. a primed infusion [ 12 ]. Methods Subjects and protocol The study design was based on the protocol used in a previous investigation in which nine HD-patients were studied during a dialysis-session using conventional heparin as anticoagulant [ 12 ]. The present study, on the same HD-patients, was performed three months later with a LMW heparin (dalteparin, Pharmacia, Stockholm, Sweden) as anticoagulant. In all other respects the dialysis regime, as well as medication and diet recommendations, was kept unchanged. The median age was 73 years and the median BMI was 24.7. The diagnoses were diabetes nephropathy (BE), chronic pyelonephritis (AJ), nephrosclerosis (HB), polycystic kidney disease (MK, CH), chronic glomerulonephritis (RH, RS, KL) and in one patient the origin was unclear (BV, not biopsied). The patients had been on maintenance hemodialysis for 5–38 months and were treated with bicarbonate hemodialysis either two (RS, HB) or three (MK, BE, KL, AJ, RH, CH, BV) times a week, depending on residual renal function. All dialyses were performed with hemophan dialysers (GFS+16, GAMBRO, Lund, Sweden) and Biosol dialysis solution (Pharmalink, Stockholm, Sweden). A central dialysis catheter was used as dialysis-access in five of the patients and an arteriovenous-fistula/graft in four. The patients were treated with antihypertensive drugs (ACE-inhibitors, beta-blockers, calcium channel inhibitors), diuretics, sodium bicarbonate and phosphate-binding drugs. The diabetic patient was non-insulin dependent and was treated only by diet recommendations. One patient (RS), having a rejecting renal transplant, was treated with low doses of corticosteroids and cyclosporine. No one was treated with lipid lowering drugs. The experiments were carried out after an overnight fast, and 48–96 hours had passed since the previous hemodialysis. A loading dose of 40 IU dalteparin per kg body weight was given, followed by a continuous infusion of 1000 IU/hour, in accordance with the manufacturer's recommendations. Blood samples were drawn before start and then regularly at 15, 30, 60, 120, 180 and 240 minutes. According to existing routines, the patients had a combined breakfast/lunch, containing 25 g fat, about two hours after the dialysis was started. The ethical committee approved the study and informed consent was obtained from all patients prior to participation. Handling of samples and assay methods Blood samples for measurement of LPL activity were collected in heparinized tubes. They were immediately chilled in ice water and centrifuged in a cooling centrifuge within 15 minutes. The plasma was frozen at -20°C and then stored at -70°C until analyses. LPL activity was measured as described [ 23 ] using an emulsion containing a trace amount of [ 3 H]-oleic acid-labelled triolein, 100 mg soybean TG and 10 mg egg yolk phospholipids per mL, prepared by Fresenius-Kabi, Uppsala, Sweden. Hepatic lipase was inhibited by pre-incubation of the plasma samples with immunoglobulins from a rabbit antiserum to human hepatic lipase. The assay medium contained a relatively high concentration of heparin, and possible differences in the heparin concentration or type in the sample would not affect the activity. All assays were made in triplicate and the mean value was used. A standard sample of human post-heparin plasma was run on each assay day and the value was used to calibrate for between-assay-variations. LPL protein mass was determined with an enzyme-linked immunosorbent assay, as previously described [ 24 ], using immunoaffinity-purified chicken antibodies raised against bovine LPL for capture and the monoclonal antibody 5D2, also raised against bovine LPL, for detection (a gift of Dr. J. Brunzell. Seattle, Washington, USA). Blood samples for lipid determination were drawn in tubes without anticoagulant, immediately chilled in ice water, centrifuged and frozen as described above. Total cholesterol, HDL-cholesterol and TG were determined by routine methods on a multianalyzer (Vitros 950 IRC; Johnson & Johnson, Clinical Diagnostics Inc, New York, NY, USA). Baseline LDL-cholesterol levels were calculated using the Friedewald formula [ 25 ]. Antifactor Xa activity was determined using a chromogenic substrate (COACUTE ® , Chromogenix AB, Mölndal, Sweden). Statistics The values are expressed in terms of median and range and were examined for significant differences by paired Wilcoxon signed-rank test. Simple linear regression and the Spearman rank correlation test were used to evaluate relationships between variables. Two-tailed P values below 0.05 were considered to be statistically significant. Results Baseline data Table 1 gives baseline data for the HD-patients at the time of the dialysis-session with dalteparin. There were no significant differences compared to the values at the time of the dialysis with conventional heparin three months earlier [ 12 ]. There was no significant difference between the ultrafiltration rates during the two dialyses. Table 1 Baseline data for the HD-patients at the time for the dialysis-session with dalteparin. Median values for the dialyses with dalteparin (D) and conventional heparin (H), respectively. There were no statistically significant differences between values on the two occasions. HD-patients Gender Age (years) Dbw* (kg) Ultra-filtration(L) BMI (kg/m 2 ) TG (mmol/L) Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) MK F 53 71.5 3.0 24.7 3.41 4.5 0.65 2.3 BE F 64 81 2.2 34.2 2.55 5.4 1.24 3.1 KL F 79 76 2.8 28.3 2.14 6.3 0.76 4.5 CH M 55 62.5 3.5 19.5 1.21 3.6 0.99 2.1 BV M 70 77.5 1.3 26.2 1.47 4.6 0.99 2.9 RS M 73 65 2.7 23.3 2.86 7.1 0.96 4.9 RH M 78 62 2.7 21.5 1.09 4.6 1.26 2.9 AJ M 78 73 3.0 26.5 2.84 5.0 0.74 2.9 HB M 90 70 3.0 24.2 1.38 4.4 1.30 2.5 median D 73 71.5 2.8 24.7 2.14 4.6 0.99 2.9 median H 73 70.5 2.2 24.6 1.84 5.6 1.09 3.7 *Dry body weight Anticoagulation effect During the dialysis with dalteparin, the anti-Factor Xa activity was between 0.52 and 0.87 IU/mL. The target value recommended by the manufacturer is 0.5–1.0 IU/mL. Hence, the plasma dalteparin concentration remained well within the range for effective anticoagulation throughout the dialysis-session. LPL activity and mass The LPL-activity rose rapidly when dalteparin was administered. The highest value was at 15 minutes, median 15 mU/mL (range 9–32). The activity remained high at 30 minutes, but then decreased so that at 120 minutes the median was only 9 mU/mL (range 5–15) (Fig 1 ). The activity then remained essentially unchanged to the end of the dialysis at 240 minutes (6 mU/mL, range 5–11). The shape of the curve resembled that during the earlier dialysis with conventional heparin (Fig 1 inset), but the values were much lower during the dialysis with dalteparin (p < 0.01). The area under the curve (AUC) for LPL activity during the peak period (0–180 minutes) was 1774 mU/mL × minutes (range 1116–3001). This is only 27% of the AUC observed during the earlier study with conventional heparin [ 12 ] (p < 0.01). For the plateau period (180–240 minutes) the AUC was 390 mU/mL × minutes (range 308–618) which is 36% of the corresponding AUC during the dialysis with conventional heparin (p < 0.01). Figure 1 Plasma LPL activities during infusion of dalteparin. The figure shows individual curves for the nine subjects in the present study. The inset compares median values from the present study (□) with those from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin [23] (●), control subjects given dalteparin [22] (○), HD patients given conventional heparin [12] (■). The inset in Fig. 1 compares the median values for LPL activities in the present study to the LPL activities observed in earlier studies with age and gender matched healthy subjects given conventional heparin [ 23 ], or dalteparin [ 22 ], and with HD patients given conventional heparin [ 12 ]. The values during dalteparin infusion were lower in both controls and in HD patients. Values in HD patients were lower than in controls both with dalteparin and with conventional heparin. Thus, the highest values were for controls given conventional heparin and the lowest values were those in the present study with dalteparin in HD patients. The differences were remarkably large. These studies have been carried out on separate occasions over several years, but duplicate samples had been saved frozen. To check the consistency of the values, the duplicate samples from the 0, 15 and 30 min time points were thawed and assayed for LPL activity and mass. There was good agreement between the activities from the earlier and the repeated assay for all four studies (Fig 2A ). Hence, the large differences between the LPL activities registered for controls and HD patients and for the two different heparin preparations were real. As an additional test of the consistency of the data, we plotted the increase in LPL activity and LPL mass from basal (before heparin) at the 30 min time points (Fig 2B ). The basis for this is that previous studies have indicated that heparin releases mainly the active form of the lipase [ 26 ]. For regression analysis, changes in LPL mass of less than 100 ng/mL were excluded because heparin may release some inactive LPL [ 26 ] and because of the uncertainty in calculating small differences between pre- and post-heparin mass. The analysis returned a slope of 0.46 ± 0.05 mU/ng (r = 0.94, p < 0.001), consistent with the expected specific activity of human LPL (around 0,4 mU/ng under our assay conditions). Figure 2 Evaluation of the consistency of data from four separate studies by repeated assay of frozen samples. Samples had been obtained and treated as described in the methods section and then stored frozen at -70°C in three earlier studies of plasma LPL during infusion of conventional heparin or dalteparin in control subjects or in dialysis patients [12,22,23] and the present study. Samples from the 0, 15 and 30 min time points were thawed and assayed for LPL activity and mass. Panel A shows the LPL activities at 15 and 30 min recorded on the second assay, as a function of the value recorded on the original assay. Regression analysis gave a slope indicating that the repeated value was 113 % of the original (r = 0.94, p < 0.0001). Panel B shows the increase of LPL activity over the baseline samples plotted against the increase of LPL mass for the 30 min samples. For this, values from the second assay were used (LPL mass was not determined in some of the earlier studies). A regression analysis, excluding samples for which the increase in LPL mass was less than 100 μg/mL, returned a slope of 0.46 ± 0.05 mU/ng LPL (r = 0.94, p < 0.001). Same symbols as in Fig 1. Triglycerides TG remained essentially unchanged for the first two hours and then increased. The change from 2.14 mmol/L (range 1.09–3.41) at the start, to 2.59 mmol/L (range 1.49–5.04) at the end of the dialysis represents a 21 % increase (p < 0.01). Compared to the values during the earlier dialysis with conventional heparin, there was no statistically significant difference at the start of dialysis, but from 30 minutes and through the remaining session TG values were significantly higher during the dialysis with dalteparin (p < 0.05) (Fig 3 ). There was no drop in TG at 30 to 60 min like that found using conventional heparin. There was no correlation between LPL activity and changes in TG during any of the dialysis-sessions. To further illustrate the changes in TG concentrations we have set the baseline value for each individual to 100% and calculated the changes from this. Median values for the changes are plotted in Figure 4 which reinforces the conclusion that there was no significant decrease of TG during the first two hours during the dialysis with dalteparin, in contrast to the marked drop observed during dialysis with conventional heparin [ 12 ], and during infusion of either conventional heparin [ 23 ] or dalteparin in control subjects [ 22 ]. Figure 3 Plasma TG concentrations during infusion of dalteparin. The figure shows individual curves for the nine subjects in the present study. The inset compares median values from the present study (□) with those from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin (●), control subjects given dalteparin (○), HD patients given conventional heparin (■). P < 0.05 for dalteparin compared to conventional heparin given to HD patients. Figure 4 Changes in plasma TG concentrations during infusion of dalteparin or conventional heparin to HD patients or matched controls. For this, the basal TG concentration for each individual was set to 100% and the concentrations observed at the subsequent time points were calculated relative to this. The figure shows median values from the present study (□) and from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin (●), control subjects given dalteparin (○), HD patients given conventional heparin (■). Cholesterol Total cholesterol increased from 4.6 mmol/L (range 3.6–7.1) at start, to 6.1 mmol/L (range 3.8–8.4) at the end (32%, p < 0.05). This differs from the earlier dialysis with conventional heparin, when total cholesterol did not change from baseline [ 12 ]. HDL-cholesterol did not change from baseline. Again, this differs from the dialysis with conventional heparin when HDL-cholesterol increased from 1.09 mmol/L (range 0.67–2.06) at start to 1.19 mmol/L (range 0.67–2.31) at the end (p < 0.05). No correlation was found between LPL activity and total or HDL-cholesterol changes during any of the dialysis-sessions. The median LDL-cholesterol, calculated by the Friedewald formula, was 2.9 mmol/L (range 2.1–4.9) before the dialysis and increased to 3.75 (2.1–5.6) at 240 min. This corresponds to an increase of 29% (p < 0.05). Discussion This study shows the same pattern of plasma LPL activity in HD patients given dalteparin as observed in previous studies with control subjects given dalteparin [ 22 ] or conventional heparin [ 23 ] and in HD patients given conventional heparin [ 12 ]. The novel aspect is how remarkably low the plasma LPL activities were in the HD patients. This prompted us to re-analyze frozen samples from the earlier studies to rule out the possibility of assay variations as a confounding factor. The increase in LPL mass was also low in the HD patients given dalteparin, and corresponded well to the increase of LPL activity. This further supports the conclusion that infusion of dalteparin to HD patients resulted in low levels of LPL in the circulating blood, compared to what was seen when conventional heparin was infused [ 12 ] or when controls were given dalteparin or conventional heparin [ 22 , 23 ]. There are several earlier studies that show that administration of LMW heparin results in lower plasma levels of LPL than conventional heparin [ 27 - 30 ]. LMW heparin preparations differ considerably in their molecular characteristics and caution should be exercised when extrapolating from one preparation to another. The comparisons to our earlier study with conventional heparin [ 12 ] are based on clinically relevant doses during HD, as recommended from manufacturer's and clinical guidelines, not on a molecule-for-molecule basis. In a study with dalteparin Persson et al . found that the early LPL activity was only about half as high compared to values observed after conventional heparin [ 27 , 29 ]. This is similar to the difference between the AUCs for the early peak of plasma LPL activity observed here and in an earlier study with conventional heparin in HD patients [ 12 ]. The LPL activities were lower in HD patients than observed in controls given dalteparin [ 22 ]. A similar difference was earlier found for infusion of conventional heparin in HD patients [ 12 ] compared to controls [ 23 ]. One possible explanation is that the depletion of LPL stores during the dialysis-sessions is not fully restored between the sessions. Arnadottir et al . have, however, found that the amount of LPL released by a bolus of heparin is restored within 24 hours [ 30 ]. In rats, it takes about four hours after a single bolus of LMW heparin before the LPL stores are replenished [ 31 ] and chylomicron catabolism occurs at normal rate [ 32 ]. A more likely explanation is that the kidney dysfunction as such causes an impairment of the LPL system [ 33 ]. Yet another possibility, that has not been explored, is that the kidney itself makes an important contribution to overall LPL stores. This is the case in mink, where kidney has a higher LPL activity than any other tissue, and in mice [ 34 , 35 ]. Administration of heparin causes a temporary derangement of lipoprotein metabolism. In our studies with control subjects given conventional heparin or dalteparin the TG concentration decreased after heparin and then gradually increased again so that at the end of the study period TG exceeded the baseline level (see inset in Fig 3 ). This probably reflects that LPL first becomes more available for lipoprotein catabolism as it circulates in blood but then becomes less available when the lipase is removed from blood by the liver and the tissues stores become depleted. This is in accord with observations in animal experiments. Chevreuil et al . found that the clearance of injected radioactively labeled chylomicron TG was dramatically increased five minutes after rats had been given conventional heparin or LMW heparin [ 32 ]. This was associated with an increased appearance of label in plasma FFA, supporting the view that the rate of lipolysis was increased. In contrast, injection of chylomicrons one hour after the heparins resulted in substantially slower clearance compared to saline-treated controls. Appearance of label in plasma FFA was also decreased, suggesting that impaired lipolysis was responsible, at least in part, for the impeded chylomicron clearance. The decrease of plasma TG was small and statistically not significant in the HD patients given dalteparin. This may, at least in part, be explained by the relatively low plasma LPL activities. In addition, there are reports that in patients with nephrosis there are inhibitors of LPL in the circulating blood and that VLDL isolated from such patients are lipolyzed slowly by LPL [ 36 ]. On the other hand, the rise of TG from two hours was pronounced in the HD patients. This indicates that the LPL stores were depleted in these patients even though the plasma LPL activities were low. This is in accord with results from animal experiments. Chevreuil et al. injected decasaccharides to rats [ 20 ]. This resulted in only a small and short-lived increase of LPL activity in blood. Nonetheless, the decasaccharides had apparently removed most of the functional LPL from peripheral tissues. The LPL activity that could be released from isolated hearts by single-pass perfusion with heparin ("functional LPL") was decreased by 75% one hour after the rats had been injected with decasaccharides. The catabolism of chylomicron TG by perfused hearts was delayed to a similar extent. The clearance of labeled chylomicrons injected to rats was markedly delayed from 30 minutes to 2 hours after a decasaccharide injection. After one hour, the fractional catabolic rate was only one-third of the control value. All of this suggests that dalteparin causes a profound depletion of functional LPL even though the plasma levels of LPL activity are relatively low. Conclusions The peak level of LPL in plasma after injection of dalteparin is less than half of that after conventional heparin. This was shown here for a group of HD patients, but a similar difference has earlier been observed for healthy controls. The peak level of LPL is lower in HD patients than in controls both after dalteparin and after conventional heparin. This is probably a consequence of the kidney disease but the detailed mechanism is not known. These two effects compound to an almost ten-fold difference in peak LPL activity comparing the present HD patients given dalteparin to a group of healthy controls given conventional heparin. Analysis of LPL activity and mass in frozen samples from our earlier studies ruled out the possibility of assay variation as a confounding factor. Prior in vitro studies of the LPL-heparin interaction, and animal experiments, indicate that decasaccharides, or longer heparins, release the enzyme efficiently from its binding sites at the vascular endothelium. The difference in plasma levels of LPL is probably due mainly to a difference in how much the respective heparin retards the uptake of LPL by the liver. Immediately after heparin, plasma LPL activity is high and catabolism of TG-rich lipoproteins is accelerated. This acceleration was not evident during infusion of dalteparin to HD-patients. Then follows a period when tissue stores of LPL are depleted and lipoprotein metabolism is retarded. This depletion was at least as marked after dalteparin as after conventional heparin, despite the lower plasma LPL levels. In the present study, the TG level increased significantly more after dalteparin than after conventional heparin. Our results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does. Abbreviations AUC – area under the curve; HD – hemodialysis; HDL – high density lipoprotein; LMW heparin – low molecular weight heparin; LPL – lipoprotein lipase; TG – triglycerides; VLDL – very low density lipoprotein Competing interests The author(s) declare that they have no competing interests. Authors' contributions BN participated in the design of the study, carried out the patient studies, assembled the data, did the statistical analyses, and participated in writing of the manuscript; BS and GO conceived of the study and coordinated the work. TO participated in the design of the study and in writing of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC534103.xml
548291	Human cytomegalovirus plasmid-based amplicon vector system for gene therapy	We have constructed and evaluated the utility of a helper-dependent virus vector system that is derived from Human Cytomegalovirus (HCMV). This vector is based on the herpes simplex virus (HSV) amplicon system and contains the HCMV orthologs of the two cis -acting functions required for replication and packaging of HSV genomes, the complex HCMV viral DNA replication origin (oriLyt), and the cleavage packaging signal (the a sequence). The HCMV amplicon vector replicated independently and was packaged into infectious virions in the presence of helper virus. This vector is capable of delivering and expressing foreign genes in infected cells including progenitor cells such as human CD34+ cells. Packaged defective viral genomes were passaged serially in fibroblasts and could be detected at passage 3; however, the copy number appeared to diminish upon serial passage. The HCMV amplicon offers an alternative vector strategy useful for gene(s) delivery to cells of the hematopoietic lineage.	Background HCMV is a member of the betaherpesvirus family [ 42 , 48 ]. Other members of this family include human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and all are widely distributed in human populations. During productive replication, the 230 kilobase pair (kbp) viral genome replicates by a rolling circle mechanism, which generates long head-to-tail concatemers that are cleaved to unit length and packaged in capsids. The state of the HCMV genome during latency remains unidentified and is likely to be circular and extrachromosomal [ 6 ]. The HCMV genome has been detected in cells within the hematopoietic lineage as early as CD34+ progenitors and up through differentiated macrophages [ 23 , 29 , 38 , 54 ]. Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level [ 13 , 18 , 31 , 43 , 52 , 67 ]. Naturally occurring defective HSV viruses and laboratory derived HSV amplicon vectors are composed of head-to-tail tandem reiterations of subgenomic regions containing a functional origin of DNA replication (Ori S or Ori L ) and a DNA cleavage/packaging signal [ 3 , 4 , 30 , 57 , 60 - 62 ]. These two cis -acting functions can be relatively small ranging from ca . 90–150 base pairs (bp) for the ori and ca . 250–300 bp for the a sequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp [ 1 , 2 , 24 , 37 ]. HCMV is unique among the herpesviruses in not having an origin binding protein homolog that is required for DNA replication [ 45 ]. The HCMV a sequence varies in size from ca . 550 bp to 762 bp, however, the conserved pac-1 and pac-2 cis -elements which determine the sites for cleavage of replicated viral DNA are present [ 15 , 28 , 58 , 64 , 65 ]. In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses [ 45 ]. However two reports described the identification of what may potentially be HCMV defective viruses created by serial high multiplicity passage [ 47 , 59 ]. These reports characterized HCMV defectives as very large subgenomic DNA molecules, in some cases extending over two thirds of the genome. In addition to these replication defective HCMV viruses, a recent report by Borst et al . 2003 [ 7 ], described the construction of an HCMV amplicon. In this report we further utilized the HCMV amplicon for gene delivery to human CD34+ cells. HCMV infects cells of the hematopoietic lineage [ 34 , 38 , 39 , 55 , 68 ]. Viral genomes can be found in CD34+ cells from seropositive individuals and granulocyte-macrophage progenitors and differentiated macrophages can be infected experimentally [ 56 ]. We were interested in determining whether the tropism of HCMV can be exploited to construct defective HCMV virus vectors (amplicons) targeted to hematopoietic stem cells. The general feasibility of such an approach for other cell types has been shown using other herpesviruses, e.g. HSV, EBV, and HHV-7 [ 20 , 25 , 26 , 30 , 35 , 49 , 70 , 71 ]. Methods Cells and virus HCMV Toledo (passage 8, from Dr. S. Plotkin, Aventis Pasteur, Doylestown, PA), and HCMV Towne var RIT (passage 134, from Dr. Plotkin via Dr. Ed Mocarski, Stanford University), were propagated in human fibroblasts (HF) cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, Kans.). Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) (Clonetech, Palo Alto, CA), under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo (G.M. Duke, unpublished data). Plasmid constructions Plasmid pON205 (Spaete and Mocarski, 1985), contains the Towne strain a sequence, was obtained from Ed Mocarski (Stanford University). pEAG2.7EGFP was derived by cloning the EGFP gene from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA) between the Eag I and Sma I site of the β2.7 gene taken from Toledo (G.M. Duke, unpublished data). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb Dra I fragment of Towne var RIT (corresponding to nucleotides 91,166 – 95,909 relative to AD169) (Figure 1B ), spanning the HCMV oriLyt into the Eco RI site of pON205. Tn9-8 was partially sequenced by single-cycle and multicycle dideoxy-nucleotide chain termination method of Sanger et al ., [ 51 ]. The plasmids designated Tn9-8GF5 and Tn9-8GF7 incorporating the EGFP gene with the HCMV Major Immediate Early (MIE) promoter and SV40 poly A sequence was isolated as a 2,334 bp Nsi I fragment from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA), and cloned into the HCMV amplicon Tn9-8 at the Pst I site in both orientations. The gpt gene in Tn9-8- gpt was derived by cloning a PCR fragment from Escherichia coli DH5α using the primer pairs 5'CTGCAGCTAGTCTAGACTGGGACACTTCACATGAGC3'and 5'CTGCAGCTATGTATCTAGAGCCAGGCGTTGAAAAGATTA3'. Figure 1 Schematic representation of amplified region near ori Lyt of the HCMV genome. A . Restriction enzyme map of minimal ori Lyt and adjacent region of heterogeneity (block). B . Region of heterogeneity shown as a dimer. Arrow indicates junction of the repeat segment. The number 91,166 to the left of the restriction map corresponds to the nucleotide position of the AD169 genome (EMBL accession number X17403). C . Autoradiograph of Southern blot utilizing a minimal ori Lyt probe, pON2623 (Kemble et al., 1996). Monomers and dimers are depicted with one and two arrows, respectively. Trimers and tetramers can be seen in the Towne var RIT viral stock. Generation of viral stocks containing amplicons Plasmid DNA was transfected by CaPO 4 precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 10 6 passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously (Spaete and Frenkel, 1982). Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1. Southern blot analysis Viral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), (Maniatis et al., 1989), and immobilized with a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, CA). DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described (Spaete and Mocarski, 1985). Isolation of CD34 + cells and infection with CMV The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc. (Berkeley, CA) using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA). The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was >95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove's modified Dulbecco's Minimal Essential Medium containing 5% fetal bovine serum. 2 × 10 5 CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18–72 hours. At the end of the incubation the cells were harvested for CD34 staining. EGFP expression and immunostaining for flow cytometric analysis Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody (Becton Dickinson, San Jose, CA). Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), at 18 and 36 hours post infection. Results In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and a sequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection. Heterogeneity at oriLyt During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the Eco RI E fragment of Towne that was not present in the Toledo strain [ 27 ]. The Eco RI E region spans in part the complex oriLyt region [ 1 , 2 , 24 , 37 ]. Sequences in a 1.2 kbp repeat fragment were shown to give rise to the heterogeneity observed at this locus in the Towne genome (Figure 1 ). The coordinates of a single repeat unit starts at nucleotide 94,561 relative to the AD169 sequence and end at nucleotide 95,807 [ 10 ]. This segment can repeat at least three times in Towne strains from different passage histories (Fig. 1 ). This heterogeneity is different from the 189 bp repeat region previously described for the Towne strain oriLyt which occurs near the Bam HI sites in Figure 1 (nt 93337–93525 relative to AD169), [ 11 , 12 ]. Since Towne replicates to relatively high titers in cell culture, it was deemed advantageous to incorporate this heterogeneity in the origin containing sequences to be used in the amplicon construct. Construction of the HCMV amplicon As a test of the feasibility of the system, an HCMV amplicon was constructed which incorporated the two cis -acting functions required for the propagation of the defective virus genomes in the presence of helper virus (Figure 2 ). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb Dra I fragment of Towne (corresponding to nucleotides 91,166–95,909 relative to AD169) [ 10 ] (Figure 1B ), spanning the HCMV oriLyt into the Eco RI site of pON205. The resulting amplicon was designated Tn9-8 (Figure 2 ), and was partially sequenced to verify its structure. Figure 2 Schematic representation of the HCMV amplicon plasmids Tn9-8 gpt , Tn9-8GF7 and Tn9-8GF5. The EGFP expression cassette was cloned in two orientations in the unique Pst I site in Tn9-8. The gpt expression cassette was cloned between the unique Pst I and Hind III sites. Generation of viral stocks containing amplicons As a test of the ability of this construct to function as an amplicon, plasmid Tn9-8 was transfected in human fibroblast (HF) cells, and subsequently infected with HCMV Towne strain at an MOI of 5 to provide helper virus replication functions. Seven days later, infected cells were harvested, sonicated, and viral stocks were prepared for passage to fresh HF cells. Fresh HF cells were infected with the progeny of the transfection/infection and incubated for 7 days. The DNA from these infected cells was harvested (designated passage 1), restricted with Hind III and Dpn I, and Southern blotted [ 36 ]. Southern blot analyses of DNA demonstrated that Tn9-8 was susceptible to digestion with Dpn I, consistent with replication of the plasmid in bacteria (Figure 3 , lane 2). In contrast, Tn9-8 in infected cells was resistant to Dpn I demonstrating that it had replicated in eucaryotic cells (Figure 3 , lanes 3–5). This observation is consistent with replication and packaging of Tn9-8 into infectious virions. These results demonstrate that foreign DNA sequences, exemplified by the plasmid pUC9, can be introduced into defective genomes that are packaged and propagated in serially passaged virus stocks. To examine whether these results were the consequence of amplicon replication and packaging or integration of the amplicon plasmid into the helper virus, DNA prepared from passage 1 infected cells was digested with Cla I, Xba I, Afl II and Dpn I. These enzymes digested the Towne helper virus DNA to fragments no larger than 13.6 kbp but do not cut within Tn9-8. The amplicon DNA was significantly larger than 23 kbp consistent with the amplicon being replicated as a concatamer (Figure 4 ). This result indicated that the high molecular weight DNA containing plasmid sequences was packaged independently and was not integrated into helper virus. Figure 3 Southern blot analysis of passage 1 of HCMV amplicon DNA probed with plasmid pUC9. Lane 1. Plasmid Tn9-8 linearized with Hind III serves as a marker for correct migration of monomeric repeats. Lane 2. Plasmid Tn9-8 restricted with Hind III and Dpn I as a control for non-replicating DNA. Lanes 3–5. Infected cell DNAs restricted with Hind III and Dpn I. The signal in lanes 3–5 (arrow) demonstrates authentic replication and packaging of amplicon Tn9-8 in eucaryotic cells. Figure 4 Southern blot analysis of high molecular weight HCMV amplicon DNA at passage 1 probed with plasmid pUC9. DNA prepared from passage 1 infected cells was digested with Cla I, Xba I, Afl II and Dpn I, Southern blotted and probed with plasmid pUC9. The samples are from those shown in Fig. 3, lanes 3 and 5. The high molecular weight DNA containing plasmid sequences (arrow) demonstrates the major hybridizing species migrating slower than the 23 kbp lambda DNA Hind III digest indicated as the marker on the left of the autoradiograph. Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8- gpt ), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8- gpt amplicons did not enhance recovery. Rescue of monomeric repeat units in bacteria Concatemeric DNA was prepared from passage 2 and 3 virus stocks containing the defective virus genomes (Tn9-8- gpt ), digested with Pst I and Hind III, respectively, in order to analyze monomeric repeat units. The Hind III-digested DNA was circularized by ligation and used to transform E. coli bacteria to analyze structure and to demonstrate shuttle vector capability between eucaryotic and bacterial hosts. A number of plasmids prepared from the rescue attempt had a restriction enzyme pattern indistinguishable from the input (Fig. 5A , lanes 1 and 2). Other plasmids however exhibited the expected restriction pattern consistent with a head-to-tail amplification of the a sequence (lanes 3 and 4). Digestion with Nae I produced a fragment of the predicted size of a unit length a sequence (762 bp), and this product hybridized with an a sequence specific probe ( Pst I- Sgr AI fragment from Tn9-8), (Figs. 2 and 5B , lanes 3 and 4). This type of amplification has been readily seen in restriction enzyme digested DNA preparations of parental genomes of both HSV and HCMV [ 31 , 40 , 41 , 58 , 64 , 69 ] and has also been observed in HSV amplicons using Southern blot hybridizations [ 15 ]. Figure 5 Tn9-8- gpt was rescued from concatamers following serial passage in HF cells. (A) Tn9-8- gpt was passaged in HF cells and monomer units were recovered by linearizing concatemeric DNA from serial passage 3 with Hind III and cloning in bacteria (lanes 2 and 4) and also by linearizing passage 2 DNA with Pst I and cloning in bacteria (lane 3). Lane 1 represents the unpassaged clone for comparison. Following rescue in bacteria, DNA was prepared and cut with Nco I. Fragments were separated on an agarose gel and visualized with ethidium bromide staining (left panel). The gel was transferred to a nylon membrane and probed with an a sequence specific probe (right panel). (B) Fragments from an Nae I digest were also separated on an agarose gel and visualized as in panel A. The gel was transferred to a nylon membrane and probed with an a sequence specific probe. Lane 5 shows a 100 bp ladder. Lanes 3 and 4 show a ca . 800 bp fragment that hybridizes to a sequences. Expression of heterologous genes in an HCMV amplicon To demonstrate that the HCMV amplicon could be used as a vector system to support the expression of a foreign gene, EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter was used as test reporter gene. Two resulting amplicon plasmids designated Tn9-8GF5 and Tn9-8GF7 both expressed EGFP following transfection of HF cells in the absence of helper virus, as expected (not shown). Packaged amplicons were generated by introduction of Tn9-8GF5 into cells and infecting with HCMV 24 hours later at an MOI of 5. Transfection-derived viral stocks were passaged onto fresh HF cells supplemented with Towne helper virus at an MOI of 1. Viral stocks prepared from passage 1 were used to infect HF cells and grown on 12-well tissue culture plates. A limited number (ca. 0. 1%) of brightly fluorescing cells could be seen by microscopic examination at 24, 72 and 96 hours post-infection (Figure 6 ). This demonstrates that a foreign gene can be expressed in the context of a HCMV amplicon viral stock in infected HF cells. Figure 6 Fluorescent Microscopic Analysis of TN9-8GF5 amplicon infected cells. Human fibroblast cells (HF) or human cord blood CD34 + cells were infected with TN9-8GF5 amplicon-containing stocks, or mock infected. Cells were observed at different time-points 24, 72 and 96 hrs post infection with TN9-8GF5 amplicon under the fluorescent microscope (Nikon TE2000 microscope). EGFP expressing fluorescent cells were observed in the TN9-8GF5 amplicon infected human fibroblast cells or human CD34+ cells at different time-points. Control uninfected cells were negative (not shown). To test the utility of the HCMV amplicon in gene therapy or gene delivery, we used packaged amplicons in viral stocks to infect and deliver an expressed gene into human CD34+ progenitor cells. Viral stocks containing amplicons carrying EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated (Figure 6 ). In a separate experiment, at 36 hours post infection, the cells were stained with PE-labeled anti-CD34 and analyzed for the CD34 marker and EGFP expression. EGFP expression was observed in the CD34+ population in the TN9-8GF5 amplicon (0.3%, 0.1%) (Figure 7a , & 7b ) or the CMV-EGFP virus (0.6%) (Figure 7c ) at 36 hours post infection. The CMV Towne control-virus infected cells or uninfected CD34+ cell control were negative for EGFP expression (Figure 7d , & 7e ). A small population (7–12%) of the cells lost expression of the CD34+ marker upon in vitro culture. EGFP expression was also observed in a CD34(-) population infected with either TN9-8GF5 amplicon containing viral stocks (0.8%, 0.1%) or with the CMV-EGFP virus, RC2.7EGFP (3.6%) (Figure 7a, 7b & 7c ). The CMV Towne infected cells or uninfected control cell also had a significant CD34 negative population but were negative for EGFP expression (Figure 7d , and 7e ). These results clearly demonstrate that CD34+ cells can be infected with replication competent or incompetent CMV vectors expressing a foreign gene. Figure 7 Flow cytometry analysis of human cord blood CD34 + cells infected with CMV amplicon containing stocks, virus, or uninfected cell control. TN9-8GF5 amplicon ( a,b ), CMV-EGFP (RC2.7EGFP) virus ( c ), CMV (Towne) infected ( d ), or control uninfected human cord blood CD34 cells ( e,f ), were stained 36 hours post-infection with PE-antiCD34 antibody ( a-e ), or were left unstained ( f ), and were analyzed for two-color cytometry analysis using a FACS Calibur instrument. The dot-plots are generated using Cell Quest software and reveal the EGFP + cells populations. Numbers in the upper right and lower right quadrants indicate percentage of the EGFP + CD34 + and EGFP + CD34 - cells respectively. A frequency lower than 0.01% is considered negative. Conclusions We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells. Advantages for use of the HCMV amplicon Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells (CD34+) and other dividing and non-dividing cell types which may support HCMV infection [ 34 , 38 , 39 , 55 , 68 ]. Genetic hematological disorders such as thalassemias and sickle-cell anemia and other hemaglobinopathies could therefore be targeted for therapy with this strategy. Another potential advantage for the system is that vector DNA could possibly be maintained as an episome with minimal concern for the potential consequences of random integration of vector DNA (i.e. activation of oncogenes or inactivation of tumor suppressor genes). In order to insure efficient segregation as an episome, the EBV latent replication origin, oriP, and the transactivator, EBNA-1, could be added as was previously shown for another hybrid herpesvirus vector [ 71 ]. However such a modification may not be necessary because HCMV genomes appear to be carried continuously in cells of hematopoietic origin in infected individuals. Yet another potential advantage as with other herpesviral vectors, is that the HCMV vector system should have the capacity for very large inserts. Infection of CD34+ cells with HCMV The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vivo and in vitro [ 53 ]. Furthermore, hematopoietic stem cells are also reported as a site for HCMV latency. Efficient transduction of human CD34+ cells with retroviral and non-viral vectors has been unsatisfactory due to the lack of maintenance of high levels of expression of the transgene following engraftment of the engineered cells [ 16 ]. The HCMV MIE promoter may not be the right promoter for optimal expression in a CD34+ cells, since it has been shown that in the context of a lentiviral-based gene transfer system this promoter appeared to function less efficiently due to a cell-type specific expression defect [ 16 ]. The approaches to improving the efficiency of gene transfer into human cells have focused on improving gene delivery vectors and optimizing ex vivo culture conditions, which preserve the developmental properties of the stem cells [ 14 , 22 ]. Umbilical cord blood is recognized as a rich source of hematopoietic CD34+ stem cells [ 33 ]. In our experiments we used cord blood derived CD34+ cells for infection with HCMV amplicon containing stocks or HCMV-EGFP virus. However, bone marrow derived CD34+ cells have also been shown to be infectable in vitro with HCMV [ 34 ]. Gentry & Smith [ 21 ], reported a progressive loss of primitive cell properties including a reduction of CD34 expression upon in vitro culture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 in vitro showed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population [ 9 ]. In our experiments with >95% pure cord blood derived CD34+ cell population, a loss of CD34 expression in a small percent population (9–12%) of stem cells upon in vitro culture has been observed. EGFP expression was also seen in the CD34(-) population (Fig 7a, 7b , & 7c ). It is possible that HCMV infection of CD34 cells could induce cell differentiation and loss of primitive properties including reduction of CD34 expression. Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines [ 5 , 8 , 19 , 32 , 44 , 46 , 50 , 66 ]. The HCMV amplicons contain only the cis -acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks [ 17 , 63 ]. These preparations would be useful for therapeutic applications in immuno-compromised patients. Competing Interests The authors of this publication are supported financially by salary and shares of MedImmune Inc., during the completion of this work. A patent application has been filed with the United States Patent Office relating to the content of this manuscript. The authors have assigned all rights of ownership to MedImmune Inc. The authors declare that they have no other competing interest. Authors' Contributions KM carried out the expression analysis in human fibroblasts and CD34 cells. MNP & GMD generated amplicon stocks and MNP did the ' a ' sequence analysis. GWK provided critical intellectual input. RRS provided the original idea and experimental design as well as cloning of the amplicon, generation of amplicon stocks and Southern analysis.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC548291.xml
514562	Intrathecal baclofen withdrawal syndrome- a life-threatening complication of baclofen pump: A case report	Background Intrathecal baclofen pump has been used effectively with increasing frequency in patients with severe spasticity, particularly for those patients who are unresponsive to conservative pharmacotherapy or develop intolerable side effects at therapeutic doses of oral baclofen. Drowsiness, nausea, headache, muscle weakness, light-headedness and return of pretreatment spasticity can be caused by intrathecal pump delivering an incorrect dose of baclofen. Intrathecal baclofen withdrawal syndrome is a very rare, potentially life-threatening complication of baclofen pump caused by an abrupt cessation of intrathecal baclofen. Case presentation A 24-year-old man with a past medical history of cerebral palsy and spastic quadriparesis developed hyperthermia, disseminated intravascular coagulation, rhabdomyolysis, acute renal failure and multisystem organ failure leading to a full-blown intrathecal baclofen withdrawal syndrome. Intrathecal baclofen pump analysis revealed that it was stopped due to some programming error. He was treated effectively with supportive care, high-dose benzodiazepines and reinstitution of baclofen pump. Conclusion The episodes of intrathecal baclofen withdrawal syndrome are mostly caused by preventable human errors or pump malfunction. Educating patients and their caregivers about the syndrome, and regular check-up of baclofen pump may decrease the incidence of intrathecal baclofen withdrawal syndrome. Oral baclofen replacement may not be an effective method to treat or prevent intrathecal baclofen withdrawal syndrome. Management includes an early recognition of syndrome, proper intensive care management, high-dose benzodiazepines and prompt analysis of intrathecal pump with reinstitution of baclofen.	Background Baclofen is a gamma-aminobutyric acid (GABA) analog that has inhibitory effects on spinal cord reflexes and brain. Intrathecal baclofen (ITB) therapy consists of long-term delivery of baclofen to the intrathecal space. Intrathecal baclofen has been used to treat spasticity due to cerebral palsy, brain or spinal cord injury, multiple sclerosis, dystonia, stroke and stiff-man syndrome, particularly for those patients who are unresponsive to conservative pharmacotherapy or develop intolerable side effects at therapeutic doses of oral baclofen [ 1 ]. Side effects such as drowsiness, nausea, headache, muscle weakness and light-headedness can occur as a result of the pump delivering an incorrect dose of baclofen. Sudden cessation of ITB administration can cause mild symptoms like reappearance of baseline level of spasticity associated with pruritis, anxiety and disorientation [ 2 ]. These mild symptoms represent "loss of drug effect". All patients experience "loss of drug effect" when ITB is discontinued, only a small (but unknown) proportion of patients develop a full-blown potentially life-threatening withdrawal syndrome. We report a case of ITB withdrawal syndrome developing hyperthermia, severe spasticity, disseminated intravascular coagulation, rhabdomyolysis, acute renal failure and multisystem organ failure. Case presentation A 24-year-old man was admitted to our intensive care unit (ICU) with a possible diagnosis of seizure disorder and sepsis. He had a past medical history of cerebral palsy and spastic quadriparesis. Three years ago, he had an ITB pump implanted for spasticity refractory to the high doses of oral baclofen. He had a significant improvement in spasticity, social and functional capacity in the past three years. Later, he developed some disorientation and increased spasticity. He was taken to a local physician who prescribed oral baclofen (120 mg daily in four divided doses) for his increased spasticity. He also advised him to have his ITB pump checked immediately. The following day, his spasticity increased even after taking oral baclofen. He developed multiple seizures and respiratory distress in the next 24-hour period. Subsequently, he was admitted in a local hospital where he was orally intubated and transferred to our ICU for aggressive management. On presentation, his temperature was 104.6°F (40.3°C), heart rate 127 beats per minute, and the blood pressure was 85/45 mm/Hg. His ventilator settings were: assist-control ventilation mode; respiratory rate, 15 breaths per minute; tidal volume, 650 mL; positive end expiratory pressure (PEEP), 5 cm H 2 O; and FiO 2 , 60%. His spontaneous respiratory rate was 18 breaths per minute and an oxygen saturation of 100% was noted on pulse oximetry. In the local hospital, he was documented to have a high fever of 107°F (41.6°C) and he had received intravenous lorazepam, phenytoin, pantoprazole, piperacillin/tazobactem and dopamine. On physical examination, neurologically he was unconscious with decerebrate posturing and his Glasgow coma scale was 6. He had an absent corneal and gag reflexes. He was moving all four limbs in response to noxious stimuli. He was also noted to have an extreme spasticity in all four limbs. Lung examination revealed decreased breath sounds in the left lower base. Cardiac examination was unremarkable. He had a palpable baclofen pump on abdominal wall and bowel sounds were heard. The differential diagnoses were septic shock, meningitis, neuroleptic malignant syndrome and malignant hyperthermia. The initial laboratory results showed serum creatinine phosphokinase (CPK) 5250 U/L (Normal, 25–235 U/L) and CPK-MB fraction 12.1 ng/ml (Normal, 0.5–6.3 U/L). Serum chemistry revealed sodium 142 mmol/L, potassium 5.1 mmol/L, chloride 120 mmol/L, bicarbonate 13 mmol/L, and creatinine 2.1 mg/dl. Hemogram showed white blood cell count 12.2 K/UL, hemoglobin 16.5 g/dl and platelet count 9 K/UL (Normal, 130–400 K/UL). Liver function test showed aspartate aminotransferase (ALT) 1128 U/L, alanine aminotransferase (AST) 1140 U/L, alkaline phophatase 90 U/L, total bilirubin 1.2 mg/dl, conjugated bilirubin 0.7 mg/dl, prothrombin time 20.2 seconds (Normal, 10–12.5 seconds), and INR 2.0 (Normal, 0.9–1.1). Blood and urine cultures were obtained. Chest radiograph was normal. A computed tomography (CT) scan of the chest revealed atelectasis of the left lung base. His CT scan of head did not show any acute infarct or bleeding. His initial management included intravenous fluids, norepinephrine, platelet transfusion, phenytoin, propofol and broad-spectrum antibiotics (vancomycin, ceftriaxone) for suspected meningitis and septic shock. He received intravenous lorazepam (4–8 mg every four hours) for his spasticity. Next day, his spasticity improved and an ITB specialist investigated his baclofen pump. His baclofen pump analysis revealed that it was stopped due to some programming error, which was restarted at a previously prescribed baclofen rate (260 μg/day). On third hospital day, his serum CPK was 15,878 U/L, AST was 2566 U/L, ALT was 2993 U/L, while CPK-MB fraction came down to 3.4 ng/ml. His urine output decreased (<400 ml/ day) and serum creatinine increased in the range of 5–6 mg/dl. Later, he was hemodialyzed few times during the course of hospitalization due to acute renal failure. His echocardiogram showed left ventricular ejection fraction of 20–25% and severe global hypokinesis. His electroencephalogram did not reveal any epileptogenic activity. He developed full-blown multisystem organ failure with an evidence of shock liver, renal failure, respiratory failure, disseminated intravascular coagulation and myocardial depression. His nutrition was started on nasogastric tube feedings, and proper ventilator care was taken through a tracheostomy tube. His serum baclofen obtained at the time of admission was less than 0.02 μg/ml (Expected values, 0.08–0.4 μg/ml). After a three-week course of aggressive management in ICU, he was weaned off from the ventilator and his multiple organ shock resolved. At a six-month follow-up, he was observed in a nursing home with his baseline functional, social, and family activities. Discussion Intrathecal baclofen provides an effective improvement in the spasticity of patients whose spasticity is not sufficiently managed by oral baclofen or other oral anti-spastic medications [ 1 ]. Regardless of the cause of spasticity (cerebral or spinal), anti-spastic effects of baclofen occur at spinal level. Poor response of oral baclofen in many patients can be explained by the fact that the spinal cord represents only about 2% mass of the brain, and receives proportionately a lower blood flow as a fraction of cardiac output. Therefore, cerebral side effects often occur before therapeutic anti-spastic effects of oral baclofen are observed. A programmable ITB pump provides a direct, pattern-controlled delivery of baclofen to the spinal cord. Precise delivery of the ITB pump yields better spasticity reduction at 1000 times lower than the doses of oral baclofen. In addition, the adverse effects are minimized as compared with oral baclofen. ITB provides reduced tone, spasms and pain, improves speech, mobility, sleep quality and bladder control, with a response rate up to 97% in adults and children [ 3 , 4 ]. The ITB pump is approximately 3 inches wide and 1 inch thick. It is surgically implanted in the subcutaneous tissue of anterior abdominal wall. Baclofen is delivered via a silicone rubber catheter into the lumbar subarachanoid space. The ITB pump delivers approximately 100–900 μg/day of baclofen, and it is titrated for the desired clinical response. It is also equipped with an alarm that signals low volume, low battery, or malfunction. Nine years ago, catheter or pump-related malfunction that leads to an overdose or withdrawal has been reported in 40 % of the patients with ITB pump [ 5 ]. Catheter system, operative techniques and programming system of the ITB pump have now been improved significantly to reduce the incidence of an overdose or withdrawal. The precise mechanism of action of baclofen as a muscle relaxant and anti-spasticity agent is not fully understood. Baclofen inhibits both monosynaptic and polysynaptic reflexes at the spinal cord level [ 6 ], possibly by decreasing excitatory neurotransmitter release from primary afferent terminals, although actions at supraspinal sites may also contribute to its clinical effects. Baclofen also causes enhancement of vagal tone and inhibition of mesolimbic and nigrostriatal dopamine neurons (directly or via inhibiting substance P) [ 7 ]. Baclofen is a structural analog of the inhibitory neurotransmitter GABA, and may exert its effects by stimulation of the GABA B receptor to cause muscle relaxation. Baclofen reduces increased muscle tone, Babinski sign, tendon reflexes, ankle clonus and sometimes decreases muscle force. Long-term ITB infusion causes down-regulation of GABA B receptors in the CNS and spinal cord. Down-regulation of GABA B receptors accounts for the decreased sensitivity to the baclofen over time. Although GABA B receptors are down-regulated, it is the baclofen itself that causes increased inhibitory tone in the CNS and spinal cord [ 8 ]. Therefore, abrupt ITB withdrawal results in a predominance of excitatory effects and simulates other conditions that are associated with CNS hyperexcitability and severe spasticity. Sudden cessation of ITB administration can cause mild symptoms like reappearance of baseline level of spasticity associated with pruritis, anxiety and disorientation. These mild symptoms represent "loss of drug effect". All patients experience "loss of drug effect" when ITB is discontinued. However, more severe symptoms like hyperthermia (109.4°F), myoclonus, seizures [ 9 - 12 ], rhabdomyolysis, disseminated intravascular coagulation, multisystem organ failure [ 10 ], cardiac arrest, coma and death [ 9 , 12 , 13 ] have been well reported, and represents a full-blown life-threatening ITB withdrawal syndrome. Food and drug administration (FDA) of USA has included a drug label warning for baclofen withdrawal syndrome in April 2002 [ 1 , 13 ]. Differential diagnoses include malignant hyperthermia, neuroleptic-malignant syndrome, autonomic dysreflexia, sepsis and meningitis. ITB withdrawal syndrome has been fatal in some cases. Six patients have died out of 27 cases reported to FDA [ 13 ]. Most reported episodes of ITB withdrawal were caused by preventable human errors or oversights. However, catheter dislodgement, catheter migration and kinks, and other catheter-related issues might be more common than pump-related malfunctions [ 1 , 14 ]. Close attention to pump refilling and programming procedures may reduce the incidence of ITB withdrawal syndrome. Benzodiazepines are helpful in controlling spasticity and seizures during ITB withdrawal syndrome [ 1 , 10 ]. Benzodiazepines activate central receptors and GABA A receptors of spinal cord by different mechanisms [ 1 ]. Therefore, ITB induced down-regulation of GABA B receptors do not interfere with benzodiazepine's mechanism of action. During a planned removal of ITB pump due to infection or other causes, premedication with high doses of benzodiazepines and augmented oral baclofen is usually administered in the hospitals to prevent spasticity. Similarly, high doses of oral baclofen is also tried in some cases of ITB withdrawal syndrome [ 15 , 16 ]. But failure of high doses of oral baclofen (80 mg three times daily) have been reported recently [ 17 ]. High doses of oral baclofen may not be adequate to treat or prevent ITB withdrawal because of down-regulation of central GABA B receptors due to chronic ITB administration. Moreover, it has been suggested that it may take many hundreds of grams of oral baclofen to achieve a therapeutic baclofen level in the cerebrospinal fluid, compared to the patients who had effective spasticity control with an ITB pump [ 17 ]. Although, our patient received oral baclofen (120 mg daily in four divided doses) initially but these doses may be low enough to prevent ITB withdrawal syndrome. Failure of high doses of oral baclofen suggests that resumption of GABA B receptor agonist by prompt restoration of ITB pump and proper supportive care might be the best treatment. Similarly, high-dose benzodiazepines may be effective because of similar mechanism of action on widespread CNS GABA A receptors. High-dose benzodiazepines could be an initial life saving strategy even before analysis and restoration of ITB pump is achieved, or in cases, where resumption of ITB administration is not as simple as correcting a programming error. Intrathecal baclofen bolus is appropriate, but due to the risk of inadvertent overdose, an experienced physician should immediately perform reinstitution of ITB pump. We had restarted ITB in our patient at a previously prescribed dose. However, a much higher dose of baclofen could have safely been given to overcome the spasticity since the patient was in an ICU. High-dose dantrolene (a direct muscle relaxant that acts on sarcoplasmic reticulum of skeletal muscle) has been tried to reduce spasticity and fever in ITB withdrawal syndrome [ 18 ]. Reduction in fever may be due to cessation of repetitive and thermogenic contractions of muscle fibers. It is unlikely that dantrolene has any GABA agonistic effects, and administration of dantrolene may not be accompanied by any correction in anomalies of CNS functions. Therefore, seizures, autonomic instability and death may occur in ITB withdrawal syndrome even after controlling spasticity with dantrolene. Cyproheptadine (a non-selective serotonin antagonist) has also been used postulating that ITB withdrawal may be a form of serotonergic syndrome that occurs from the loss of GABA B receptor-mediated presynaptic inhibition of serotonin [ 19 ]. We did not consider dantrolene or cyproheptadine in our patient due to lack of sufficient clinical support in the treatment of ITB withdrawal syndrome. There was a three-day period of delay in diagnosing the ITB withdrawal syndrome leading to deterioration and multisystem organ failure. However, our patient had a successful recovery in response to restoration of baclofen pump and adequate intensive care management. Conclusions Baclofen withdrawal syndrome is a potentially life-threatening complication of intrathecal baclofen pump. Empty pump reservoir, catheter leaks or displacement, pump malfunction, programming error and refill of pump with improper drug concentration are the possible mechanisms which could lead to an ITB withdrawal syndrome. Regular check-up of the ITB pump by a specialist, educating patients and their caregivers may decrease the incidence of ITB withdrawal syndrome. Oral baclofen replacement may not be an effective method to treat or prevent ITB withdrawal syndrome. Early recognition of syndrome, high-dose benzodiazepines, prompt analysis of the ITB pump with reinstitution of baclofen, and proper intensive care management are mainstays for the management of ITB withdrawal syndrome. List of abbreviations GABA -gamma amino butyric acid, ITB – intrathecal baclofen, CPK – creatinine phosphokinase, ALT – aspartate aminotransferase, AST – alanine aminotransferase, CT – computed tomography. Competing interests None declared. Authors' contributions IM: Direct patient care, article conception, critical and extensive revision of article for important intellectual content, review and drafting of the original article. AH: Literature search, case review and summary, drafting of the original article. Both authors read and approved the final manuscript and contributed equally to the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC514562.xml
535553	AIDS-defining illnesses among patients with HIV in Singapore, 1985 to 2001: results from the Singapore HIV Observational Cohort Study (SHOCS)	Background The objective was to describe the causes of initial and overall AIDS-defining disease episodes among HIV patients in Singapore. Methods A retrospective observational cohort study was performed of all adult patients seen at the national HIV referral center between 1985 and 2001. Data were extracted from the patients' records by ten trained healthcare workers. AIDS-defining conditions were established using predefined criteria. Results Among 1504 patients, 834 had experienced one or more AIDS-defining diseases. The most frequent causes of the initial AIDS-defining episode were Pneumocystis carinii pneumonia (35.7%), Mycobacterium tuberculosis (22.7%) and herpes simplex (7.4%). In total 1742 AIDS-defining episodes occurred. The most frequent causes were Pneumocystis carinii pneumonia (25.1%), Mycobacterium tuberculosis (16.2%) and cytomegalovirus retinitis (9.5%). Conclusions The most frequent causes of AIDS-defining illnesses in Singapore are similar to those reported in the West, prior to the introduction of anti-retroviral therapy. Opportunistic infections remain the most frequent AIDS-defining illnesses.	Background Cohort studies have provided valuable information on the clinical course of HIV infection in patients from Europe [ 1 - 16 ], North America [ 3 , 17 - 21 ], South America [ 22 , 23 ] and Africa [ 24 - 32 ]. The introduction of antiretroviral therapy has dramatically altered the incidence of AIDS-defining illnesses in the West. In the EuroSIDA study there have been substantial falls in the incidence and total number of AIDS-defining episodes between 1994 and 1998 [ 10 ]. During this period the proportions of reported AIDS-defining illnesses due to cytomegalovirus retinitis and Mycobacterium avium have decreased from 9% to 2% and 8% to 3% respectively [ 10 ]. The proportion due to non-Hodgkin's lymphoma has increased from 4% to 16% [ 10 ]. Substantial decreases in the incidence of disease caused by cytomegalovirus, Pneumocystis carinii , Mycobacterium avium and other opportunistic infections have also been observed in the United States [ 20 , 21 ]. Relatively few data are available on the course of HIV infection in Asian populations. In Bangkok between 1987 and 1993 the most frequent AIDS-defining diagnoses were extrapulmonary tuberculosis (22.8%), Pneumocystis carinii pneumonia (7.0%) and cryptococcal meningitis (10.9%) [ 33 ]. Little is known regarding whether the pattern of HIV-related conditions has changed following the introduction of antiretroviral therapy in Asia. The Communicable Diseases Centre (CDC) in Singapore is the national reference centre for adult patients with HIV and AIDS and nationwide 94.9% of all Singaporean adult residents, who have ever been diagnosed, have been referred there. Underreporting of HIV does not occur as reporting occurs automatically from the reference laboratory. Records have been kept since the first case was identified in Singapore in 1985. Therefore the Singapore HIV Observational Cohort Study (SHOCS) contains almost the entire country's HIV experience. Nucleoside analogues have been available in Singapore since the early 1990s and protease inhibitors and non-nucleoside reverse transcriptase inhibitors since 1997. The usage of these drugs has been increasing annually and during 2000 and 2001 approximately 70–80% of patients regularly attending CDC were taking some form of anti-retroviral therapy. Methods The details of the SHOCS cohort have been described previously [ 34 ]. The study was approved by the Ethics Committee of Tan Tock Seng Hospital. Data were extracted from the case notes of all Singaporean residents who were seen at CDC on or before 31 st December 2001, by ten trained healthcare workers. Probable and confirmed criteria were developed for the diagnosis of category C [ 34 ] and category B (AIDS-related complex) conditions, based on the 1993 guidelines of the United States' Centers for Disease Control and Prevention [ 35 ]. All disease episodes were initially determined by the data extractors and then checked by an infectious disease physician (RB). Diagnoses were not included if they did not fulfill the specified criteria even if there was a strong clinical suspicion of a particular condition. If there was insufficient evidence to satisfy the criteria for a specific diagnosis, a more general diagnosis was assigned, for example cerebral lesion (cause unknown) would be used if there was insufficient evidence to support a diagnosis of toxoplasmosis of the brain or primary cerebral lymphoma. Median CD4 counts were calculated based on the sample taken nearest the time of diagnosis of the initial AIDS-defining condition. CD4 counts from 893 patients were used, as in 37 (4.0%) cases no suitable count was available. Data were entered into a computer database (Microsoft Access™) and checked for errors and inconsistencies. Statistical analyses were performed using Stata 7.0™. Each new or recurrent AIDS-defining condition counted as one event (i.e. a patient with three diagnoses would contribute three events). An AIDS-defining condition was counted as a separate additional event if it recurred six or more months after the initial diagnosis or in the case of tuberculosis, six or more months after treatment completion. To examine the effects of highly active anti-retroviral therapy (HAART) on the proportions of AIDS-defining illnesses due to each of the three most common infections ( Pneumocystis carinii pneumonia, Mycobacterium tuberculosis and cytomegalovirus retinitis), comparisons were made between the pre-HAART era (1986 to 1995) and the established HAART era (2000 to 2001) using χ 2 tests. Results Among the 1504 patients infected with HIV who were seen at CDC between 1985 and the end of 2001, 834 developed one or more AIDS-defining conditions. The most frequent initial AIDS-defining disease episodes were due to Pneumocystis carinii pneumonia (35.7% of total diagnoses), M. tuberculosis (22.7%), herpes simplex (chronic mucocutaneous) (7.4%) and candidiasis (esophageal or tracheobronchial) (6.9%) (table 1 ). The median CD4 count at which the initial AIDS-defining condition occurred was 27 (inter-quartile range 11 – 63). The median CD4 count was higher for M. tuberculosis infection (52, IQR = 18 – 110) and Kaposi's sarcoma (46.5, IQR = 23.5 – 227.5) than for Pneumocystis carinii pneumonia (16.5, IQR = 9 – 40), herpes simplex (25, IQR = 9 – 63) and extrapulmonary cryptococcosis (13, IQR = 6.5 – 39) (table 1 ). 1742 AIDS-defining disease episodes were recorded. The 10 most frequent AIDS-defining conditions were all of infectious etiology. There were 1658 first episodes of disease and 84 recurrences. The most frequent AIDS-defining diseases were Pneumocystis carinii pneumonia (25.1% of total diagnoses), M. tuberculosis (16.2%), cytomegalovirus retinitis (9.5%), candidiasis (7.8%), chronic mucocutaneous herpes simplex (8.2%) and disseminated M. avium (8.2%) (table 2 ). Several additional conditions could have included undiagnosed AIDS-defining diseases. The most common of these infections was pneumonia of unknown cause (117 cases), which is likely to include some patients with Pneumocystis carinii pneumonia (table 3 ). Weight loss of >10% body weight was a frequent problem and affected 383 (25.5%) patients (table 3 ). Between 1996 and 2001 there was a continuous increase in the total number of AIDS-defining disease episodes occurring each year, due to the increase in the number of patients being followed up. There was no significant change in the overall proportion of AIDS-defining episodes caused by infections. Pneumocystis carinii and M. tuberculosis remained the two commonest causes. Between 1986 and 1995 they respectively accounted for 26.7% and 10.8% of all AIDS-defining disease episodes. During 2000 to 2001 the percentage of episodes caused by Pneumocystis carinii was not significantly different at 26.3% (Yates' χ 2 = 0.01, 2df, P = 0.94), but that due to M. tuberculosis had increased to 18.0% (Yates' χ 2 = 8.10, 2df, P = 0.004). There was no significant change in the percentage of episodes due to cytomegalovirus retinitis (9.7% between 1986 and 1995 and 8.5% during 2000 and 2001; Yates' χ 2 = 0.25, 2df, P = 0.62) (table 4 ). Discussion Comparison of HIV-associated morbidity with other populations In the SHOCS cohort the proportion of conditions which were of infectious etiology remained very high throughout the study period. The distribution of conditions was similar to that seen in Western countries prior to the introduction of anti-retroviral therapy [ 1 ]. However the proportion of conditions caused by mycobacteria was higher than in Europe [ 1 , 12 ]. In Bangkok the most common AIDS-defining conditions were infections. Between 1986 and 1993 the most frequent AIDS-defining illnesses in Bangkok were extra-pulmonary tuberculosis (22.8%), cryptococcal meningitis (10.9%) and Pneumocystis carinii pneumonia (7%) [ 33 ]. Between 1993 and 1996 they were extrapulmonary cryptococcosis (38.4%), tuberculosis (37.4%), wasting syndrome (8.1%) and Pneumocystis carinii pneumonia (4.8%) [ 36 ]. Pneumocystis carinii pneumonia caused a much higher proportion of AIDS-defining diseases in Singapore than in Bangkok and cryptococcal meningitis occurred less frequently in Singapore. Potential sources of bias The SHOCS cohort provides data on the experience of 94.9% of Singaporean residents who have been diagnosed with HIV since the epidemic began. There are good diagnostic facilities in Singapore and patients with HIV-related illness receive extensive investigations. Therefore Singapore offers an ideal location for collecting information on the causes of morbidity and mortality in persons with HIV in Asia. Medical record keeping is of a high standard in our hospital and this allowed accurate diagnoses to be assigned for AIDS-defining conditions which occurred throughout the AIDS era. To ensure consistency, data was extracted by carefully trained healthcare workers and diagnoses were assigned using predefined criteria. All AIDS-defining diagnoses and all causes of death were reviewed by the same infectious diseases physician (RB). Follow-up rates in our cohort are very high with only 5.8% of patients lost to follow-up at 12 months and 9.3% after 3 years. The SHOCS cohort therefore provides a unique source of information on HIV infection in an Asian country since the start of the epidemic. In a retrospective study it is impossible to ensure that all patients received a full series of investigations in order to satisfy the study criteria for diagnosis of HIV-related conditions. As a consequence the criteria for making a probable diagnosis are unavoidably biased against certain conditions. For example if a patient undergoes a therapeutic trial of tuberculosis treatment he or she can be given the diagnosis of "probable Mycobacterium tuberculosis ". However if a patient receives a successful trial of treatment for disseminated M. avium , the diagnosis is "probable mycobacteriosis (species unidentified)". Differences in physicians' awareness of the link between HIV and opportunistic infections can influence diagnosis rates. For example, the increase in the proportion of AIDS-defining disease episodes caused by tuberculosis between 1986–1995 and 2000–2001 may have been due to greater awareness of the link between HIV and tuberculosis. Diagnosis rates for M. avium and M. tuberculosis may also have been affected by improvements in mycobacterial culture techniques. Conclusions Despite the availability of anti-retroviral medication, opportunistic infections remain a common problem among HIV patients in Singapore. This may partly be explained by the high proportion of Singaporean patients, who present with advanced disease and very low CD4 counts. These patients often already have one or more AIDS-defining opportunistic infections at first presentation. Opportunistic infections may also occur before adequate immune-reconstitution can occur when HAART is commenced when the patient has a very low CD4 count. Studies in the West have shown that AIDS-defining illnesses occur more frequently among patients who have a CD4 count below 50 when anti-retroviral therapy is commenced than among those with higher CD4 counts [ 37 , 38 ]. Immune-reconstitution may also be impaired by difficulties in maintaining high levels of adherence, sub-optimal treatment regimens and unplanned treatment interruptions (due to adverse effects, financial or logistical reasons). The current reductions in the cost of anti-retroviral therapy (Eg. due to generic manufacture) may enable high levels of anti-retroviral use to be achieved in many Asian countries. The experiences of the SHOCS cohort are likely to be repeated throughout Asia. Our results suggest that avoidable opportunistic infections may continue to occur even when high levels of anti-retroviral use are achieved. To reduce the burden of morbidity and mortality caused by these infections, efforts must be made to diagnose HIV infection earlier and to make the treatment of HIV and its associated opportunistic infections more affordable. List of abbreviations SHOCS = Singapore HIV observational cohort study, CDC = Communicable Diseases Centre, AIDS = acquired immunodeficiency syndrome, HIV = human immunodeficiency virus, HAART = highly active anti-retroviral therapy. Competing interests The authors declare that they have no competing interests. Authors' contributions RB participated in the design of the study, supervised data extraction, performed the statistical analysis and wrote the manuscript. SS designed the database and supervised data extraction. NP participated in the design of the study and contributed to the statistical analysis and production of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC535553.xml
544355	Intestinal parasites prevalence and related factors in school children, a western city sample-Turkey	Background Intestinal parasitic infections are amongst the most common infections worldwide. Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites. Previous studies in Turkey revealed a high prevalence of intestinal parasitic infection. The objectives of the current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7–14 years old school children and to identify associated socio-demographic and environmental factors, behavioral habits and also related complaints. Methods Multistage sampling was used in the selection of the study sample. A questionnaire, cellulose adhesive and a stool specimen examination were done. Results A total of 456 stool specimens were collected. 145 students (31.8%) were infected with one or more intestinal parasites. 29 (6.4%) of the students were infected more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites. The three most common were E. vermicularis , G. intestinalis and E. coli . Intestinal parasite prevalence was higher in rural area, in children with less than primary school educated mother, in children who use hands for washing anal area after defecation, and in children who use toilet paper sometimes or never. The relation between child health and mother education is well known. Children were traditionally taught to wash anal area by hand. Toiler paper usage was not common and might be due to low income or just a behavioral habit also. Most of the complaints of the study population were not significantly related with the intestinal parasitic infection. Conclusions Intestinal parasitic infection is an important public health problem in Aydin, Turkey. Rural residence, mother education less than primary school, sometimes or never usage of toilet paper, and washing anal area by hands after defecation were the significant associations. Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required. The ratio of uneducated women should be declined with specific programs. A multisectoral approach is needed.	Background Intestinal parasitic infections are amongst the most common infections worldwide. It is estimated that some 3.5 billion people are affected, and that 450 million are ill as a result of these infections, the majority being children. These infections are regarded as serious public health problem, as they cause iron deficiency anemia, growth retardation in children and other physical and mental health problems [ 1 ]. Epidemiological research carried out in different countries has shown that the social and economical situation of the individuals is an important cause in the prevalence of intestinal parasites. In addition, poor sanitary and environmental conditions are known to be relevant in the propagations of these infectious agents [ 2 - 4 ]. Previous studies at various institutions in Turkey revealed a high prevalence of intestinal parasitic infections among the following populations: 6–16 year olds (10.8%), 12–16 year olds (48.0%), 7–15 year olds (55.1%), and 6–12 year olds (88.0%) [ 5 - 8 ]. However, almost all of the studies were performed in isolated groups, such as collecting all samples from children attending the same school. Furthermore the majority of the studies were performed in the eastern part of Turkey. They have limitations in regards to giving an idea about all age groups in the region. Although there are studies on interactions between infection and socio-demographic, environmental factors, and behavioral habits from eastern regions, to our knowledge, there is lack of adequate information for the western part of Turkey [ 7 , 9 ]. The objectives of this current study were to determine the prevalence of intestinal parasitic infections in Aydin among 7–14 year old school children and its relation to socio-demographic factors, environmental factors, behavioral habits and complaints related to intestinal infections. Methods Study population The data for the present study was acquired from the primary schools in urban and rural areas of Aydin, a city in the western part of Turkey. The study design was cross-sectional. The sample size was calculated on a prevalence of 30%, d = 0.05 at a confidence level of 95%. A design effect of 2 was used to allow for multistage sampling [ 10 ]. The calculated study population size was 639. Multistage sampling was used in the selection of the study sample. Aydin was separated into four regions according to the socio-economic and health data taken from Directorate of Health. From these regions two schools were selected randomly, one from the urban communities and one from rural. The children were selected from the classes randomly based on the age, gender and weight for the population. First to eighth graders were included in the study, and the total numbers of the students enrolled in the classes were 74, 60, 70, 72, 72, 107, 104, and 81 students, respectively. Permission was obtained from Directorate of Education. The questionnaire and family information form The questionnaire contained four sections: 1. Socio-demographic data: age, gender, residence, education and occupation of parents, number of adults and children in the family and birth order of the child; 2. Environmental factors: housing conditions (ownership of the house, number of rooms and bathrooms) and water supply; 3. Behavior habits: type of toilet commonly used, hand washing (no washing/washing with only water/washing with soap), washing anal area by hands after defecation (yes/no), usage of toilet paper (always/sometimes/never); 4. Complaints: abdominal pain, nausea/vomiting, lack of appetite, abdominal distention, intestinal dismotility, salivation during sleeping, headache, irritability in sleeping, perianal itching, teeth grinding, and history of parasitic infections. An informational document about the study, including how to supply a stool specimen and a cellulose tape slide, was given to each participant for their family. Intestinal parasitic examination Mothers were asked to perform one cellulose tape test on their child who was participating in the study. Laboratory slides were provided with cellulose tape attached to them. The mother collected material for examination in the early morning prior to bathing or defecation. On the same morning a field worker collected these slides to be microscopically examined. The stool specimens (0.5–1.5 gr) were collected in labeled plastic vials without preservatives and transported to the laboratory within four hours after collection. They were examined for the presence of parasites by direct wet mount, Lugol's iodine solution and modified formaline-ethyl acetate sedimentation techniques. The presence of parasites was confirmed when observed by any of the methods above. Statistical analysis A computer program was used for data analysis. The descriptive data was given as a mean ± standard deviation (SD). The chi-squared test and Student t-test were used for the analytic assessment. The differences were considered to be statistically significant when the p-value obtained was less than 0.05. Results A total of 456 (71.4%) samples for both stool specimens and cellulose tape slides were collected. The response rate to the questionnaire was lower with 367 (57.4 %). The mean age was 10.34 ± 2.27, 10.17 ± 2.30 for girls and 10.51 ± 2.23 for boys. Important socio-demographic characteristics, housing conditions and hygienic habits of children are given in Table 1 . Table 1 Important socio-demographic characteristics, housing conditions and hygienic habits of children Characteristics No % Socio-demographic characteristics Residence Urban 258 56.6 Rural 198 43.4 Gender Female 232 50.9 Male 224 49.1 Education of mother No education/primary school incomplete 68 19.0 Primary/secondary school 269 75.4 High school and more 20 5.6 Education of father No education/primary school incomplete 21 5.8 Primary/secondary school 292 81.4 High school and more 46 12.8 Housing conditions Owner 239 66.0 3 rooms and less 154 43.5 4 rooms and more 200 56.5 1 toilet 180 49.9 2 and more toilet 181 50.1 5 and less people living in 267 74.4 6 and more people living in 92 25.6 Municipal water network 232 68.6 Hygienic habits Type of toilet commonly used Modern style 86 26.5 Traditional style 135 41.5 Both 104 32.0 Toilet paper Always 206 57.1 Sometimes 105 29.1 Never 50 13.9 Washing anal area by hands after defecation Yes 138 39.4 No 212 60.6 Washing hands with soap after toilet Always 308 85.3 Sometimes 51 14.1 Never 2 0.6 Taking a bath Once a day 18 5.2 Three times a week 161 46.1 Once a week or less 170 48.7 In all, 145 students (31.8%) were infected with one or more intestinal parasites. 29 (6.4%) of the students were infected with more than one parasite, 26 (5.7%) with two parasites and 3 (0.7%) with three parasites. The most common was Enterobius vermicularis (E. vermicularis ) with 63 (13.8%) pure and 20 (4.4%) with multiple infections, in total 83 (18.2%) infected children. The second was Giardia intestinalis ( G. intestinalis ) with 28 (6.1%) pure and 21 (4.6%) with multiple infections, in total 49 (10.7%) infected children. The third one was Entamoeba coli ( E. coli ) with 21(4.6%) pure and 15 (3.3%) multiple infections, in total 36 (7.9%) infected children. G. intestinalis was the most commonly found parasite in multiple infections. The distribution of parasites is given in Table 2 . Table 2 The parasites distribution of the study population Parasites No. % Single E. vermicularis 63 13.8 G. intestinalis 28 6.1 E. coli 21 4.6 H. nana 4 0.9 Total 116 25.4 Multiple E. vermicularis+G. intestinalis 11 2.4 E. vermicularis+E. coli 8 1.8 G. intestinalis+E. coli 4 0.9 G. intestinalis+H. nana 2 0.4 G. intestinalis+Taenia spp. 1 0.2 G. intestinalis+E. coli+H.nana 2 0.4 E. vermicularis+G. intestinalis+E. coli 1 0.2 Total 29 6.4 Overall total 145 31.8 No statistically significant difference was observed between presence of intestinal parasites and gender, (p = 0.805), and also age (p = 0.916). The prevalence of intestinal parasites were significantly higher (p = 0.042) in the rural area (36.9%) than in the urban area (27.9%). A summary of significant relations observed in overall intestinal parasitic infections for the study population are given in Table 3 . Table 3 Significant relations for the intestinal parasitic infection in the study population Risk Factor Overall infection χ 2 p n % Residence Urban 72 27.9 4.415 0.042 Rural 73 36.9 Mother education Less than primary school 29 42.6 4.436 0.035 Primary school and more 85 29.4 Toilet paper Always 50 24.3 13.596 0.000 Sometimes/never 66 42.6 Washing anal area by hands after defecation Yes 55 39.9 5.503 0.019 No 59 27.8 Parasite-specific significant relations were the following: The prevalence of E. coli infections was significantly higher (p = 0.010) in the rural area (11.1%) than in the urban area (5.0%). Mother education less than a primary school education (p = 0.012), washing anal area by hands after defecation (p = 0.013) were the significant relations for G. intestinalis , where sometimes or non-usage of toilet paper was significant for G. intestinalis (p = 0.008) and for E. vermicularis (p = 0.024) both. Family size was larger in the group infected with G. intestinalis (p = 0.029). The prevalence of intestinal parasitic infection was 31.0% in the group using a municipal water network and 34% in the group lacking a municipal system (p = 0.592). The prevalences of G. intestinalis in these two groups were 9.9% and 16.0% (p = 0.106) respectively. No further significant relationships were found between intestinal parasitic infection and environmental or behavioral factors. The most frequent complaint related with any parasite infection was intestinal dismotility (40.0%). Nausea/vomiting (37.7%) was second and abdominal distention (37.1%) was third. All of the complaints were seen in higher prevalence for E. vermicularis infections than G. intestinalis and E. coli . Discussion It was found that approximately one-third (31.8%) of the students, ages 7 to 14, in Aydin were infected by intestinal parasites. In a sample within the same age group in Izmir, a city also in the western part of Turkey, the prevalence for intestinal parasites was 22.4%, with E. vermicularis (16.0%) and G. intestinalis (11.9%) being the two most common infections, as was observed in Aydin [ 11 ]. In another study performed in Izmir, the prevalence of infection was 45.3% for E. vermicularis , 21% for G. intestinalis , 10% for H. nana , 4.3% for E. coli , 0.03% for Ascaris lumbricoides ( A. lumbricoides ), and 0.03% for Trichuris trichiura ( T. trichiura ). No Taenia was found [ 12 ]. In this current study; the most frequently observed parasites were E. vermicularis , G. intestinalis , and E. coli , 18.2%, 10.7% and 7.9%, respectively. Higher prevalence was found in the studies from the eastern part of Turkey, where the socio-economic and environmental conditions were lower. Additionally, it was observed that the types of parasites found in this study where different than those found in the eastern part of Turkey. A survey conducted among 1001 children in four elementary schools in Sanliurfa found parasites in 88% of the stool samples examined (50% A. lumbricoides , 53% T. trichiura , 22% G. intestinalis , and 11% E. coli ); unfortunately, no data on E. vermicularis was given, because samples with cellulose tape slides were not taken [ 7 ]. In an another study from the eastern region, in an elementary school age group, 48.12% A. lumbricoides , 4.43% T. trichiura and 15.35% G. intestinalis were found [ 13 ]. In the last study, samples with cellulose tape slides were not taken. Geohelmint infections ( Ascaris , Strongyloides and Trichuris ) were of lower prevalence in the western part of Turkey, but of higher prevalence in the eastern part. This difference may be due to improper toilet facilities which require individuals to defecate in areas around their homes as well as the use of fecal material for fertilizer in gardens in the eastern parts of Turkey [ 13 - 15 ]. In a study from the eastern region, 42.2% of children were found to be working in gardens watered with contaminated sewage and eating the vegetables of those gardens. There were 44.7% A. lumbricoides , 11.7% T. trichiura infections in these children while there was 12.2 % A. lumbricoides and 6.6% T. trichiura infections in the control groups [ 14 ]. Enterobiasis occurs worldwide, usually involving school-aged children [ 16 ]. In general E. vermicularis infection is transmitted by hand to mouth and/or person to person directly. High prevalence of E. vermicularis in the current study might be due to improper hygiene including not washing hands with soap after defecation, before eating and preparing foods. In the study area, there are two traditional methods of cleaning anal areas after defecation: (1) washing the anal area by hand with tap water (2) a piece of cloth is used to clean the anal area after defecation. The cloth is used multiple times until the person decides that it has become too dirty after which it is washed and reused. These improper cleaning practices after defecation could be the probable causes behind autoinfection. The higher prevalence of E. vermicularis could also be explained by the highly infectious nature of the parasites. G. intestinalis and E. coli were the most common intestinal protozoa among the study population. Both can be transmitted orally by drinking infected water and both are environmental contaminants of the water supply. The water supply is really an important risk factor for the Giardiasis, and several large outbreaks of giardiasis have resulted from the contamination of municipal water supplies with human waste [ 17 ]. The ingestion of contaminated water is a common problem in Turkey countrywide due to the lower quality of water and faulty sewage lines. The problem is greater in the rural areas that do not have a municipal water network or sewage system [ 18 ]. Contamination of drinking water with Giardia spp . has been increasingly recognized over the past 10 years as a cause of water-borne diseases in humans [ 19 ]. Giardia cysts have been isolated from water supplies in different parts of the world [ 20 , 21 ]. Epidemic giardiasis may be related to drinking water [ 22 ]. G. intestinalis and E. coli are most common in the western part of Turkey [ 11 ]. In a study assessing giardiasis cases in Turkey published within the last 15 years, the prevalence of G. intestinalis was found 11.6% in the western part of Turkey [ 23 ]. From the study, 68.6% of the study group uses municipal water, while the others either use a chlorinated collection tank with a crude water network or purchase bottled water. Although no statistically significant difference was observed, the prevalence of G. intestinalis was lower in the group using municipal water (9.9%) than the other group (16.0%) in the current study. It is thought that an in-depth assessment should be done on the ways that drinking water becomes contaminated. In this current study, there were no cases of Taenia spp . Since the consumption of pork and pork products are forbidden for Muslims this may account for the absence of T. solium cases in the population. It is also a common practice to eat uncooked meat in the eastern part of Turkey, but not in the western part. T. saginata infections are observed in the eastern studies, with prevalence of 13.8% and 12.9% [ 19 , 24 ]. However, in the western region infections by this parasite were not observed in this or in another previous study [ 12 ]. Differences due to gender were not observed in this current study. These results were similar to a study conducted in the central part of Turkey [ 24 ]. In this current study; the prevalence of intestinal parasitic infection was higher in the rural areas. A similar result was found in the central region of Turkey where the prevalence of intestinal parasites was higher in the rural area [ 25 ]. In this current study, most of the complaints by the study population were not significantly related with the intestinal parasitic infection. For example, perianal itching was noted in 15.8 % of the study population. There was no significant difference in the prevalence of this symptom in pinworm infected and non-infected children. Furthermore, no association was found between the prevalence of pinworm infection and a history of teeth grinding, colic, enuresis, and irritability. The complaints may not have been assessed effectively through the use of only a questionnaire without an interview, or they might not be useful for every individual's diagnosis. The prevalence of intestinal parasites was higher in groups where the mother in the household had less than a primary school education, where the hand is habitually used for the cleaning of the anal area and where toilet paper is seldom or never used. The relation between a child's health and the mother's education is well known. Health indicators of children whose mother's education level is lower are always worse [ 26 ]. In the last two groups, the habits of the children are a factor, along with a cultural dimension. They were taught to clean the anal area by washing with the hand. Toiler paper usage was not common, possibly due to low income. Usage of a piece of cloth instead of toilet paper was also common. The major limitation of the current study was a low response rate. The assessment would have been more valuable if a higher response rate could have been obtained. But, it was thought that the results were still important because there is little knowledge on the data of the region. Additionally, the current study was the first population-based study for the region. Although an important number of risk factors were discussed, a few risk factors (e.g.: shoe wearing) were not evaluated in the current study. This might have been another limitation of the study. The important risk factors for the region were evaluated in the study. Conclusion As a conclusion, intestinal parasitic infection is an important public health problem in Aydin, Turkey. Rural residence, households where the mother has less than a primary school education, periodic or non-usage of toilet paper, and the washing of the anal area by hand after defecation were the significant associations. Interventions including health education on personal hygiene to the students and to the parents, especially to mothers are required. The ratio of uneducated women should decline with specific programs. A multisectoral approach is needed. Competing interests The author(s) declare that they have no competing interests. Authors' contributions PO planned the research, performed the sampling and statistical analyzes and wrote draft and final version of the manuscript. SE contributed in planning the research, performed parasitic examinations and contributed discussing the results and writing manuscript. BG performed parasitic examinations and contributed discussing the results. OO organized the work in the schools and participated in its design. EB participated in initial study design, coordinated the study and revised the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544355.xml
546230	Free does not mean affordable: maternity patient expenditures in a public hospital in Bangladesh	Objective This study investigated a) the amount and types of out-of-pocket expenditures by patients for nominally free services in a large public hospital in Bangladesh, b) the factors influencing these expenses, and c) the impact of these expenses on household income. Methods Eighty-one maternity patients were interviewed during their hospitalization in the Dhaka Medical College Hospital. Patients were selected by quota sample to match the distribution of maternity patient categories in the hospital. Patients were interviewed with a semi-structured, in-depth questionnaire. Results All interviewees incurred substantial out-of-pocket expenditures for travel, hospital admission fees, medicine, tests, food, and tips. Only two of the expenditures, travel expenses and admission fees, were not supposed to be provided free of charge by the hospital. The median total per-patient expenditure was $65 (range $2–$350), equivalent to 7% (range 0.04%–225%) of annual household income. Half of all patients reported that their families had to borrow to pay for care at interest rates of 5%–30% per month. A third of these families reported selling jewelry, land or household items to moneylenders. The rural patients reported more difficulty in paying for care than the urban patients. Factors increasing the expenditures were duration of hospitalization, rural residence, and necessary (e.g. C-section, hysterectomy) and unnecessary (e.g. episiotomy) medical procedures. Conclusion Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities.	Background In developing countries governments often subsidize services at public health care facilities and provide them free of charge to users. However, evidence suggests that users still incur large expenditures using the 'free' services for such things that are supposedly provided without charge. Studies have found that patients incurred substantial out-of-pocket expenditures for medicine, food and travel from the use of 'free' public health facilities [ 1 - 3 ]. A study in Vietnam found that out-of-pocket payments can cause serious equity problems such as the poor becoming poorer without greatly affecting the non-poor [ 4 ]. Household difficulty in payment of health care expenses can result in the 'distress sale' of property, delay or abandonment of treatment, and sacrifice expenditures on food and education [ 3 , 5 ]. Other studies have found that introducing or increasing user fees negatively affect the utilization of public health facilities [ 6 - 9 ]. Three previous studies have explored issues related to patient expenditures in Bangladesh [ 3 , 10 , 11 ]. Nahar et al. enumerated the patient expenditures and affordability of free maternity services for normal delivery and caesarean section. Killingsworth et al. explored the linkage between official and unofficial fees in public health facilities, and concluded that these fees had income and equity effects. Stanton et al. reviewed literature on user fees and pointed out the need to further investigate the factors and practices causing patient expenses before institutional implementation of user fees. Thus, this study examined the type, amount and household financial results of out-of-pocket expenditures by patients for nominally free services in a large government hospital in Dhaka. The study also identified the factors and medical practices producing and influencing the out-of-pocket expenditures. Plans to begin fees for service in Bangladesh make it important to document the amount of money actually being paid by the patients under the present system. If current expenditures are large, fee for service may have serious negative impacts on utilization and on the economic well-being of Bangladeshi households. If current expenditures are modest, it is possible that such fees will have a lesser impact. Methods Study site The study was conducted in the Department of Obstetrics and Gynaecology (ObGyn) of the Dhaka Medical College Hospital (DMCH). DMCH is the largest teaching hospital in Bangladesh with 850 beds located in the capital city. DMCH is government funded and provides a wide range of out- and in-patient services. Public hospitals have two payment categories for in-patients: non-paying and paying. Patients first go to an out-patient unit for diagnosis where they are categorized as out- or in-patient. Those categorized as in-patient are then classified as paying or non-paying by observing the clothes and general appearance of the woman and any accompanying relatives. Non-paying patients pay only the hospital admission fee. Paying in-patients are charged the fees for hospital admission, bed, and surgery. The various fees are: hospital admission fee: $0.23, bed fee: $1.34–3.50 per day, surgery fee: $12.50–125. Taka was converted into US dollars using the 1994 exchange rate of US$1.00 = Taka 40.00. Neither patient category is supposed to pay for medicine, tests, food, nursing and other support services during hospitalization; these commodities and services are theoretically provided free by the hospital. Study population, sampling and sample size The study interviewed 81 non-paying in-patients hospitalized for reproductive health conditions (about two thirds were for maternity conditions). Patients were selected by quota sample matching the distribution of the patient categories in the hospital i.e. the selected medical conditions accounted for the greatest number of ObGyn admissions reported for the hospital, and also reflect the causes associated with high maternal mortality and morbidity in Bangladesh [ 12 ]. These included normal vaginal delivery (NVD), caesarean section (C-section), abortion, and hysterectomy. NVDs included cases with episiotomy, without episiotomy, and with eclampsia. C-sections included elective and eclamptic cases. Abortion included non-septic and septic abortions. Hysterectomies included abdominal and vaginal hysterectomies for treating fibroid, prolapsed uterus, and pelvic inflammatory disease. Table 1 illustrates the distribution of the selected cases for this study. Table 1 No. of in-patients surveyed by medical condition No. of patients Normal Vaginal Delivery 19 with episiotomy 5 without episiotomy 5 with eclampsia 9 Caesarean section 20 elective 10 eclamptic 10 Abortion 20 non-septic 10 septic 10 Hysterectomy 22 prolapsed uterus 10 fibroid 9 pelvic inflammatory disease 3 Total 81 Variables Information was collected on various characteristics of the study participants. Demographic characteristics included age, education, marital status, and residence. Socio-economic characteristics included occupation and annual household income. Information was also collected on underlying medical condition. Out-of-pocket expenditure related information included types and amounts of expenses incurred during hospitalization such as those for travel, medicine, food, fees, etc. Factors influencing expenditures included type of treatment received and duration of hospitalization. Sources of funds included amount borrowed and interest charged for borrowed amount. Data collection tools and technique Data were collected from patients and their relatives with semi-structured open-ended questionnaires between January – June 1994. The interviewers were physicians employed in DMCH. The interviewers selected the cases by diagnosis from patient admission records. To minimize possible selection bias the first case was selected randomly from the records and then every third case was selected. The selected patients were interviewed a minimum of three times to minimize recall error. Recall error was also minimized as information was collected while patients were still hospitalized. During the first interview demographic and socio-economic information was collected with structured questions. During the second and third interviews information related to expenditures was collected with open-ended questions. To illustrate the data collection process a description of an interview with a typical C-section patient follows. C-section patients are usually hospitalized for two weeks in DMCH. On the first day of hospitalization an interviewer collected information on patient's age, education, marital status, etc. On the eighth day of hospitalization the second interview collected information on treatment received, treatment related out-of pocket expenditures, annual household income, amount of money borrowed to pay for treatment, source of borrowed money, and interest rate charged. On the fourteenth day the third interview collected more monetary information on out-of-pocket expenditures, and on expected expenditures immediately after leaving the hospital. This survey did not cover the expenditures for the full course of the treatment. Expenditure estimates were derived for the duration of the current hospitalization only, i.e. from the day of admission until the day of discharge. Expenditures immediately before admission and after discharge from the hospital included only travel expenses to and from the hospital for the patient and her accompanying relatives. Results Socio-demographic characteristics of study participants The median age of the study participants was 26 years (range 15–60 years). The majority (88%) of the patients were married, the rest were separated (4%), divorced (2%), and unmarried (1%). Forty-four percent of the patients lived in rural areas. The median annual household income was $750 (range $3–$6000) per respondent. The annual household income was higher for the urban (median $900; range $150–$6000) than the rural (median $615; range $3–$6000) respondents. Patient out-of-pocket expenditures All 81 patients interviewed reported incurring substantial out-of-pocket expenditures during their hospitalization. These out-of-pocket expenditures were for travel, hospital admission fee, medicine, tests, food, tips, and other items. As expected there were expenditures related to travel and admission fees which the hospital is not supposed to subsidize. But there were also expenditures for medicine, tests, food, tips, and other items which were supposed to be provided free from the hospital but were not. The median total expenditure for hospitalization was $65 (range $2.15–$350) per patient. On average, 61% of these expenditures ($49) were for services and commodities that were supposed to be provided free from the hospital but were not. The per patient median expenditure for the various expense categories were: medicine $26, tests 0, tips $1.25, food $1.25, other items $4.38, travel $22.25, and hospital admission fee $0.25. On average, medicine constituted 42%, travel 38%, tests 5%, food 4%, tips 2%, admission fees <1%, and others 8% of the total expenditures. C-section and hysterectomy cases had the highest median expenditures. Table 2 illustrates the out-of-pocket expenditures by items not supposed to be provided free by the hospital and items supposed to be given free from the hospital. A description of the expenses follows. Table 2 Distribution of the out-of-pocket expenditures by medical condition (in US$) in 1994 Expenditures on items NOT supposed to be provided from hospital Expenditures on items supposed to be provided free from hospital Travel Fee Medicine Food Tips Other Tests Total NVD (n = 19) median 12.50 0.25 11.25 0.88 1.25 3.88 0.00 62.50 mean 29.72 0.23 18.35 2.24 2.11 5.99 3.42 62.04 range 1–127 0.10–0.33 1–70 0–23 0.75–5 0–25 0–23 6–225 C-section (n = 20) median 36.70 0.10 51.88 2.50 2.06 11.25 0.00 118.75 mean 44.32 0.20 63.52 4.25 2.56 16.79 1.88 133.50 range 5–150 0.10–0.75 25–160 0.50–16 0.25–7 2–75 0–25 41–350 Abortion (n = 20) median 3.08 0.18 12.06 0.84 0.63 0.00 0.00 15.56 mean 11.67 0.17 18.93 1.40 0.77 0.00 0.00 32.94 range 1–63 0–0.30 1–75 0–5 0–2 0.00 0.00 2–125 Hysterectomy (n = 22) median 23.25 0.25 36.25 2.50 1.25 5.00 0.00 75.50 mean 32.04 0.38 30.89 4.84 2.38 4.02 10.15 84.70 range 1–86 0.10–4 1–50 0–19 0–10 0–10 0–75 2–178 Total (N = 81) median 22.25 0.23 26.25 1.25 1.25 4.38 0.00 65.25 mean 29.50 0.24 33.05 3.23 1.96 6.64 4.02 78.65 range 1–150 0–4 1–160 0–23 0–10 0–75 0–75 2–350 NVD: normal vaginal delivery Expenditures on items supposed to be provided free from hospital Medicine All patients were supposed to be provided required medicines free from the hospital but were not. Medicines included antibiotics, analgesics, syringe, catheter, blood, and so forth. Medicine was usually bought when patients were admitted at night. The medicine required for treatment is ordered by the on-duty physician but it takes several hours for the hospital management to process the order. Thus, no free medicine is available immediately. To start the treatment, the on-duty physician requests the patient's relatives to buy the medicine which is purchased from nearby private pharmacies. Tests All tests (e.g. pathology, radiology) are supposed to be provided by the hospital but sometimes the patients had the tests done in a private laboratory because waiting time for tests is very long in the DMCH due to the high patient load. Food Food is provided by the hospital but the interviewees found the hospital food of poor quality or totally lacking (liquid food such as soup or horlicks had to be bought for patients who had undergone surgery since these were not provided by the hospital). Relatives usually stayed with the patient in the hospital because of lack of ayahs (cleaning ladies) or nurses to provide necessary services. Thus, food was usually bought from a vendor or brought from home for both patient and relatives. Tips Tips ( bakshish ) are payments made to ayahs and guards. Ayahs were given tips for routine services such as pushing the patient's trolley to and from the labour/operation room, shaving the patient before delivery/surgery, giving enemas, etc. Guards at the gates were tipped each time a relative came to visit the patient during non-visitor hours. However, ayahs and guards are salaried hospital employees and are supposed to provide these services free of charge. The patients were reluctant when talking about the tips probably because they were still hospitalized and depended on these employees for access to certain services. Other items The other expenditures included items for the patient (e.g. hot water, bucket for hot water) and the newborn baby (e.g. blanket) that were supposed to be provided by the hospital free of charge but were not. Expenditures on items not supposed to be provided by the hospital Travel Travel expenses are not supposed to be provided by the hospital. Travel expenses consisted of travel to and from the hospital by the patient and any accompanying relatives, and travel expenditures of relatives during hospitalization for purchasing medicine and food for the patient. The patients came to DMCH because they expected 'free' and 'affordable' services compared to private clinics, or they were referred from a primary/secondary level facility, or to get better treatment here. Patients from rural/peri-urban areas took longer to reach DMCH than those from urban Dhaka (range half an hour to two days). Hospital admission fee This expense is also not supposed to be covered by the hospital. The official price of admission was $0.23, but it was zero for two patients and more than the official price for half of the patients interviewed. The study could not elicit the reason the patients paid more than the official price. When probed the patients could not or would not elaborate beyond the amount paid. The patients were very reluctant when talking about paying more than the official price for the admission fee. Factors increasing out-of-pocket expenditures Duration of hospitalization and rural residence of the patients increased the out-of-pocket expenditures. Rural residence increased the travel expenses and thus the total expenditures. Longer duration of hospitalization increased virtually all expenditures. The median duration of hospitalization was 8 days (range 1–34 days) per patient. Duration of hospitalization was the longest for hysterectomies followed by C-sections. Duration of hospitalization was related to severity of medical condition (e.g. eclampsia), necessary medical procedures (e.g. hysterectomy), and unnecessary medical procedures (e.g. episiotomy). One day of extra hospitalization increased expenditures by $2.30 per patient. Choice of medical procedures increased the patient expenditures. Episiotomy increased expenditures as patients were hospitalized for a longer duration and resulted in the purchase of more medicine. Episiotomy increased expenditures for both uncomplicated NVD (by 37%) and eclamptic NVD (by 84%) compared to cases where no episiotomy was performed (data not shown). The medical reason for performing episiotomies is the prevention of perineal tearing but because of a high case load at DMCH physicians perform episiotomies to reduce the length of delivery time, effectively turning hospital expenditures into patient expenditures. Eclampsia increased the expenditures for NVD by 180% (data not shown). Eclampsia is not under the control of the health system or the patient, and procedures used for treating eclampsia are unavoidable. C-sections caused higher patient expenditures compared to NVDs (median $119 and $63 respectively) because C-sections had a longer duration of hospitalization and required more medicine. Elective C-sections and eclamptic C-sections incurred similar expenses because elective C-sections were hospitalized for a longer duration even though there were no complications. Vaginal hysterectomies were 25% less expensive than abdominal hysterectomies because they required less invasive procedures, used local anaesthesia, and had a shorter duration of hospitalization. Sources of funds for patient expenditures The respondents said that they were willing to pay for care. However, rural households reported more difficulty in paying for care than urban households. Difficulty was inferred from the number of households who borrowed to pay for care, and the ratio of the amount borrowed to the annual household income. The median patient expenditure was equivalent to 7% (range 0.04%–225%) of annual household income, and was higher for rural (median 10%; range 1%–225%) than urban (median 7%; range 0.04%–78%) respondents. Half (n = 40) of the households reported borrowing to pay for care. The patient who spent 225% of her annual household income was a rural patient who had a hysterectomy for prolapsed uterus. Surgical patients like her are usually hospitalized for a month as they require more tests than non-surgical patients. This patient's total expenditures were not higher than the others who also had a hysterectomy, however, her annual household income was much lower than that of the others. More urban (n = 23) households borrowed than rural (n = 17) households but the amount borrowed was higher for rural households. The median amount borrowed per household was $38 and was equivalent to 8% (range 0.58%–208%) of the annual household income. On average, the rural households (median 14%; range 2%–208%) borrowed almost double the amount than the urban households (median 6%; range 0.58%–28%). Most often (n = 30) money was borrowed from friends and relatives without interest. When borrowing was from money-lenders (n = 10), households reported interest rates of 5%–30% per month. Three households put up security such as jewelry, land and household goods when borrowing money from moneylenders. The highest reported percentage of money borrowed to income for a rural household was 208% compared to 28% for an urban household. Finally, greater amounts of money were borrowed by C-section and hysterectomy patients than the other categories of patients. Table 3 illustrates the duration of hospitalization, total patient expenditures, annual household income, and amount borrowed by type of residence. Table 3 Distribution of median duration of hospitalization, expenditures, income, and amount borrowed by residence Urban Rural % of patients 56% (n = 45) 44% (n = 36) Duration of hospitalization (day) 7 (1–33) 9 (1–34) Total patient expenditures (US$) 59.25 (2.15–350) 79.25 (2.40–250) Annual HH income (US$) 900 (150–6000) 615 (2.50–6000) Borrowed amount (US$) 37.50 (6.25–250) 52.50 (12.50–200) Parenthesis shows range HH: household Discussion and conclusions The study findings indicate that all the surveyed patients incurred substantial out-of-pocket expenditures for a one time hospitalization. The median per patient expenditure was $65, and two-thirds of these expenditures were for commodities and services that were supposed to be provided free by the hospital but were not. Half the households borrowed to pay for care since they did not have the ability to pay (a finding similar to those found by Nahar et al.). A third of these households sold jewelry, land or household items to moneylenders. The rural households reported more difficulty in paying for care than the urban households. The rural patients had lower income but incurred higher expenditures and borrowed larger amounts than the urban patients. The study data are a decade old but worth presenting because Bangladesh is only recently beginning health sector reform and fee for service. Also, the hospital practices with regard to providing maternity care remain the same, in the hospital studied and in other public hospitals in Bangladesh. Other limitations of the study are its small sample size and that all the interviewees came from only one hospital. The small sample size is the norm when doing in-depth interviews. When a paper is qualitative not quantitative no statistical tests are customarily done. The results are striking despite the limitations. The costs related to NVD and C-section were much higher in this study than that estimated by Nahar et al. (NVD: $62 and $32 respectively; C-section: $133 and $118 respectively). Possible reasons for the differences are: a) recall error higher in the Nahar et al. study as they interviewed post-partum mothers, whereas, this study interviewed patients during hospitalization; b) the Nahar et al. study included only uncomplicated cases, whereas, this study included both uncomplicated and complicated cases. The annual household income was lower in this study compared to the Nahar et al. study ($750 and $1476 respectively). This may be attributable to having rural patients in this study whose income is much lower than urban patients, whereas, Nahar et al. studied only urban patients. Changing current practices regarding the length of hospitalization and medical procedures (e.g. episiotomy) will reduce patient expenditures. This can be achieved by having shorter duration of hospitalization for elective C-section and elective hysterectomy, and limiting the use of episiotomy. Limiting these practices may also lead to lower provider expenditures. Medicine constituted almost half of the total patient expenditures. However, lack of access to medicine resulted from an inefficient management system not from unavailability. Taking less time to process orders would make medicine available quicker to patients and so reduce their expenditures. Hospital management needs to ensure that patients pay only the official rate of admission fees. Hospital management also needs to ensure that patients do not pay tips to salaried hospital staff for routine services. The hospital could arrange for liquid food and hot water for surgical patients in addition to the other services it already provides, i.e. provide a more comprehensive hotel service. Alternatively the hospital could subcontract out its hotel services to those employees who extract tips from patients to provide such services. This may act as an incentive for eliminating the unofficial fees from tips. Making some services such as eclampsia and hysterectomy available at secondary level facilities will benefit the rural patients by reducing the travel expenses. Government hospitals can generate revenue by introducing or increasing some fees for medicine and tests for the paying category of patients but clearly exempting the poorest. How would the system determine who was the poorest? One option would be to let all rural women use hospital for free and have all urban women pay some fee. For service fees not to become a serious barrier to use current systems will have to eliminate or reduce practices that already result in high user expenditures from unofficial payments. Otherwise the added fees may lead to more borrowing from moneylenders, putting lands and goods at risk, and potential impoverishment of more households. A fee for service system needs to be based on information, and more than just setting a price. This can be facilitated by knowing what the patients were already spending in the current system and who were the most at risk of impoverishment. The challenge is to focus on realistic, short term changes that can reduce patient expenditures and inequities. Most of the recommendations of this study depend more on the will of the physicians and the hospital administrators than on the infusion of resources per se. Free maternity services in Bangladesh impose large out-of-pocket expenditures on patients. Authorities could reduce the burden by reducing the duration of hospital stays, limiting use of medical procedures, eliminating tips, and moving routine services closer to potential users. Fee for service could reduce unofficial expenditures if the fee were lower than and replaced typical unofficial expenditures, otherwise adding service fees without reform of current hospital practices would lead to even more burdensome expenditures and inequities. Competing interests The author(s) declare that they have no competing interests.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546230.xml
517719	Lactobacillus casei strain GG in the treatment of infants with acute watery diarrhea: A randomized, double-blind, placebo controlled clinical trial [ISRCTN67363048]	Background Adjuvant therapy to ORT with probiotic bacteria for infants with acute watery diarrhea has been under active investigation. Most studies have been done in the developed world showing benefit only for viral mild gastroenteritis. We evaluated the effect of a milk formula containing one billion (10 9 ) cfu/ml of Lactobacillus casei strain GG (LGG) upon duration and severity of diarrhea in infants in an environment with more severe acute diarrhea, where etiologic agents other than rotavirus are involved more frequently, and where mixed infections are more prevalent. Methods Male infants aged 3–36 months brought for treatment of acute watery diarrhea of less than 48 hours were eligible. After rehydration was completed with the WHO's oral rehydration solution, patients were randomly assigned to receive a milk formula either containing LGG or not. Stool volume was periodically measured using a devise suited to collect stools separate from urine. Duration of diarrhea was estimated based on stools physical characteristics. Results Eighty nine patients received the placebo milk formula and ninety received the LGG containing formula. Both groups were comparable in their baseline characteristics. Total stool output was significantly larger (p = 0.047) in the LGG group (247.8 ml/kg) than in the placebo group (195.0 ml/kg). No significant differences were found in duration of diarrhea (58.5 hours with LGG vs. 50.4 hours with placebo), rate of treatment failure (21.1% with LGG vs. 18.0% with placebo), and proportion of patients with unresolved diarrhea after 120 hours (12.2% with LGG vs. 12.5% with placebo). The rate of stools with reducing substances after 24 hours of treatment increased significantly in both groups (from 41.4% to 72.2% with LGG and from 45.9% to 68.0% with placebo). Conclusion This study did not show a positive effect of LGG on the clinical course of acute watery diarrhea. Positive beneficial effects of LGG, as had been reported elsewhere, could have been masked in our study by worsening diarrhea due to transient lactose malabsorption. Further studies with low-lactose or non-lactose conveyors of LGG are desirable.	Background Diarrheal diseases in the developing world continue to cause significant morbidity and mortality, especially when associated with malnutrition [ 1 ]. Dehydration, potassium depletion and acidosis are the main life-threatening complications of the acute losses during watery diarrhea. More than 25 years of extensive use has demonstrated that these complications could be effectively treated or prevented by oral rehydration therapy [ 2 ]. However, the oral glucose-electrolyte rehydration solution such as that recommended by WHO and Unicef neither shortens the duration of the illness nor reduces the stool loss and may cause an increase in stool volume at least during the first hours in children with acute diarrhea [ 3 ]. Optimization of the standard WHO-ORS solution, by reducing its osmolarity, has been shown to reduce diarrhea duration, total stool output, and the need for unscheduled intravenous therapy [ 4 ]. Adjuvant therapy to ORT, based on oral administration of live probiotic bacteria aimed to improve recovery of infants from acute watery diarrhea, has been under active investigation [ 5 ]. These studies, done in the developed world, have shown a benefit only for viral mild gastroenteritis. Isolauri et al have shown that oral bacterial therapy with Lactobacillus casei strain GG (LGG) reduces both the severity and duration of acute non dehydrating rotavirus enteritis [ 6 ]. Another trial in Karelia republic, LGG was shown to decrease the duration of acute diarrhea in children with viral acute diarrhea but not in those with bacterial diarrhea [ 7 ]. In a multicenter study carried out in Europe, LGG was administrated in a hypotonic oral rehydration solution to children with acute diarrhea, showing that it was safe and resulted in shorter duration of diarrhea, less chance of a protracted course, and faster discharge from the hospital [ 8 ]. Limited information is available on the potential role of LGG in infants living in the developing world, with more severe acute watery diarrhea [ 9 ], where etiologic agents other than rotavirus are involved more frequently and where mixed infections are more prevalent [ 10 ]. We conducted this study aimed to assess the effect of a milk formula containing Lactobacillus casei strain GG (LGG) on the duration and severity of diarrhea in children with acute watery diarrhea from our hospital. The study was conducted in the Rehydration Unit of Cayetano Heredia Hospital in Lima, Peru from September 1991 to June 1992. The study protocol and Consent Form were reviewed and approved by the Ethical Committee of Cayetano Heredia University in compliance with the Helsinki Declaration. Methods Study population Male infants 3–36 months of age brought for treatment because of acute diarrhea to the Rehydration Unit of Cayetano Heredia Hospital, Lima during September 1991 to June 1992 were eligible for the study if they met the following criteria: (a) a history of three or more watery stools per day for less than 48 hours, (b) no bloody stools at the moment of first examination, (c) clinical signs of dehydration, (d) no clinical features of hypovolemic shock, (e) no clinical signs of a coexisting acute systemic illness (i.e., meningitis, sepsis, pneumonia) or a recognized chronic disease such as pulmonary tuberculosis, and (f) no history of current antibiotic or antidiarrheal medication use. Patients 3–6 months old were considered for the study only if they were not on exclusive breastfeeding. Patients were not selected if their weight for age was less than 60% of the median weight for age established by the National Center for Health and Statistics (NCHS) [ 11 ]. Only patients whose parent or legal guardian was willing and able to give written informed consent were admitted to the study. Only male patients were selected in order to facilitate separate collection of stools and urine. The study size was calculated expecting a 33% reduction in duration of diarrhea and mean volume of diarrhea stool for a significance level of 0.05 and a power of 80%. Study design Infants fulfilling the admission criteria were randomly assigned to a double-blind comparison of a milk formula containing 10 9 cfu/ml of Lactobacillus casei strain GG (LGG) or a similar milk formula not containing LGG. Both formulas were of identical looking and tasting. Both milk formulas were provided by the manufacturer (Valio Ltd, Helsinki, Finland) as milk powder to reconstitute with water. They were given in bottles, appropriately labeled and number-coded for each child to secure the double-blind design. The actual assignment of children to their study numbers was made at the time of their enrollment into the study. Each child admitted into the study was allocated to either treatment group by restricted randomization using random permuted blocks. The codes were prepared in Finland and kept in sealed envelopes in Lima. Baseline assessment A clinical history and physical examination was completed upon admission to identify the patient, to determine the duration, type and severity of his diarrhea, to assess associated clinical features (fever, vomiting, dehydration, abdominal pain, distension), and to establish the nutritional status and previous dietary regimen of the subject. Blood for determination of serum sodium, potassium, chloride, glucose and bicarbonate, blood urea nitrogen, microhematocrit and plasma specific gravity was drawn from each patient before starting treatment. A routine urine analysis was also made. Fecal samples or rectal swabs taken at admission were transported in Cary-Blair transport medium to Cayetano Heredia University Microbiology Laboratory for primary isolation and identification of bacterial enteropathogens. A separate vial containing phosphate buffer saline was used to transport fecal specimens for rotavirus examination. Microbiologic methods Specimens were cultured for Salmonella , Shigella , E. coli and Vibrios by standard direct and enrichment enteric media. Campylobacter was selectively cultured on sheep blood agar containing Butzler's antibiotic supplement and Aeromonas on ampicillin blood agar preceded by overnight enrichment in alkaline peptone water. Five lactose fermenting colonies of E. coli isolates per patient were serotyped according to standard methods using polyvalent sera for the recognition of enteropathogenic E. coli . Detection of Rotavirus was made by ELISA method using Rotazime ® . Identification of enterotoxigenic strains of E. coli was not done. Details of the microbiologic methods used in this study are described in detail elsewhere [ 10 ]. Rehydration The degree of dehydration was assessed clinically. Dehydration was corrected and then fluid balance maintained using the oral rehydration salts (ORS) in its standard WHO's recommended formulation, following the WHO Guidelines [ 12 ]. Briefly, each child was given approximately 100 ml/kg of ORS during the first four hours. The ORS was administered in frequent sips using a spoon or by nasogastric tubes if vomit or stool output rates were high. Children were not given other fluid or foods during this period. Upon completion of this period, on-going fecal losses were replaced with the same solution, on a volume to volume basis until diarrhea ceased. Administration of the study formula Once dehydration was corrected, feeding, including administration of the assigned study formula was initiated. The first dose of the assigned study formula was administered as soon as rehydration was completed and subsequent doses were given every four hours until cessation of diarrhea or for a maximum of five days. The formula was prepared immediately before administration of each dose by a specially trained nurse that was exclusively working for the study. Measuring spoons provided by the manufacturer were used and the formula powder was kept refrigerated all the time once the container was opened. Before use, the sachets with the study formulas were kept in a -20°C refrigerator. The formula powder was diluted in warm boiled water as to provide 670 Kcal per liter. The amount of formula given to each child was calculated based on the child's body weight, each child being given 150 ml/kg/day to a maximum of 1000 ml/day. On average, each serving of 100 ml will supply 10 11 cfu of LGG for those receiving the formula containing LGG. Administration of the formula was by bottle and not compulsive. If the child refused to drink the formula after reasonable attempts, no extraordinary methods, like delivery via nasogastric tubing, were used; in this situation the formula was offered again four hours later. Other foods given Patients under six months of age were fed only the randomly assigned study formula to provide a daily caloric intake of at least 100 Kcal/kg (in a volume of 150 ml/kg/day). No other foods were given for these under six months patients. Older patients were fed the assigned formula ad libitum for a maximum of 1000 ml/day. Caloric requirements were completed with a blended soft baby food prepared from fresh ingredients (chicken breast, rice, carrots, squash, potatoes, and vegetable oil), by the Hospital's Nutrition Department. This soft baby food provides 1 Kcal per gram (approximately, 10% of its calories as protein, 45% as carbohydrates and 45% as fats). A total of at least 100 Kcal/kg/day (including the calories provided by the study formula) were offered to these children. Measurement of clinical outcomes All oral intake (ORS, study formula, breast milk, plain water, soft baby food) as well as urine, stool and vomitus volume were strictly measured and recorded during hospitalization. For this purpose patients were put on metabolic beds (which have a hole in the mattress were a container was placed to allow direct stool collection). A urine collection bag was fitted to avoid contamination of stools with urine and to measure the urine volume. After the first 72 hours, differential weight of dry and soiled diapers was used instead the metabolic bed to determine stool weight if the previous observed purging rate was below 5 ml/kg/hour. Blood analyses taken at admission were repeated at 24 hours. Stool examination for pH, glucose, reducing substances, leukocytes and microscopic fat were performed at admission, 24, 72 hours and at discharge. Urine specific gravity was determined every 24 hours until discharge. Criteria for stopping treatment and discharging the patient Patients were discharged from the study 24 hours after cessation of diarrhea, or at the end of five days from admission, or at the time treatment failure occurred. At discharge each patient was categorized as have completed the trial, as a treatment failure, as an unresolved diarrhea situation, or as a withdrawal. Primary outcomes • Rate of treatment failure: proportion of patients in each study group who have recurrence or continued presence of more than 5% dehydration, worsening electrolyte abnormalities, no weight gain since admission, developing of ileus or severe diarrhea defined as a purging rate in excess of 10 ml/Kg/hr in two consecutive 4-hour periods. • Rate of unresolved diarrhea after five days of treatment: proportion of patients in each study group with continuing diarrhea after five days of treatment. • Duration of diarrhea: time in hours from admission until cessation of diarrhea • Duration of hospitalization: time in hours from admission until discharge from hospital. Patients with treatment failure are excluded. • Total stool output: volume of diarrheic stools collected from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. • Total intake of oral rehydration solution: volume of ORS taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. Secondary outcomes • Total study formula intake: volume of study formula taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in milliliters per kilogram of body weight. • Total energy intake: energy from all sources (ORS, formula) taken from admission until cessation of diarrhea or for a maximum of 120 hours if diarrhea continues, expressed in kilocalories per kilogram of body weight. • Total volume of vomitus: volume of vomitus collected each day from admission until cessation of vomitus or for a maximum of 120 hours, expressed in milliliters per kilogram of body weight. • Total volume of urine: volume of urine collected each day from admission until cessation of diarrhea or for a maximum of 120 hours, expressed in milliliters per kilogram of body weight. Working definitions The following definitions were used for this study: • Carbohydrate malabsorption: stool sample giving a positive reaction for reducing substances (0.75 g % or above). • Cessation of diarrhea: passage of formed stool or passage of no stool for 12 consecutive hours. • Early withdrawal: patient who (a) developed a complicating illness, (b) presented bloody diarrhea after admission and before five days of treatment, (c) was prematurely discharged under request of his parent or legal guardian, or (d) had any protocol violation or non-compliance. Statistical analysis The data, initially collected in pre-coded forms, were entered in a database organized with a relational model under Fox Pro ® v. 1.02 (Dbase ® compatible formats) using a data entry program with on-line checking. The data was checked again in batch mode and finally transferred to EpiInfo ® for Windows ® v. 3.01 (November 2003) where the tabulations and statistical analysis were performed. Letters (A or B, maintaining blindly the treatment group assignment) identified treatment groups until all statistical analysis was completed. Only after completing the analysis investigators unblinded the treatment assignment. Baseline and outcome two-way comparisons were made between the two treatment groups. A 5% level of significance for statistical tests was set in advance for all comparisons. Chi-square Yates corrected was used to compare discrete variables. One way analysis of variance was performed for continuous variables. Data from patients prematurely withdrawn from the study was included in the analysis up to the time of withdrawal (intention-to-treat analysis). Results A total of 179 patients were admitted into the study, 90 in the LGG group and 89 in the control group. Nineteen patients, eight from the LGG group and 11 from the control group, were prematurely withdrawn from the study (figure 1 ) because of either one of the following reasons: bloody stools within the first 24 hours after admission (11 patients); parental non-compliance (four patients); no diarrheal stools passed within the first 24 hours since admission (two patients); typical severe cholera-like diarrheal disease improperly included (one patient); and severe systemic infection present but not recognized at admission (one patient). Baseline characteristics Table 1 presents the baseline characteristics of patients upon admission. The treatment groups were comparable in age, nutritional status, and other clinical and laboratory variables. Marginal differences were found, however, in stool output during the first four hours of the rehydration phase, before administration of the study formula. The LGG group was thus comprised by slightly more severe cases. Table 2 shows stool microbiology and parasitology at admission. The distribution of enteropathogens was similar between both groups, except for rotavirus which was found more frequently in the placebo group (39.3%) than in the LGG group (24.4%) with a marginal statistical significance for the difference (p = 0.052). Table 1 Clinical and laboratory features on admission Placebo group N LGG group N P Age (months) 14.7 ± 6.4 89 14.9 ± 7.5 90 0.878 Duration of diarrhea before admission (h) 29.5 ± 14.9 89 28.8 ± 14.9 90 0.733 N° of stool motions 24 h before admission 6.6 ± 3.2 89 7.5 ± 3.9 90 0.101 N° of patients with vomitus 24 h before admission 64 (71.9%) 89 66 (73.3%) 90 0.963 N° of patients with fever 24 h before admission 39 (43.8%) 89 43 (47.8%) 90 0.703 Clinical dehydration 89 90 0.165 Mild 48 (53.9%) 36 (40.0%) Moderate 40 (44.9%) 52 (57.8%) Severe 1 (1.1%) 2 (2.2%) % of weight gain after rehydration 3.0 ± 2.2 89 3.1 ± 2.3 90 0.763 Fever at admission (≥ 37.5°C) 16 (18.0%) 89 14 (15.9%) 88 0.868 Weight for height* 94.8 ± 9.3 89 94.1 ± 7.6 90 0.551 Height for age* 88.9 ± 12.1 89 97.2 ± 3.7 90 0.546 Weight for age* 88.9 ± 12.1 89 88.9 ± 10.8 90 0.952 Stool output (ml/kg) first 4 h (rehydration phase) 14.4 ± 12.4 89 18.1 ± 16.1 90 0.085 Hematocrit (%) 35.0 ± 4.2 89 35.5 ± 3.8 90 0.426 Plasma specific gravity 1.028 ± 0.002 89 1.028 ± 0.002 90 0.769 Serum Na + (mEq/l) 136.6 ± 4.7 89 136.4 ± 4.6 87 0.770 Serum K + (mEq/l) 4.0 ± 0.7 89 4.0 ± 0.7 87 0.985 Serum Cl (mEq/l) 102.5 ± 5.2 89 102.6 ± 5.4 87 0.874 Serum total CO2 (mmol/l) 14.0 ± 3.9 88 14.0 ± 3.9 87 0.971 Blood urea nitrogen (mg/dl) 13.1 ± 10.5 70 11.4 ± 6.6 73 0.253 Blood glucose (mg/dl) 107.5 ± 33.4 88 106.5 ± 35.9 88 0.848 N° of patients with fecal leukocytes (> 20 PMN/hpf) 30 (34.5%) 87 21 (23.6%) 89 0.154 N° of patients with microscopic fat 56 (64.4%) 87 52 (58.4%) 89 0.513 Fecal pH 6.3 ± 1.1 87 6.6 ± 1.1 89 0.126 N° of patients with CHO malabsorption 39 (45.9%) 85 36 (41.4%) 87 0.659 * % of NCHS median Values are mean ± SD or n (%) Table 2 Stool microbiology and parasitology at admission Placebo group N LGG group N P Rotavirus 33 (39.3%) 84 22 (24.4%) 90 0.052 EPEC 12 (17.6%) 68 9 (13.2%) 68 0.635 Vibrio cholerae 7 (8.0%) 88 11 (12.4%) 89 0.471 Campylobacter sp . 6 (6.8%) 88 9 (10.1%) 89 0.605 Shigella sp . 9 (10.2%) 88 4 (4.5%) 89 0.241 Aeromonas sp . 2 (2.3%) 88 1 (1.1%) 89 0.992 Salmonella sp . 0 (0.0%) 88 1 (1.1%) 89 0.995 Negative microbiology 25 (36.8%) 68 32 (47.1%) 68 0.452 Cryptosporidium parvum 6 (7.0%) 86 2 (2.2%) 89 0.256 Giardia lambliae 2 (2.3%) 86 3 (3.4%) 89 0.969 Strongyloides stercolaris 1 (1.2%) 86 0 (0.0%) 89 0.986 Trichomonas sp . 0 (0.0%) 86 2 (2.2%) 89 0.492 Negative parasitology 77 (89.5%) 86 82 (92.1%) 89 0.738 Negative microbiology and parasitology 22 (35.5%) 62 30 (44.8%) 67 0.371 No patients were found positive for the following parasites: Ascaris, Enterobius, Trichuris, E. hystolytica , Ancylostoma, Necator. ETEC identification was not done Values are n (%) Stool purging reduction The mean total stool output (Table 3 ) was lower in the placebo group (195.0 ml/kg) than in the LGG group (247.8 ml/kg) with a marginal statistical significance for the difference (p = 0.047). The difference between means in total stool output was -52.8 ml/kg, with 95% confidence limits between -105.4 to -0.2 ml/kg. Table 3 Clinical outcomes Placebo group N LGG group N P Difference between means Discharge condition 89 90 Early withdrawal 11 (12.4%) 8 (8.9%) 0.609 Treatment failure 16 (18.0%) 19 (21.1%) 0.734 Cessation of diarrhea 51 (57.3%) 52 (57.8%) 0.931 Unresolved diarrhea 11 (12.45%) 11 (12.2%) 0.842 Mean duration of diarrhea (hours)* 50.4 ± 28.0 51 58.5 ± 30.2 52 0.157 -19.6 to 3.4 Mean duration of hospitalization (hours)** 74.7 ± 33.7 62 81.2 ± 32.6 63 0.280 -18.4 to 5.4 Total stool output (ml/kg) 195.0 ± 171.9 89 247.8 ± 180.2 90 0.047 -105.4 to -0.2 Total oral rehydration solution intake (ml/kg) 236.5 ± 195.6 89 272.8 ± 188.1 90 0.208 -93.7 to 21.1 Total study formula intake (ml/kg) 215.5 ± 136.3 89 231.5 ± 136.6 90 0.435 -56.8 to 24.8 Total energy intake (Kcal/kg) § 220.7 ± 140.4 89 247.4 ± 143.0 90 0.209 -69.1 to 15.7 Total volume of vomitus (ml/kg) 27.6 ± 45.5 89 27.4 ± 33.9 90 0.968 -11.8 to 12.2 Total volume of urine (ml/kg) 81.5 ± 65.2 89 87.1 ± 69.4 90 0.579 -25.7 to 14.5 * For patients who ceased diarrhea before 120 hours. ** Patients with treatment failure or early withdrawals are excluded. § Calculated from calories provided by study formula, soft foods, breastmilk, and oral rehydration solution. Values are mean ± SD, n (%), or 95% confidence limits. Duration of diarrhea The proportion of patients with unresolved diarrhea after 120 hours was similar in both treatment groups (table 3 ). For those patients that ceased diarrhea during the study period the mean duration of diarrhea was similar between groups (table 3 ). The difference between means in duration of diarrhea was 8.1 hours with 95% confidence limits between -19.6 hours to 3.4. Blood chemistry No patient had to be withdrawn from the study because of worsening blood chemistry abnormalities. Carbohydrate malabsorption The proportion of patients with carbohydrate malabsorption 24 hours after admission into the study was high although not different between the treatment groups (table 4 ). However, the rate of carbohydrate malabsorption after 24 hours of treatment increased significantly in both treatment groups with respect to admission (table 4 ). The pH of stools at 24 hours after admission is also significantly lower in both groups compared to the pH at admission, a result consistent with worsening carbohydrate malabsorption (table 4 ). Table 4 Comparison of stool characteristics at admission and 24 hours after Admission N 24 h later N P Fecal pH Placebo group 6.3 ± 1.1 87 5.9 ± 0.8 76 0.014 LGG group 6.6 ± 1.1 89 6.1 ± 0.9 74 0.004 CHO malabsorption Placebo group 39 (45.9%) 85 51 (68.0%) 75 0.008 LGG group 36 (41.4%) 87 52 (72.2%) 72 < 0.001 Values are mean ± SD or n (%) No adverse effects due to the study formula were notice in either group during the study. Discussion Children included into this study probably represent the type of patients with diarrheal disease most frequently seen at a health service in Peru before the cholera epidemic of 1991. They were infants under two years of age, with an episode of acute watery diarrhea, presenting with signs of mild to moderate dehydration and a slight compromise of the nutritional status. Thus, we can conclude that the results of this study can be applied to most cases of acute watery non-cholera diarrhea seen in this population setting. The comparison of baseline characteristics showed no significant differences between groups. Marginal differences (not statistically significant) were observed in two variables that measure severity of illness: stool frequency 24 hours before admission and stool output during rehydration phase. Both were higher in the LGG group. Other indicators of severity, like clinical estimate of dehydration, percentage of weight gain after rehydration and proportion of patients with vomitus before admission, were similar between both groups. On the other hand, there were more cases (not statistically significant) of rotavirus in the placebo group than in the LGG group. In order to make sure that the groups were in fact comparable, we perform the statistical adjustment for these variables using multiple regression analysis and found no association with the principal outcomes (results of this analysis are not shown in the report). In more than 55% of patients an enteropathogen could be identified. This figure could be higher because studies for ETEC were not done. The most prevalent agents were rotavirus (30.7%), EPEC (11.7%), Vibrio cholerae (10.1%) and Campylobacter sp . (8.4%), with an important proportion of patients having mixed infections. These findings differ considerably from those of the Finnish clinical trial in which LGG was also tested for efficacy in reducing acute infantile diarrhea [ 6 ]. In the Finnish trial, rotavirus accounted for more than 80% of diarrheal cases, with no cases of bacterial diarrhea. A setting of greater diversity of etiologic agents and frequently co-pathogen association is perhaps more representative of developing countries [ 10 ]. The first topic to be discussed here is the high proportion of patients that did not recovered optimally during the trial, a condition that did not depend on the assigned treatment group. About 12% of patients had an unresolved diarrhea and an additional 20% were classified as treatment failures, mostly due to severe diarrhea. In two clinical trials conducted at our unit similar rates were found. For instance, in a clinical trial designed to test the clinical efficacy of a citrate-based ORS in children with acute watery diarrhea, patients who received standard therapy (WHO-ORS) had a treatment failure rate of about 24% [ 13 ]. This rate was 40% if only rotavirus associated diarrhea was considered. In another study, in which a chicken soup-based ORS was evaluated for children with similar characteristics (not published), 15.4% treatment failure rate for all subjects standard and 20% for those with rotavirus was observed. In the Finnish study, all children recovered before 5 days and no treatment failures were reported [ 6 ]. This striking difference could be explained in part by dissimilar etiologies as was mentioned above. Bacterial diarrhea and infection with more than one pathogen might produce a more severe illness. The nutritional status is also important to explain the difference. It has been well established from studies in developing countries that diarrhea lasts longer in poorly nourished children [ 14 ]. This difference seems to be important even when only slight malnutrition is present, as is the case for the patients admitted into this study. Except for total stool output (which showed a marginal statistical significance), no significant differences in the principal outcomes between groups were found. Duration of diarrhea, duration of hospitalization, and total ORS intake were not significantly different between the experimental and placebo group. The mean volume of formula (and of lactose) administered to children was the same for both groups. The confidence limits of the difference between the two groups indicate that a maximum reduction on duration of diarrhea of 6.7% attributable to the LGG formula could have been detected with the sample size of this study. These numbers indicate that the study does have a sample size sufficient to reject a type II error. No positive effects of treatment could be demonstrated as opposed to Isolauri's experience in Finland [ 6 ]. The dose factor could not explain the difference between the two studies. The dose of LGG in Isolauri's study was 2 × 10 10–11 cfu per day [ 6 ]. The dose of LGG in our study was 6–8 × 10 11 cfu per day, well above the threshold dose of 10 billion cfu suggested as most effective [ 15 ]. Viability of the Lactobacillus GG in the sachets while stored in Lima was not confirmed bacteriologically. There were slightly fewer cases of rotavirus in the LGG group than in the placebo group (24.4% vs. 39.3%), although the difference was not statistically significant. LGG appears to be more effective in viral than in bacterial diarrhea [ 8 ]. These facts could explain in part the lack of beneficial effect of LGG in our study. It is possible that LGG was not able to colonize the gut of our patients in the presence of bacterial pathogens. Although colonization has been documented during rotavirus diarrhea in children [ 16 ], to our knowledge this ability has not been demonstrated when other intestinal pathogens are involved. Colonization properties of LGG in patients with bacterial enteritis would deserve further studies. Our study clearly shows that the rate of carbohydrate malabsorption raised in both treatment groups from 43.6% at admission to 70.1% 24 hours later. The amount of lactose offered to patients in this study was much higher than in the usual feeding regimen at our unit (10.5 g/kg/day vs. 3.7 g/kg/day, respectively). Although to some investigators lactose intolerance is becoming a decreasing problem [ 17 ], it is clear from these data that lactose malabsorption as a factor cannot be dismissed. We had consistently found a high proportion of patients with carbohydrate malabsorption in all similar studies conducted in our unit. Thus, one possible explanation for the lack of efficacy of LGG in our patients is that worsening diarrhea due to lactose malabsorption could have masked any beneficial effect of the LGG. If this is true, then reducing the lactose content of the formula might reveal a positive effect. Another option is to deliver the LGG in suitable non-lactose containing infant food that could be given to diarrheic children as part of their treatment. Carefully conducted studies might prove this hypothesis. Conclusions Both groups were comparable in their baseline characteristics. Marginal differences were found in variables that measured severity of illness before treatment. Total stool output was slightly higher in the LGG group reaching a marginal statistical significance. No significant differences in the other principal outcome variables were found between treatment groups. The proportion of patients with unresolved diarrhea after 120 hours was similar in both treatment groups. For those patients that ceased diarrhea during the study period the mean duration of diarrhea was similar between groups. The rate of carbohydrate malabsorption after 24 hours of treatment increased significantly in both treatment groups. Competing interests None declared. Authors' contributions ES-L participated in the design of the study, organized and coordinated the study, participated in the statistical analysis and drafted the manuscript. PM-L carried out the clinical work, participated in the statistical analysis and the drafting of the manuscript. MC-S carried out the statistical analysis. EC-W participated in the clinical work. RBS conceived of the study, and participated in its design and drafting of the manuscript. All authors read and approved the final manuscript Figure 1 Diagram showing the flow of study subjects throughout the study Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517719.xml
520823	Immunological evaluation of the new stable ultrasound contrast agent LK565: a phase one clinical trial	Background Ultrasound contrast agents (UCAs) allow the enhancement of vascular definition, thereby providing more diagnostic information. LK565 is a new second-generation UCA based on synthetic polymers of aspartic acid which is eliminated from the blood stream via phagocytosis. LK565 forms very stable air-filled microspheres and is capable of repeated passage through the pulmonary capillary bed after peripheral intravenous injection. This characteristic allows examination of the cardiac function or extracardiac vessel abnormalities up to 15 minutes. Methods A phase one clinical study was conducted on 15 healthy volunteers to identify the development of an undesirable immune response. Phagocytosis capacity, TNF-α secretion, and MHC class II upregulation of monocytes was monitored, as well as microsphere specific antibody development (IgM, IgG). Furthermore, the kinetics of the activation surface markers CD69, CD25, CD71, and CD11b on leukocytes were analyzed. Results Due to LK565-metabolism the administration of the UCA led to saturation of phagocytes which was reversible after 24 hrs. Compared to positive controls neither significant TNF-α elevation, neither MHC class II and activation surface markers upregulation, nor specific antibody development was detectable. Conclusion The administration of LK565 provides a comfortable duration of signal enhancement, esp. in echocardiography, without causing a major activation cascade or triggering an adaptive immune response. To minimize the risk of undesirable adverse events such as anaphylactoid reactions, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent should be encouraged as a safe means to provide additional diagnostic information.	Background Echocardiography allows the analysis of ventricular motion and heart-valve morphology. The discovery that injection of small air bubbles dispensed in saline are capable of causing an opacification of the heart [ 1 , 2 ] started an enormous effort to develop contrast agents to assist in diagnosing coronary artery disease and myocardial infarction [ 3 ]. Early developments led to stabilized bubbles in hyperosmolar solutions [ 4 ]. Albumin-stabilized bubbles were one of the first contrast agents to allow examination of both ventricles [ 5 ], but there are stability problems in patients with elevated pulmonary pressure [ 6 ]. Second-generation contrast agents improved the contrast especially in patients with subnormal echo signals [ 7 - 9 ]. LK565 and comparable substances represent a new concept in contrast agents as it consists of small, stable, air-filled particles [ 10 ] that provides a good contrast in both ventricles (figure 1 , 2 ). Due to its high stability, LK565 improves examination of cardiac morphology and function as well as extracardiac vessel abnormalities for at least 15 minutes [ 11 ] (figure 3 ). Figure 1 Electron micrograph of LK565 Electron micrograph of the particles of the new contrast agent LK565 Figure 2 Apical 4 chamber view with LK565 Apical 4 chamber view before (a) and 1 minute after (b) intravenous LK565 application (4 mg/kg BW). Device: SONOS 5500, S3 Probe, harmonic Figure 3 Apical 4 chamber view with LK565 Apical 4 chamber view before (a), 1 minute (b), and 30 minutes (c) after intravenous LK565 application (4 mg/kg BW) and intermittent ultrasound exposure. Device: SONOS 5500, S3 Probe, harmonic The particles consist of a synthetic polymer of aspartic acid. Via peptide bonds, the molecules form bold, stable beads filled with air. The average size of LK565 particles is 3 μm in diameter (figure 1 ). Because of their size and even polymeric surface the beads have similarities to microorganisms. This circumstance implies the induction of an immune response, which needs to be considered. Results from previous studies indicate that phagocytes are responsible for eliminating the contrast agent from the blood stream [ 11 ]. In vitro, it has been shown that macrophages and neutrophils quickly devour the LK565 particles. Preliminary studies proved that the LK565 particles are phagocytosed by macrophages, resulting in slight activation with an increase in the intracellular calcium content. However, there were no symptoms of a respiratory burst [ 12 ]. The activation of macrophages can lead to different conditions: no activation, normal activation, and overreaction. In the last case, over-expression of tumor necrosis factor alpha (TNF-α) can lead to a septic shock by induction of intravascular blood clots apart from other effects, which may result in disseminated intravascular coagulation [ 13 , 14 ]. Furthermore, macrophages act as antigen-presenting cells which are highly important to induce an adaptive immune response, i.e. specific antibody production. It is probable that the LK565 particles attract antigen-presenting cells to present LK565 fragments as antigens. This may induce the production of specific antibodies against the contrast agent, which in rare cases could result in pathogenic immune reactions. The aim of this study was to examine a possible immune response to LK565 considering phagocytosis, TNF-α secretion by macrophages, lymphocyte activation and specific antibody production. Methods Volunteers The experiment was approved by the local ethics committee (registration number 708/98). After medical examination, 15 healthy male volunteers under the age of 30 were enrolled in this phase one clinical study. Each volunteer was exposed to a single dose of the contrast agent LK565 applied intravenously. Four participants were exposed to the contrast agent LK565 for the second time because they had already been involved in a prior study [ 11 ]. The 15 volunteers were divided up into 3 groups (n = 5). The first group received 0.15 mg/kg bodyweight, the second group 0.4 mg/kg bodyweight and finally the third group 0.7 mg/kg LK565. Contrast agent Prior to application, the contrast agent LK565 was dissolved in 10 ml physiological sodium chloride solution (37°C) under sterile conditions. To eliminate aggregations, the solution was filtered using a 20 μm mesh just before intravenous injection. Sampling Heparinized blood samples (2 ml) were taken before application (t = 0), after 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 1 ml after 10 d, 12 d and 14 d. For routine analysis, additional blood samples were drawn before and 2 hours after intravenous contrast agent injection. The volunteers were under medical care throughout the whole experiment. Phagocytosis Phagocytosis capacity was determined in vitro before (t = 0) and after 2 h, 6 h and 24 h with Phagotest™ (Becton Dickinson, Germany) and analyzed via flow cytometry (FACScalibur™, BectonDickinson, Germany). FITC-labeled E. coli, which were opsonized with antibodies and complement factors, were incubated with leukocytes. After incubation, the increased fluorescence of the phagocytes was due to the uptake of bacteria. The fluorescence intensity is dependent on the amount of phagocytosed bacteria. A sample chilled on ice was used for control. Tumor necrosis factor alpha (TNF-α) Intracellular TNF-α production in monocytes and macrophages was analyzed before (t = 0) and after 2 h, 6 h, and 24 h. For detection, an anti-human TNF-α Pycoerythrin (PE) monoclonal antibody (mAb) was applied (Pharmingen, Germany). The specificity of the antibody had been validated in blocking experiments. To 40 μl blood, 145 μl RPMI and 15 μl 40 μM Monensin were added (Monensin prevents cytokine release by blocking the golgi apparatus). Cells were incubated for 2 h at T = 37°C and 5% CO 2 atmosphere. After washing, monocytes were surface stained by anti-human CD14 TriColor mAb (Medac, Germany). Red blood cells were lysed using FACS™ Lysing Solution (Becton Dickinson, Germany). Cells were fixed and permeabilized by addition of 250 μl Cytofix/Cytoperm™ (Becton Dickinson, Germany). Finally, the retained intracellular TNF-α was stained by the anti-TNF-α PE mAb. For a positive control, cells were stimulated with 0.25 μg Lipopolysaccharide (LPS), following the same procedure as mentioned above. Surface activation markers Surface activation markers were analyzed before (t = 0) and after 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h. For the detection of activation markers, anti-human CD69 PE (Holzel Diagnostika, Germany), CD25 Fluoresceinisothiocyanat (FITC), CD71 FITC, CD11b PE (Diaclone Research, Germany) and HLA-DR PerCP (Becton Dickinson, Germany) mAbs were used. For a positive control, cells were stimulated with 10 ng/ml Phorbol-12-Myristat-13-Acetat (PMA) and 1 μg/ml Ionomycin for 2 h, while macrophages/monocytes were stimulated with 1.25 μg/ml LPS for 2 h. To distinguish between leukocytes, additional surface markers, anti-human CD45 Allophycocyanine (APC), CD19 APC (Caltag Laboratories, Germany), CD3 Tri/Color (DAKO, Germany) and CD14 FITC (Diaclone Research, Germany) mAbs were used for detection. Blood samples (40 μl) were stained for 4-color flow cytometry analysis. After staining, red blood cells were lysed, subsequently leukocytes were analyzed. Antibody specificity was validated by isotype controls (Becton Dickinson, Germany). Specific antibody development Serum samples were analyzed before (t = 0) and after 24 h, 48 h, 72 h, 96 h, 10 d, 12 d, and 14 d. Specific anti-LK565 antibodies were determined via indirect ELISA. Indirect ELISA was performed in a 96-well plate by LK565 immobilization overnight. Optimal concentrations had previously been determined by common cross-testing. Serum was obtained from blood samples after centrifugation at 400 G. After incubation for 2 h at room temperature, detector antibodies (Biozol Diagnostica GmbH, Germany) were added and incubated at 4°C overnight. Washing was carried out with phosphate buffered saline (PBS) and blocking buffer. For detection, alkaline phosphatase (AP)-linked goat-anti-human IgG and IgM antibodies with para-Nitrophenylphosphate (p-NPP) as substrate (Biozol Diagnostica GmbH, Germany) were used. After development, the enzyme reaction was stopped with 0.5 M NaOH and measured at λ = 405 nm. For the functional (positive) control of the indirect ELISA, total immunoglobuline was captured polyclonally. For a negative control, the serum of a person who had not been exposed to the contrast agent was obtained. Results We investigated 15 volunteers in a clinical phase one trial over a period of 14 days. The kinetics of activation surface markers CD69, CD25, CD71 and CD11b on leukocytes, the phagocytosis capacity, and TNF-α production in monocytes were analyzed, as was the specific antibody production after LK565 application. The expression of CD69 [ 15 ], CD25 [ 16 ], CD71, HLA-DR [ 17 ], and CD11b [ 18 ] is increased after activation [ 19 ]. CD69 will be presented quickly after activation on almost all leukocytes normally after some hours but on neutrophils even after a few minutes due to mobilisation of intracellular storages [ 20 ]. The α-chain of the IL-2 receptor CD25, also known as the high-affinity receptor on lymphocytes, is developed after 6 hours at the earliest and up to 1–2 days. Transferrin receptor CD71 correlates directly with cell proliferation and a late marker which can be expected after 3–4 days similar to antigen presentation via MHC-II analyzed as HLA-DR. Integrin expression (CD11b) on monocytes and neutrophils is associated with phagocyte migration into tissue [ 21 ]. Independent of dosage or repeated contact, the contrast agent LK565 was well tolerated by all volunteers. After exposure to LK565 no erythema, no drop in blood pressure, neither significant change of the heart frequency nor fever occurred (data not shown). The volunteers did not complain of relevant clinical symptoms such as chest discomfort or asthenia. It exhibited good opacification of both ventricles a few seconds after application (figure 2 ). While the minimum dosage of 0.15 mg/kg LK565 was sufficient for just one echocardiogram, a suitable duration of echo contrast was obtained at a 0.4 mg/kg dose. A further increase in dosage provided no improvement in quality or contrast duration (data not shown). The uptake of LK565 led to the saturation of phagocytes. The uptake capacity of the phagocytes from blood samples 6 h after injection was exhausted (figure 4 ). Neither macrophages nor neutrophils phagocytosed any more labeled bacteria after LK565 uptake, the observed effect was reversible. After 24 h the phagocytic capacity reached almost the starting level. Figure 4 Phagocytosis capacity after LK565 exposure Phagocytosis capacity refer to opsonized E. coli of neutrophils (a) and monocytes (b) in vitro illustrated in percent of control after LK565 exposure (n = 15, error bars show standard error). During the first 24 h an increase in intracellular TNF-α production was detected in monocytes and macrophages (figure 5a ). However, in relation to the positive control after 2 h of LPS stimulation, the slight increase in intracellular TNF-α was negligible. No connection between repeated exposure and dosage was found. The amount of intracellular TNF-α decreased to starting levels after 24 h. Figure 5 TNF-α and specific antibody production after LK565 application (a) intracellular TNF-α (Monocytes) after LK565 exposure relative to starting value and (b) LK565 specific antibody production (IgM, IgG) relative to positive control (n = 15, error bars show standard error). An increase in integrin expression CD11b was detected on monocytes and macrophages 6 hours after application. Compared to the positive control, the upregulation of the integrin was only poor. In all cases, the upregulation of the integrin was only of brief duration and returned to normal levels after 24 h (figure 6d ). Only a slight and short increase in CD69 after 6 h was determined in neutrophils in a few cases. Most volunteers exhibited no CD69 increase in neutrophils. Even less CD69 expression was found on monocytes and macrophages where CD69 stimulation is associated with the production of prostaglandines, leukotriens and TNF-α. Only minor CD69 expression was observed on T-cells and on B-cells (figure 6c ). No increase of CD25 and CD71 (also on monocytes) was detected (figure 6a,6b ). Figure 6 Activation marker expression after LK565 application CD25, CD71, CD69 and CD11b expression on different cell types after LK565 exposure relative to starting value (n = 15, error bars show standard error). Discrete MHC class II (HLA-DR) upregulation on macrophages and lymphocytes was observed (figure 7a ). We found no further evidence of an adaptive immune response via antibody production. None of the serum samples during our clinical trial exhibited any development of specific antibodies (IgM, IgG) versus the contrast agent (figure 5b ). Figure 7 HLA-DR expression after LK565 application HLA-DR expression relative to starting value and differential cell count after LK565 application (n = 15, error bars show standard error). Discussion The results show that there is only a slight activation of leukocytes esp. of phagocytes after application of LK565, a particular contrast agent based on a polymer of the naturally occurring amino aspartic acid. No specific antibody development against the contrast agent was detected. Thus, the risk of a major immunological activation cascade after repeated dosage can be regarded as minor. Since LK565 does not lead to an adaptive immune activation in this study, the possibility of an overexpression of IgE, which plays a key role in allergic diseases, also becomes more unlikely. Although none of the 15 volunteers enrolled in this phase I study developed an allergic-type reaction, such reactions could, however, occur in further phases of clinical testing or application. An allergic reaction against a pharmaceutical drug is a rare event which is impossible to exclude even in large clinical studies. An increase in CD11b/CD18 is connected with phagocytosis and the diapedesis readiness of phagocytes. LK565 is eliminated from the blood stream via phagocytosis, therefore neutrophils exhibited an increase in CD11b expression and phagocytosis activity. This is also connected to a temporary higher intracellular TNF-α level in monocytes. On lymphocytes, especially T-cells, the cascade of CD69, CD25 and CD71 is closely related to an immune response. After expression of CD69, the interlinkage of the receptor leads to proliferation and secretion of IL-2, IFN-γ and TNF-α. After effective activation, the T-cells secrete IL-2 which activates the development of the high-affinity IL-2 receptor CD25 and therefore launches cell proliferation with an expression of CD71 after 3 – 4 days. During our study after LK565 application no major activation cascade was detected on either T-cells or B-cells. As mentioned above, the best contrast performance for echocardiography was obtained at a LK565 dosage of 0.4 mg/kg. Opacification of both ventricles was not enhanced by a further increase in dosage. Bearing in mind the fact that the contrast agent is phagocytosed and uptake capacity is limited, it is recommended that the daily dosage does not exceed 90 mg, which is equivalent up to three echocardiograms per day. Conclusions To minimize the risk of an undesirable adverse event such as an anaphylactoid reaction, immunological studies should be included in clinical trials for new UCAs. The use of LK565 as another new ultrasound contrast agent (UCA) with a comfortable duration of signal enhancement esp. in echocardiography should be encouraged as a means to provide additional diagnostic information without causing a major activation cascade or triggering an adaptive immune response. This can be an advantage for the "difficult-to-image" patient with adverse reactions related to other UCAs. Competing interests H.K. Maerz received salary for 6 months from Dr. F. Koehler Chemie GmbH, Alsbach-Haehnlein, Germany, the developer/manufacturer of LK565. R. Zotz is co-author of one of several patents related to LK565 (Zotz R, Erbel R, Krone V, Magerstädt M, Walch A (1992): Ultrasonic contrast agents, processes for their preparation and the use thereof as diagnostic and therapeutic agents. United States Patent 5.137.928). Authors' contributions BF: Prepared and edited the manuscript as well as the figures und performed echocardiographic evaluations; HKM: Drafted the manuscript and participated in the design of the study esp. immunological aspects; SO: Carried out TNF-α evaluations, recruited and coordinated the clinical management of the patients; SP: Carried out expression pattern analysis, recruited and coordinated the clinical management of the patient; IL: Participated in the study design; US: Reviewed the manuscript according to study design and immunological aspects PW and TG: Reviewed the manuscript and edited the figures; RZ: Participated in the development of LK565, the study design and coordination. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC520823.xml
449895	Talking Science	The Cold Spring Harbor Laboratory Oral History Collection presents interviews with over 80 leading scientists, drawn from the fields of molecular biology and genetics	“…where people do come with a sense that there's going to be openness. That there's going to be a great comfort with people standing up and challenging a particular perspective. That young people are welcome. That old codgers are welcome as well. This is sort of the idyllic way that science is supposed to happen. And Cold Spring Harbor [Laboratory] provides that opportunity.” —Francis Collins, Cold Spring Harbor Laboratory Oral History Collection Cold Spring Harbor Laboratory (CSHL) has played a significant role in the development of genetics, a role that began at the Laboratory in 1904, just a few years after the discovery of Mendel's work. In 1908, George Shull's development of hybrid corn (maize) shaped the future of modern agricultural genetics; 55 years ago, Barbara McClintock worked on maize cytogenetics and discovered “jumping genes”; and in 1977, Richard Roberts was a codiscoverer of “split genes.” Throughout the 1950s, the Phage Course at CSHL trained many of the leaders of the then new field of molecular genetics, and the Laboratory's courses continue to train young scientists. CSHL Archives is a unique repository of rare books, manuscripts, photos, and scientific reprints that document the institution's significant contributions to molecular genetics research over the past 100 years. In 2000, the Archives received James Watson's personal collection of original materials, including manuscripts, correspondence, photographs, lab notes, lectures, memorabilia and reprints, for processing and preservation. This is an invaluable documentary resource for the history of molecular biology. While organizing over 60 years of Watson's correspondence, we realized that many of his correspondents have passed through CSHL as researchers, participants in the laboratory's leading scientific meetings, or instructors of our courses. We decided to talk to these scientists and record their commentaries, stories, and personal memories. These oral history interviews will enhance our collection by adding the voices of participants in significant scientific events. The project began in 2000. In 2002, we received a grant from the Gladys Brook Foundation that enabled us to purchase state-of-the-art recording and editing equipment for carrying out this project, and created a web site exhibiting taped conversations. During the last three years, we have interviewed over 80 scientists from several generations working in different fields of molecular biology and genetics, and have developed a video collection at CSHL's Carnegie Library. (A customized DVD of this collection is available.) The unique environment of the CSHL facilitated our task. Throughout the year, researchers from all over the world attend our scientific meetings to exchange information and ideas about their research, so most of the interviews took place at the Laboratory during these meetings. The scientists were always very pleasant and receptive to our requests for interviews. Other interviews were conducted in Cambridge and Boston, Massachusetts; La Jolla, California; Washington, DC; and abroad, in Oxford, England, and Sydney and Melbourne, Australia. Interviewing scientists was very exciting. We often wanted to come back for an additional interview because one or two hours was not enough time to collect all the memories that they wanted to share with us. It is well known that Sydney Brenner is a superb storyteller. After spending a few hours listening to Sydney in La Jolla, California, I received a call confirming my meeting with Francis Crick, which was to take place in the next 30 minutes. Despite looking forward so much to this meeting, I was torn between my present interview, in which I was deeply involved, and my next, which I was gladly anticipating. The CSHL Oral History Collection web site ( library.cshl.edu ) provides an opportunity to “meet and get to know” the leading minds of molecular biology and genetics. We hope that our site makes the history of science come to life. These interviews can be helpful to all categories of users: students, scientists, historians, writers, journalists, and others. The site is searchable and allows visitors to cross-reference people and information, and links them to the world of narratives and anecdotes, as told in the voices of those interviewed. Selected interview clips are organized into five topics: CSHL, James D. Watson, prominent scientists, scientific experience, and genome research. Each topic is divided into subtopics; for example, “Genome Research” includes: involvement in genomics, mechanism of the Human Genome Project, challenges of the Human Genome Project, dangers of genomic research, the future of genomics, and others. “Woman in Science” ( Figure 1 ) is one of the subtopics of “Scientific Experience.” Visitors also can choose a particular scientist and link to all the topics that he or she discussed. A brief biography of each participant has been included. Figure 1 Women in Science One of the pages from the “Scientific Experience” topic of the CSHL Oral History Collection. Our Web site adds a new dimension to the papers and books written by leading scientists—now you can listen to them discuss their work in their own voices! On this site you will meet a remarkable range of scientists, from friends and students of Barbara McClintock and Jim Watson, to the evolutionist Ernst Mayr, to the genome bioinformaticist James Kent. You'll hear Nancy Hopkins, Tom Maniatis, Matt Ridley, and Joan Steitz, to name just a few. We remain committed to our objective of providing an unprecedented and exciting approach for scientists, historians, scholars, and students to gain a firsthand account of the history of molecular biology and the stories behind the people who contributed to it. We have many more interviews planned, so in the future you can expect to meet even more of the fascinating figures who illuminate the glory days of the world of molecular biology.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC449895.xml
533877	Older age does not influence CD4 cell recovery in HIV-1 infected patients receiving Highly Active Anti Retroviral Therapy	Background Diagnosis of HIV infection is recently occurring with increasing frequency in middle-aged and in older individuals. As HAART became available, a minimal beneficial effect on immunological outcome in older in respect of younger subjects has been reported. In fact, both the intensity and the rapidity of the immunological response appeared to be reduced in elderly subjects. On the contrary, only few reports have indicated a similar immunological outcome both in older and younger HIV-positive subjects. Interestingly, older age did not seem to significantly affect the long-term virological outcome of HAART treated subjects. Methods To characterise epidemiological and clinical features of older HIV+ subjects, a prospective case-control study was performed: 120 subjects ≥ 50 and 476 between 20 and 35 years were initially compared. Subsequently, to better define the impact of HAART on their viro-immunological response, 81 older were compared with 162 younger subjects. Results At baseline cases presented significantly lower TCD4+ cell number and were more frequently affected by comorbid conditions. Under HAART a statistically significant increase in TCD4+ cell number was observed in cases and controls. At multivariate analysis, there was no statistically significant difference between cases and controls regarding viro-immunological response. Conclusions Although older subjects present a more severe HIV infection, they can achieve, under HAART, the same viro-immunological success as the younger individuals.	Background Early in the epidemic, Human Immunodeficiency Virus (HIV) infection primarily affected young adults; later on, it occurred with increasing frequency in middle-aged and in older individuals, too. In fact, a rate of 12.3% of AIDS patients aged 50 years or older, with subjects ≥ 60 years equalling 3.2%, has been recently reported by the Centres for Diseases Control and Prevention (CDC) [ 1 ]. Most of the known epidemiological features about older HIV-infected subjects, have been defined before the introduction of Highly Active Antiretroviral Therapy (HAART), as the standard therapy for HIV-positive subjects, in 1996. A low level of T CD4+ cells at HIV diagnosis [ 2 , 3 ], a rapid decline of T CD4+ cell count [ 4 , 5 ] and a high level of HIV viral load after seroconversion [ 6 , 7 ] have been reported in older HIV-infected subjects. In addition, these individuals presented a clinical condition of AIDS at the time of diagnosis of HIV infection more frequently than younger [ 2 ] and they were more likely to die within one month after HIV diagnosis [ 8 ]. Several studies have indicated that older patients had a shorter survival than younger [ 2 , 9 , 10 ], independently of HIV-risk behaviour [ 5 , 11 , 12 ] and AIDS-related illnesses [ 13 ]. Either the more rapid course [ 4 , 9 ] or the decreased survival [ 10 , 14 ] observed in older subjects were considered to be determined by the more frequent presence of non HIV-related comorbidities. As HAART became available, a less beneficial effect on immunological outcome in older in respect of younger subjects has been reported [ 15 - 17 ]. In fact, both the intensity [ 15 , 16 ] and the rapidity [ 17 ] of the immunological response appeared to be reduced in older subjects. On the contrary, only few reports have indicated a similar immunological outcome both in older and younger HIV-positive subjects [ 18 , 19 ]. Interestingly, older age did not seem to significantly affect the long-term virological outcome of HAART treated subjects [ 15 , 18 , 20 ]. Two were the aims of this study: 1. To characterise the epidemiological and clinical features of older subjects at the time of HIV diagnosis in the HAART era. 2. To better define the impact of HAART on virological and immunological response in a cohort of older HIV-positive patients when confounding variables such as adherence to therapy, side effects and non HIV-related comorbidities were evaluated. Methods Study setting The Catholic University teaching hospital is a 1,900-bed tertiary care centre located in Rome, Italy, with approximately 67,000 patient admissions each year. There is a 60-bed unit for the admission of HIV-infected subjects and a day-hospital for out-patient care. Study design Prospective case-control study. Study population All patients presenting the first HIV positive test from January 1997 to June 2003, admitted to our in- and out-patient care, were considered. For the first aim of the study, subjects aged ≥ 50 years (older) and between 20 and 35 years (younger) were identified. For the second aim of the study, older and younger patients who were given HAART regularly and with a follow-up of at least six months, were included as cases and controls, respectively (ratio 1:2). The control group was matched by sex, year of HIV diagnosis, presence of AIDS defining conditions and type of HAART regimen (i.e. NNRTI containing vs. PI containing regimens). A patient (either case or control) was considered as regularly HAART-treated if he/she was under antiretroviral therapy with a combination of two nucleoside reverse transcriptase inhibitors (NRTI) plus either a non nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI), as single drug or with the addiction of a low dose of ritonavir (i.e. 100 mg bid), for at least three months. Parameters evaluated Patients' records were reviewed by one of us, using a standardised data collection form with predefined criteria. For each subject enrolled in the study, the following data were obtained at inclusion after patient's informed consent: age, gender, HIV-risk behaviour, date of the first available HIV test, reason for HIV testing, CDC stage of HIV infection [ 21 ], HIV-related conditions, date of AIDS diagnosis, number of hospitalisations after HIV diagnosis, observed AIDS free interval (i.e. time from HIV infection to AIDS diagnosis), type and use of prophylaxis for Pneumocystis carinii pneumonia (PCP) and toxoplasmosis, antiretroviral therapy (type, duration, adherence and side effects) and other medications (antituberculous drugs, antivirals, corticosteroids, antacids), time of follow-up, time from AIDS diagnosis to death, cause of death. Data collected from the laboratory records included: numbers of circulating CD4+ T cells (/mm 3 ), CD8+ T cells (/mm 3 ) and plasma HIV RNA viral load (copies/ml) with a limit of detection below 50 copies/ml. The time of the beginning of HAART was considered as time zero in the analysis. All subjects underwent periodical clinical evaluations including determination of CD4+ T cells and HIV viral load every six months. Adherence was defined through a self-reported evaluation by the patient and registered as percentage by the physician. An evaluation higher than 80% was classified as "adherent", whereas lower than 80% was considered as "non-adherent". The following adverse effects were considered: anaemia (haemoglobin <9.0 gr/dl), digestive intolerance, dyslipidemia (serum cholesterol >240 mg/dl and/or triglycerides >200 mg/dl), hepatitis, hyperglycaemia (glucose >150 mg/dl), lipodystrophy, pancreatitis, peripheral neuropathy, rash, renal colic and thrombocytopenia (platelets <50,000 cells/mm 3 ). Three outcomes were considered for the study: 1. Immunological success (IS): defined as T lymphocytes CD4+ cell count >200/mm 3 at the end of the follow-up; 2. Virological success (VS) defined as a HIV viral load undetectable (<50 copies/ml) at the end of the follow-up; 3. Viro-immunological success (VIS) defined as either undetectable HIV viral load and T lymphocytes CD4+ cell count >200/mm 3 at the end of the follow-up. Evaluation of comorbidity In order to evaluate the significance of non HIV-related comorbid conditions, we used a modified version of the Charlson comorbidity index which excludes AIDS from the diagnosis [ 22 ]. Cardiovascular, pulmonary, neurological, endocrine, renal, hepatic, gastrointestinal, rheumatologic diseases, coagulation disorders and cancer were considered as comorbid non HIV-related conditions. Statistical analysis Quantitative variables were tested for normal distribution and compared by means of Student's two tailed t-test. Differences in group proportions were assessed by use of the χ 2 test or, for small numbers, Fisher's exact test. Stepwise logistic regression models were used for each factor to adjust for the effects of confounding variables. Two tailed tests of significance at the p < 0.05 level were used to determine statistical significance. Statistical analysis was performed using the software program Intercooled Stata 6.0 (Stata Corporation, Texas). Results Epidemiological and clinical features of older patients at the time of HIV diagnosis The rate of HIV diagnosis in patients aged ≥ 50 years admitted to our in- and out-patient care remained constant (about 10%) over the study-period. From a total of 1265 subjects with new HIV-positive test, 129 (10,1%) subjects aged ≥ 50 years were identified. Nine of them were not further considered because of missing data. One hundred twenty older subjects were evaluated. Data were available for 476 younger subjects. Among older subjects there were more males, less intravenous drug abusers, more advanced HIV disease, more frequent C stage of HIV infection and higher mortality, (Table 1 ). The reasons for the first HIV test were comparable both for older and younger subjects. In particular, 30 (25%) older subjects were asymptomatic. For those who were symptomatic, the more frequently observed symptoms were: fever (25%), haematological dysfunctions (17%), weight loss (14%), neurological (8%), gastrointestinal (6%) or hepatic (7%) disorders. Regarding AIDS-defining conditions, a significantly higher frequency of PCP, HIV-encephalopathy, Kaposi's sarcoma and cryptococcosis was observed in older HIV-infected patients than in younger. Matched case-control study Thirty nine out of 120 HIV-positive older subjects were not further considered: 29 because the duration of follow-up was below six months and ten because the case-control matching was not possible. A final number of 81 older patients under regular HAART and with a follow-up of at least six months (cases) were thus compared with 162 younger subjects (controls). Baseline characteristics and evolution Cases had a median age of 56.9 ± 5.2 years and controls of 31.3 ± 3.2 years. As a result of the matching, 73% of subjects in both groups were males and 49% were in CDC stage C of HIV infection. HIV infection risk factors were similar in the two groups, but eighteen (22%) cases referred an unknown risk factor compared to 26 (16%) controls (p = 0.1). The mean of CD4+ T cells was significantly lower in cases compared to controls (107 ± 109 cell/mm 3 vs. 178 ± 189 cell/mm 3 ; p < 0.01), such as the mean CD4 percentage (9% vs. 14%; p < 0.01). The mean of CD8+ T cells at baseline was similar in cases and controls (757 ± 380 cell/mm 3 vs. 818 ± 362) (p = ns). Mean of HIV viral load log was similar in the two groups (4.65 ± 0.57 vs. 4.74 ± 0.76 copies/ml; p = ns). No statistical significant difference was observed for haematological parameters, serological markers of previous viral hepatitis (type B or C) and syphilis. Laboratory tests only indicated a significant increase of glycaemia in older patients (103.3 ± 18.7 vs. 89.0 ± 10.6 mg/dl; p < 0.01). Follow-up was similar in the two groups (40 ± 13 vs. 42 ± 11 months; p = ns), ranging from 6 to 78 months. No differences were observed between cases and controls in the number of hospitalisations (1.2 ± 2.1 vs. 1.3 ± 2.8; p = ns). Seven (9%) and six (4%) patients died in the case and control groups, respectively (p = ns). All causes of death in both groups were HIV-related conditions with the exception of one case who died for an acute mesenteric vascular episode. Comorbid conditions Cases had more comorbid conditions than controls (45.6% vs. 15.9%; p < 0.01). One third of the cases suffered from cardiovascular diseases, a rare condition among controls (32.2% vs. 2.1%; p < 0.01). Gastrointestinal disorders (9.4% vs. 1.2%; p = 0.04) and diabetes (11.0% vs. 2.1%; p = 0.02) were also significantly more frequent among cases than controls. No statistically significant differences were found for cancer, pulmonary, renal or endocrine diseases, chronic hepatitis and coagulation disorders. The total comorbid points were 37 for cases and 20 for controls. Cases had also a higher mean Charlson index in respect to controls (0.57 ± 0.92 vs. 0.12 ± 0.44; p < 0.01). Antiretroviral therapy No statistically significant difference between cases and controls was observed in type, number and duration of HAART regimens. The first line antiretroviral therapy included PI in more than 80% of cases and controls (Table 2 ). The patients who required a change in antiretroviral therapy (second line therapy) received a therapeutical cocktail including PI in 40% vs. 48% (p = ns) and NNRTI in 57% vs. 50% (p = ns) of cases and controls, respectively. Similar percentages were observed in the patients who required another change of antiretroviral therapy (third line therapy) during the follow-up. In the first line drug regimen, side effects were more frequently responsible of the change of the drug regimen in cases compared with controls, although not reaching a statistically significant level (p = 0.1). A high level of adherence to HAART was observed in both groups (82% vs. 75%; p = ns). The more frequent adverse reactions were dyslipidemia, digestive intolerance and lipodystrophy in both groups (p = ns). A significantly higher frequency of hyperglycaemia and anaemia was observed in cases than in controls (Table 2 ). Immunological and virological response HAART related immunoreconstitution Comparing the mean values obtained at baseline in both cases and controls with those obtained at the end of the follow-up, a statistically significant increase in T CD4+ cell number was observed. In particular, T CD4+ cell count increased in 48 months from 107 ± 109 to 476 ± 258 for cases (p = 0.04), and from 178 ± 189 to 584 ± 252 for controls (p < 0.01). There was always a progressive linear enhancement in both groups at any individual time of the follow-up. The lower values observed in cases in respect to controls at baseline, persisted until the end of the follow-up (Figure 1 ). The increasing value of T CD4+ cell count every six months in respect to baseline was comparable in the two groups (Figure 2 ). According to the above mentioned outcome definition, IS was observed in 59 (73%) of the cases and 128 (79%) of the controls (p = ns). The following variables were significantly associated with IS at univariate analysis: CDC stage A (p = 0.01), low Charlson index (p = 0.01), CD4+ T cell count at the beginning and at 6,12,18,24,30,36 months of HAART (p < 0.01), months to achieve CD4+ T cell >200/mm 3 (p < 0.01), enhancement of CD4+ T cell count in the first six months (p < 0.01), HIV viremia at 30 months (p < 0.01). HAART related viral suppression A statistically significant reduction of HIV viral load was observed in both cases and controls when comparing baseline with the end of the follow-up values. In particular, means of HIV-RNA viral load log decreased in 48 months from 4.74 ± 0.57 to 2.01 ± 0.4 copies/ml in cases (p < 0.01) and from 4.69 ± 0.76 to 1.6 ± 0.2 copies/ml in controls (p < 0.01). HIV viral load reduced under antiretroviral therapy in both groups in a comparable manner. In fact, there was no statistically significant difference between cases and controls at any individual time of the follow-up (Figure 3 ). According to the above mentioned outcome definition, VS was observed in 66 (82%) of cases and 121 (75%) of controls (p = ns). The following variables were significantly associated with VS at univariate analysis: CDC stage A (p = 0.02), high adherence to HAART (p = 0.01), dyslipidemia as adverse effect of HAART (p = 0.01), HIV viral load at 6 (p < 0.01) and 24 months (p = 0.02). HAART related virological and immunological outcome According to our definition of the outcome, VIS was observed in 53 (66%) of cases and 100 (62%) of controls (p = ns). The following variables were significantly associated with VIS at univariate analysis: CDC stage A (p < 0.01), high adherence to HAART (p = 0.02), dyslipidemia as adverse effect of HAART (p < 0.01), CD4+ T cell number at baseline and at 6,12,18,24,30,36 months of HAART (p < 0.01), enhancement of CD4+ T cellcount in the first six months (p < 0.01), HIV viral load at 6 (p = 0.04) and 30 months (p = 0.01). No statistical difference in immuno-virological response was found between cases and controls when, as cases, only a subgroup of patients aged > 60 years was considered (data not shown). Multivariate analysis In a multivariate logistic regression model, after adjustment for sex and Charlson index, there was no statistically significant difference between cases and controls for IS, VS and VIS (p = ns). Similar results were obtained when CDC stage A and CD4+ T cell count at HIV diagnosis were added to the model. Another model including sex, Charlson index, CDC stage A, CD4+ T cell count at the beginning of HAART, adherence and dyslipidemia as adverse effect of HAART did not show any statistically significant difference between cases and controls for IS, VS and VIS. Further adjustment for sex, Charlson index, CDC stage A, adherence, dyslipidemia as adverse effect of HAART, CD4+ T cell count at beginning of HAART and after six months of therapy, enhancement of CD4+ T cell count in the first six months, months to achieve CD4 + T cell count >200/mm 3 , HIV viral load at six months did not modify the results. Discussion This study, conducted in an Italian population, shows that in the HAART era, the percentage of older patients out of the new HIV infections is approximately 10%, slightly higher than previously reported [ 23 ]. The impact of long term HAART-related immunoreconstitution in this sub-population of HIV patients could reduce the incidence of older patients with AIDS within the next years to less than the 12% recently reported [ 1 ]. The first part of our study confirms previous pre-HAART era observations, about more severe HIV infection in older subjects [ 2 - 10 ]. In fact, higher frequency of patients with AIDS at HIV diagnosis, lower T CD4+ cell count and higher mortality in older subjects in respect of younger were observed. A late diagnosis or a more aggressive impact of HIV infection in older population could explain these results. The first possibility could reflect low level information and/or physicians' insufficient suspicion [ 2 , 24 ], despite the world-wide durable informative campaigns and preventive efforts on this issue. Alternatively, a shorter or less symptomatic pre-AIDS phase, as already reported in older HIV-positive subjects, could contribute to the late diagnosis [ 2 ]. On the other hand, the more aggressive impact of HIV infection in older population has been related to the hypothesis that HIV is an additional aggravating factor to an already debilitated host potentially presenting numerous comorbidities, less physiological reserves [ 10 , 14 ] and a less responsive immune system [ 2 , 25 ]. In fact, in HIV-negative older subjects, important changes in cellular and humoral components of the immune system including defects in T CD4+ lymphocyte function, have been described [ 26 ]. Regarding HIV-risk behaviour, our data indicate the heterosexual route as the most frequent in older patients, followed by no risk reported, homosexual contacts, intravenous drug abuse. In particular, our data confirm recent reports [ 8 , 27 ] indicating an increased incidence of HIV subjects with no risk reported. This aspect could contribute to the above mentioned low level of suspicion of HIV diagnosis in older subjects. It is of interest that a large epidemiological survey in the US general population has reported a prevalence of at least one risk factor for HIV infection in about 10% of subjects aged 50 years or older [ 28 ]. To date, no studies have indicated that older AIDS patients should be treated differently for HIV disease. Clinical drug trials often exclude older patients because of multiple medical problems, comorbidities or co-existing non HIV-related medication regimens [ 29 ]. The first available literature data obtained in a large population of HIV-positive patients treated with zidovudine in the early nineties showed that age was an independent predictor of rapid progression to AIDS and shorter survival [ 30 ]. On the contrary, antiretroviral therapy was the only independent predictor of survival after the diagnosis of AIDS in older HIV-positive subjects in the pre HAART era [ 31 ]. Very little literature and study has been devoted specifically to the role of protease inhibitors and aggressive antiretroviral triple therapy in elderly patients. Few data have been published concerning the specific response of older patients to new HIV antiretroviral treatments, and currently there are no guidelines for specific antiretroviral treatment modalities for patients>50 years of age. However, a poor immunological response under HAART was observed in small groups of patients [ 15 , 16 ] possibly due to the well known depressed thymic function of the elderlies [ 15 - 17 ]. Only few reports showed a similar virological and immunological outcome in older HIV-positive compared with younger subjects [ 18 , 19 ]. This six year prospective matched case control study not only confirms this observation but indicates that older patients under HAART experienced a successful immunological response comparable to younger subjects also in a long term follow-up, in line with preliminary reports [ 32 - 34 ]. In fact, our older patients, starting HAART with a lower level of T CD4+ cells than younger, obtained a significant increment in their T CD4+ cell count, with the same rapidity, intensity and persistence in time as observed in younger patients. Meticulous adherence to HAART is necessary to achieve an optimal clinical and virological response. A variety of factors may predict medication adherence among HIV-infected adults. Although older age is usually associated with higher rates of antiretroviral adherence, older participants who were cognitively impaired could have difficulty in adequately adhering to their medication regimen [ 35 - 37 ]. Our findings indicate that this goal could be achieved independently from age. In fact, although thymic functions decline with age, substantial output of T CD4+ cells can be maintained into late adulthood, thus contributing to HAART-related immune recovery in older HIV-positive patients [ 38 - 40 ]. Early work also demonstrated a positive association between age and the plasma HIV-1-RNA copy number at the time of identification of HIV-1 serostatus among recently diagnosed older HIV-1 positive individuals [ 41 ]. However, this could have been caused by a lead-time diagnostic bias, with a greater delay among older individuals. No differences were observed in virological response as indicated by viral load reduction in older patients with respect to younger. This result is in line with previous reports [ 15 , 18 ] including a large study which indicated that older age decreases the risk to have viral load greater than 1000 copies/ml in HIV-positive subjects under HAART [ 2 ]. In a recent report, older age was associated with lower levels of HIV-1 replication, independent of antiretroviral therapy usage, regimen adherence, and diseases stage. It is suggested that the effect may be caused by changes in viral evolution or immunological monitoring specific to older individuals with HIV-1 infection [ 42 ]. In our study both the frequency and the distribution of adverse effects of HAART were similar between the two groups, with the exception of anaemia and hyperglycaemia which were more frequent in older patients. Hyperglycaemia can be explained because diabetes is a frequent comorbid condition among older. Older subjects were more susceptible than younger to adverse effects in the first line therapy; consequently they had to change therapy more frequently. Univariate analysis showed an association of low Charlson index with immunological success. However, considering that the Charlson index was higher in older patients, none of the models of multivariate analysis indicated an association between comorbidity and virological and/or immunological response. Nonetheless previous studies have suggested a more rapid course and a decreased survival in older HIV-positive patients because of the presence of comorbidities [ 10 ], our data did not confirm this association (data not shown). Interestingly, cardiovascular disease and diabetes were the more frequent comorbidities in our older population. Dyslipidemia and lipodystrophy in association with gastrointestinal disorders were the more frequent adverse reactions. In view of these results, of the recent reports of endothelial vascular damage, premature atherosclerosis and coronary events in younger HAART-treated patients [ 43 - 45 ], an important increase of cardiovascular event incidence in older HIV-positive patients under HAART could not be excluded to occur in the forthcoming years. Conclusions In conclusion, we strongly underline the need for an early diagnosis of HIV infection in older subjects through implementation of educational campaigns specifically targeted to this population. In relation to older people, physicians should consider more often the possibility of HIV diagnosis, through more screening and counselling. An early diagnosis is mandatory because, although older subjects present with more severe HIV infection, the use of HAART allow them to achieve the same viro-immunological response as younger individuals. At present, there is an increasing number of older subjects living with HIV either because of new HIV diagnosis in older population or because who have previously acquired HIV infection now become older due to HAART improved survival. It is reasonable to predict that the average age of chronically treated HIV-positive patients will progressively increase in the forthcoming years. Based on this reality, ageing with HIV is a newly manifested chronic disease with a complex long-term management in consideration also of the impact of HIV and HAART on the natural history of other chronic diseases typically associated with older age. List of abbreviations CDC Centers for Diseases Control HAART Highly Active Antiretroviral Therapy NRTI Nucleoside Reverse Trascriptase Inhibitor NNRTI Non Nucleoside Reverse Trascriptase Inhibitor PI Protease Inhibitor VIS Viro Immunological Success VS Virological Success IS Immunological Success Competing interests The author(s) declare that they have no competing interests. Authors' contributions MT conceived the study, performed the statistical analysis and wrote the manuscript RR participated in the design of the study, in the collection of the data, in the statistical analysis and in the writing of the manuscript KdGD participated in the design of the study, in the collection of the data and in the writing of the manuscript SB participated in the design of the study and in the collection of the data EM participated in the collection of the data ET participated in the collection of the data ET participated in the design of the study RC did the supervision and critically read the last version of the paper All authors read and approved the final manuscript Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC533877.xml
546224	The prognosis of women with stage IB1-IIB node-positive cervical carcinoma after radical surgery	Background Pelvic lymph nodes metastasis is an important prognostic factor for patients with cervical carcinoma. However, the relationships between the number of positive nodes, site of metastases nodes, adjuvant therapy and the prognosis is controversial. The purpose of this study was to investigate the influence of positive lymph nodes on the prognosis of Chinese women with stage IB1-IIB cervical carcinoma. Patients and methods Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery in Cancer Hospital, Fudan University. Of these sixty-six patients (16.6%) who were histologically confirmed to have positive pelvic lymph nodes were analyzed retrospectively. The survival was estimated using Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. Results The 5-year survival of the patients with pelvic lymph nodes metastases was 40.7%. Cox proportional hazard model analysis showed that cellular differentiation, the number of positive nodes and adjuvant therapy to be the independent prognostic factors ( P < 0.05). The 5-year survival of patients with one positive node was higher than that of those with two or more positive nodes (56.5% vs. 36.4%, P < 0.05). The distant metastasis rate in the former group (5.9%) was lower than the latter's (32.7%) ( P = 0.05). However, there was no significant difference of pelvic recurrence between the two groups ( P > 0.05). The number of positive nodes positively correlated with the level of positive nodes ( P < 0.01). The 5-year survival of the patients who had no adjuvant therapy (12.6%) was much lower than that (53.7%) of those with adjuvant therapy ( P < 0.05). However, there was no obvious difference between adjuvant radiotherapy, chemotherapy and chemo-radiotherapy ( P > 0.05). Conclusions The prognosis of patients with stage IB1-IIB node-positive cervical carcinoma who underwent radical surgery alone was very poor. Adjuvant therapy increases the survival rate, decreases the pelvic recurrence and distant metastasis.	Background Although radical radiotherapy (RT) and radical surgery can be the proper choices for patients with early stage cervical cancer, most of the patients in China prefer the radical surgery to RT. Hence, in China the radical surgery has been widely used as first-line therapy for this group of women. Some poor prognostic subgroups have been identified, among these the pelvic lymph node status has been considered as the most important prognostic factor. Radical hysterectomy with bilateral pelvic lymphadenectomy produces an expected 85–90% survival in women with stage IB and IIA cervical carcinoma without lymphatic spread. However, once tumors involve regional lymph nodes, 5-year survival has been reported to be only 30–60% [ 1 ]. In most of the studies the presence of pelvic lymph node metastases has been associated with increased pelvic recurrence and distance metastases, and a decrease in overall survival [ 2 - 7 ]. However, many questions such as the relationship between the numbers, the site of positive nodes, the modality of postoperative multidisciplinary therapy and the prognosis is not yet clear. This study investigated the factors that could predict the prognosis of the patients with stage IB1-IIB node-positive cervical carcinoma. Patients and methods Between January 1992 and December 1997, 398 women with International Federation of Gynecology and Obstetrics (FIGO) stage IB1-IIB cervical carcinoma underwent radical surgery at the Department of Gynecologic Oncology, Cancer Hospital of Fudan University. Of these 66 patients who had undergone Wertheims-Meigs' surgery (radical hysterectomy and pelvic lymphadenectomy) and were histologically confirmed to harbor positive pelvic lymph node were included in this study. The median age at diagnosis was 49 years (range 21 to71). Out of 66, 8 patients were in stage IB1 (12.1%), 37 patients (56.1%) in stage IIA and 21 patients (31.8%) in stage IIB. Histologically 41 women (62.1%) had squamous carcinoma, 20 (30.3%) had adenocarcinoma, 4 (6.1%) adenosquamous carcinoma and 1 patient (1.5%) had small cell carcinoma. The tumors in 4 patients (6.1%) were well differentiation, 46 cases (69.7%) moderately differentiated and 16 (24.2%) poorly differentiated. The average lymph nodes resected were 14.8 per patient while the average positive lymph nodes resected were 3.7 (1~28) per patient. The average diameter of the cervical tumors was 3.6 cm (1~7 cm). The details of the patients' clinical characteristics are listed in Table 1 . Table 1 Clinico-pathologic characteristics of patients with node-positive cervical carcinoma after radical surgery Factors n Percentage (%) Age(yrs) <40 15 22.7 ≥40 51 77.3 Stage IB 8 12.1 IIA 37 56.1 IIB 21 31.8 Tumor size(cm) <4 31 47.0 ≥4 35 53.0 Histology Squamous 41 62.1 Adenocarcinoma 20 30.3 Adenosquamous 4 6.1 Others 1 1.5 Differentiation Poor 16 24.2 Moderate 46 69.7 Well 4 6.1 Pelvic lymph node metastases 1 17 25.8 ≥2 49 74.2 Parametrial extension Negative 58 87.9 Positive 8 12.1 Vaginal margin involved Negative 64 97.0 Positive 2 3.0 Depth of stromal invasion ≤2/3 19 28.8 ≤2/3 47 71.2 Lymphvascular permeation Negative 47 71.2 Positive 19 28.8 Sixty four of these women (97.0%) had brachytherapy in either three or four fractions with a total dose of 15~20 Gy at point A, two weeks prior to radical surgery because of bulky tumor or vaginal vault involvement. Intra-arterial chemotherapy was administered to 11 patients (16.7%) before surgery because of bulky tumor or parametrial extension. The regimen based on cisplatin (CDDP) + 5-Fluorouracil(5-FU) with 2~3 cycles at 3-weeks intervals was used. All patients underwent Wertheims-Meigs' procedure that included radical hysterectomy and bilateral pelvic lymphadenectomy. Three patients had para-aortic lymph node (PALN) sampling because there was suspicion of metastasis. Postoperative external beam irradiation was delivered to 18 patients (27.3%) with 6 MV X-ray routine anterior and posterior portal at a dose of 35~45 Gy at point B with 1.8 Gy daily fraction. Two patients with positive vaginal margin were given extra brachytherapy with 15 Gy 0.5 cm below the vaginal mucosa. 12 cases with positive common iliac node and 3 with PALN metastasis received an additional 40 Gy to the PALN chain area. 10 patients (15.2%) were given postoperative chemotherapy. The regimen consisted CDDP + 5-FU + Cyclophosphamide (CTX) for squamous carcinoma, and CDDP + 5-FU + Mitomycin (MMC) for adenocarcinoma with 2~6 cycles at 3~4-weeks intervals. A total of 19 patients (28.8%) received adjuvant radiotherapy and chemotherapy as mentioned above. Follow-up Patients were evaluated every two months for the first two years and then six monthly for the additional years by clinical interview, or telephone, or letters. Disease-free survival (DFS) was defined as the time from the date of surgery to local or nodal recurrence or metastasis. Overall survival (OS) was calculated from the date of surgery to the date of death. Recurrences were defined as local if they were detected in the pelvis or vagina and distant metastases as detected in extra-pelvic locations. The median follow-up time was 32 months (range 2~108 months). Statistical analysis Data analysis was performed with Statistical package for social sciences (SPSS) version 10.0 statistical package. The survival was calculated by Kaplan-Meier method. The differences in survival were compared with Log-rank test. Multivariate analyses were performed with the Cox proportional hazard model. The correlation analysis was performed by Kendall's method. Pearson's chi-square or Fisher's exact test was used to compare the difference of proportions. A probability value of P < 0.05 was considered significant. Results The 5-year overall survival of the patients with pelvic lymph node metastasis was 40.7%. The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes ( P < 0.05). The former's distant metastasis rate (5.9%) was lower than the latter's (32.7%) ( P = 0.05). However, there was no significant difference of pelvic recurrence between them ( P > 0.05) (Table 2 , Figure 1 ) Table 2 Relationship between the number of positive nodes and prognosis Number of positive nodes n 5-year survival (%) P Recurrence rate (%) P Metastasis rate (%) P 1 17 56.5 0.033 23.5 0.807 5.9 0.050 ≥2 49 36.4 26.5 32.7 Figure 1 Overall survival according to one vs. two or more positive lymph nodes. The 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was less than that (43.1%) of patients with lower than common iliac node involvement. However, the difference was not significant (P > 0.05). The former's pelvic recurrence rate (13.3%) was lower than the latter's (29.4%) (P > 0.05). However, the patients with common iliac or paraaortic nodes had significantly higher distant metastasis (53.3%) than that of the patients with lower nodes (17.6%) (P < 0.01). The number of the positive lymph nodes was closely correlated with the level of lymph node metastasis. The relative coefficient was 0.557 (P < 0.01) (Table 3 ). Table 3 Relationship between the site of positive nodes and prognosis Site n 5-year survival rate (%) P Recurrence rate (%) P Metastasis rate (%) P Common iliac or above 15 33.3 0.086 13.3 0.318 53.3 0.005 Lower than common iliac 51 43.1 29.4 17.6 The 5-year survival (12.6%) of the patients who had no adjuvant therapy was much lower than that (53.7%) of those with adjuvant therapy (P<0.05). The 5-year survival rates of the adjuvant radiotherapy, adjuvant chemotherapy, and adjuvant radio-chemotherapy groups were 53.5%, 49.2% and 56.1% respectively (P > 0.05). The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than those with adjuvant therapeutic groups. However, the differences were not significant (P > 0.05) (Table 4 , Figure 2 ) Table 4 Relationship between adjuvant therapy and prognosis n 5-year survival (%) Recurrence rate (%) Metastasis rate (%) No adjuvant therapy 19 12.6 42.1 31.6 Radiotherapy 18 53.5 22.2 22.2 Chemotherapy 10 49.2 20.0 10.0 Radiochemotherapy 19 56.1 15.8 21.1 Figure 2 Overall survival according to adjuvant therapy [radiotherapy (RT) vs. chemotherapy (CT) vs. radiochemotherapy (RT+CT) vs. no adjuvant therapy (No)]. Multivariate analysis of prognostic factors Cox proportional hazard model analysis showed that cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors for survival ( P < 0.05) (Table 5 ) Table 5 Cox proportional hazard model analysis of variables in predicting overall survival Factors Coefficient RR 95%CI P Age -0.031 0.970 0.932~1.010 0.136 Stage 0.283 1.327 0.379~4.644 0.658 Tumor size 0.332 1.394 0.984~1.975 0.061 Histology -0.035 0.966 0.563~1.659 0.900 Differentiation 0.787 2.196 1.104~4.370 0.025* Number of positive nodes 0.076 1.079 1.006~1.158 0.034* Parametrial extension 0.484 1.623 0.584~4.508 0.353 Vaginal margin involved -1.007 0.365 0.029~4.655 0.438 Depth of stromal invasion 0.423 1.526 0.856~2.722 0.152 Lymphvascular permeation 0.270 1.311 0.500~3.437 0.582 Nerve invasion 0.101 1.106 0.092~13.337 0.937 Adjuvant therapy -1.684 0.186 0.075~0.459 0.000* * P < 0.05 Discussion Cervical cancer remains the third common cancer in women around the world, although its incidence is on the decline in North American and in Europe. It is estimated that there will be 10,370 new cases in 2005 in North America [ 8 ]. In many developing countries, not only is cervical cancer the most frequently occurring cancer among middle-aged women, but it is also a leading cause of death, partly due to the poor access to medical care and the unavailability of routine screening in many of these countries [ 9 ]. In Shanghai, the biggest city in China, there were 150 new cases in 2000. The standard incidence rate was 2.9 per 100 000 [ 10 ]. Radical surgery has been found to be very effective in patients with early stage (IB-IIA) cervical carcinoma [ 11 - 13 ]. However, the 5-year survival has been lingering at 50%-90% during the past twenty years [ 14 ]. The reported risk factors of cervical carcinoma included clinical stage, bulky tumor, histological types, cellular differentiation, deep stromal invasion, parametrial extension, vaginal margin involved, lymphvascular permeation, lymph node metastasis, race and age [ 13 , 15 - 18 ]. In most studies the presence of pelvic lymph node metastases has been associated with increased pelvic recurrence and distance metastases, and a decrease in overall survival. Our Cox proportional hazard model analysis showed cellular differentiation, number of positive nodes and adjuvant therapy to be the independent prognostic factors ( P < 0.05). The 5-year survival of patients with one positive node (56.5%) was higher than that (36.4%) of those with two or more positive nodes ( P < 0.05). There was no significant difference of pelvic recurrence between them. However, the former's distant metastases rate (5.9%) was lower than the latter's (32.7%) ( P = 0.05). In the analysis of the site of lymph node metastases, the 5-year survival (33.3%) of the patients with positive common iliac node and paraaortic lymph node (PALN) was lower than that (43.1%) of patients with lower than common iliac node involvement. Unlike the series of Tsai the difference in our series was not statistically significant [ 19 ]. This is probably due to limited number of cases with paraaortic or common iliac nodes. The number of positive nodes correlated with the height of positive nodes ( P < 0.01). The result suggested that in patients with more positive pelvic nodes there is higher chance of nodal metastasis at higher level node basins, which supported the external irradiation of PALN chain area for the multiple pelvic nodes involvement. The distant metastasis rate was 32.7% in patients with multiple positive lymph nodes, which showed the limitation of adjuvant radiotherapy and theoretically supported the postoperative combined chemotherapy. The prognosis of patients with positive lymph node was poor because of local recurrence and distant metastasis. How to improve the prognosis of these patients has been the focus of gynecological oncology. Stock et al [ 7 ] compared postoperative whole pelvic irradiation with those treated with radical hysterectomy alone, and showed a significantly improved pelvic control rate, 5-year disease-free survival (DFS) and overall survival (OS) (78% Vs 45%, 65% Vs 41%, 58% Vs 46%, respectively). Peters' study [ 20 ] demonstrated that postoperative radio-chemotherapy could greatly improve the 4-year DFS and OS compared to the adjuvant radiotherapy alone. In our study, adjuvant therapy improved the 5-year survival than the surgery alone (53.7% vs. 12.6%), decreased the pelvic recurrence and distant metastasis, which suggested the clinical importance of adjuvant therapy in patients with positive nodes. In this study the 5-year survival of adjuvant radio-chemotherapy arm was higher than adjuvant radiotherapy alone or chemotherapy alone. The pelvic recurrence (42.1%) and distant metastasis (31.6%) of the surgery alone were higher than the other three therapeutic arms. However, the differences were not significant and the limited number of cases in each arm could be the reason. It is necessary that these results are verified in prospective randomized control trials. Several randomized clinical trials [ 20 - 25 ] performed by the Gynecologic Oncology Group (GOG), the Radiation Therapy Oncology Group (RTOG) and the Southwest Oncology Group (SWOG) have demonstrated a significant advantage both in DFS and OS when cisplatin-based chemotherapy was administered during radiation for advanced stages of cervical cancer and early stage disease with poor prognostic factors. Green et al [ 26 ] did a systemic review of all known randomized controlled trials published between 1981 and 2000, 2865–3611 patients were available for analysis. The findings suggested that concomitant chemotherapy (CT) and radiotherapy (RT) improves OS and DFS, and reduces local and distant recurrence. Based on results, the US National Cancer Institute (NCI) released a clinical announcement supporting the concurrent use of cisplatin-based chemotherapy with RT for high-risk early stage and locally advanced stage cervical cancer [ 9 , 27 ]. Recently a prospective randomized trial performed by the National Cancer Institute (NCI) of Canada [ 25 ] failed to show benefit with the use of chemoradiation compared with RT alone. The potential inclusion of paraaortic nodes positive patients and the significant difference in anemia raises question about this trial result [ 28 ]. The therapy of patients with stage IIB cervical carcinoma is still a controversy [ 29 ]. Although radical radiotherapy (RT) is the proper choice for patients with stage IIB cervical cancer in general, the therapeutic effect is not as good as expected if the tumor is too bulky or is histologically adenocarcinoma. Hence a few gynecology oncologists tried brachytherapy and/or neoadjuvant chemotherapy to decrease the lesion, then performed radical surgery for some stage IIB patients with bulky tumor (>4 cm) or slight (less than 1/2) parametrial extension [ 30 , 31 ]. Postoperative adjuvant therapy would then be recommended according to risk factors [ 9 , 17 , 20 , 32 ]. In our study, the 5-year survival of the patients with stage IIB cervical carcinoma was only 54.3%, which was not at all satisfactory compared with the reported 58.9%–80.1% [ 33 - 35 ]. So we advocate that radical surgery should be taken cautiously for this group of patients. Any attempt to improve their prognosis by means of adjuvant therapy is not recommended if the parametrium can not be thoroughly dissected from the pelvic wall. Competing interests The author(s) declare that they have no competing interests. Authors Contributions XC collected the data, patients' follow-up and drafted the article. SC, ZL, MT, MX collectively designed the study, participated in collection of data and revising the article. RZ participated in collection and analysis of data, revising the article Funding Source None	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546224.xml
533863	Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines: less involvement of metallothionein	Background Heptaplatin is a new platinum derivative with anticancer activity against various cancer cell lines, including cisplatin-resistant cancer cell lines (Cancer Chemother Pharmacol 1995; 35: 441). Methods Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines has been investigated in connection with metallothionein (MT). Cytotoxicity was determined by an MTT assay. MT mRNA, was determined by RT-PCR assay. Transfection study was carried out to examine the function of MT. Results Of various gastric cancer cell lines, SNU-638 and SNU-601 showed the highest and lowest levels of MT mRNA, respectively, showing 80-fold difference. The IC 50 values of SNU-638 to cisplatin, carboplatin and heptaplatin were 11.2-fold, 5.1-fold and 2.0-fold greater than those of SNU-601, respectively. Heptaplatin was more effective against cisplatin-resistant and MT-transfected gastric cancer sublines than cisplatin or carboplatin was. In addition, heptaplatin attenuated cadmium, but not zinc, induction of MT. Conclusion These results indicate that molecular mechanisms of heptaplatin effective against cisplatin-resistant gastric cancer sublines is at least in part due to the less involvement of MT in heptaplatin resistance as well as its attenuation of MT induction.	Introduction Gastric cancer is the most frequently diagnosed and the second leading cause of cancer-related death in Korea. For many years, a few single agents such as 5-fluorouracil, doxorubicin, mitomycin C, and nitrosourea, have been considered to have significant antitumor activity in gastric cancer patients [ 1 ]. However, the response rate has been <30% and complete remission has been rare. Several combination chemotherapy regimens such as FAM (5-fluorouracil, adriamycin, and mitomycin C) have been attempted in order to improve the treatment outcomes. In a nonrandomized Phase II study for advanced gastric cancer, the FAM regimen achieved an objective partial response rate of 42% [ 2 ]. Some cisplatin-based combination chemotherapy regimens have shown high response rates [ 3 , 4 ]. Despite the efficacy of cisplatin against gastric cancer, there were two major problems with this agent. Firstly, there are significant side effects, such as severe nausea and vomiting, nephrotoxicity, and neurotoxicity [ 5 ]. Secondly, the cancer cells show a primary or acquired resistance to cisplatin [ 6 ]. Therefore, extensive effort to develop novel cisplatin analogs with equivalent or greater antitumor activity and a lower toxicity has been made. Among them, carboplatin has reduced renal and gastrointestinal side effects than cisplatin [ 7 ]. However, carboplatin has no enhanced therapeutic efficacy over cisplatin and has not circumvented the acquired resistance to cisplatin. Therefore, it is essential to develop a new platinum drug with a greater potent antitumor activity as well as being effective against resistant cells. Recently, SK Pharmaceutical (Seoul, Korea) developed many cisplatin analogs, including heptaplatin (SKI-2053R) (Fig. 1 ). Heptaplatin exhibited a greater antitumor activity against a number of human cancer cell lines including gastric cancer as well as a lower nephrotoxicity [ 8 , 9 ]. In addition, heptaplatin 2053R is also effective against cisplatin-resistant L1210 leukemia cells (L1210-CPR) [ 10 ]. Figure 1 Structure of cisplatin analogs. Metallothioneins (MT) are a family of stress-induced proteins with a wide variety of physiological functions, including protection against metal toxicity and oxidants. They may also assist in regulating cellular proliferation, apoptosis, and malignant progression. MT has a high affinity for heavy metals since it contains many cystein residues, which account for approximately 30% of the total amino acids in this protein molecule [ 11 ]. MT is a low molecular weight metal binding protein whose synthesis is induced by heavy metals, glucocorticoids and other factors [ 12 ]. Although the physiological role of MT is unclear, MT participates in detoxifying heavy metals or maintaining zinc and copper homeostasis [ 12 ]. Some reports have shown that MT has free radical scavenging ability in vitro [ 13 , 14 ] and MT expression also increases the cellular resistance to radiation damage [ 15 ]. The cells transfected with the bovine papilloma virus expression vectors containing the DNA encoding human metallothionein-IIA were resistant to cisplatin, melphalan, and chlorambucil but not to 5-fluorouracil or vincristine [ 16 ]. In this study, the effect of MT on the cisplatin analog-induced cytotoxicity was investigated in the gastric cancer cell lines. Since heptaplatin is less associated with MT, it is believed to be effective against cisplatin-resistant cells related to the high level of MT. Results MT cDNA, whose expression level was higher than the wild-type AML-2, were isolated from the paraquat-resistant AML-2 cells [ 17 , 18 ]. The nucleotide sequence of the clone, containing an almost full-length cDNA copy of the human MT mRNA, was determined. Molecular cloning and nucleotide sequence analysis revealed that the hMT transcript is a novel hMT-I isoform, which was designated hMT-Ip, and exhibits a 98.4% homology with the hMT-Ie isoform, whereas it shows 86.9% homology with hMT-II, 83.8% homology with hMT-III and 62.9% homology with hMT-IV [ 18 ]. MT mRNA expression in the gastric cancer cell lines Using the RT-PCR assay, the MT mRNA was determined in the 8 human gastric cancer cell lines, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668. The gastric cancer cell lines showed differential expression level of MT mRNA, in which the SNU-601 cells expressed the lowest MT mRNA level whereas SNU-638 expressed the highest level (Fig. 2 ). The MT mRNA level of SNU-638 was 80 times higher than that of SNU-601. Figure 2 (A) MT mRNA expression in the gastric cancer cell lines. The expression level was determined by RT-PCR assay. β-actin was used as a control for RNA. The cDNA reverse-transcribed from the mRNA was individually amplified with each primer pair for the MT and β- actin genes. Aliquots of each PCR reaction mixture were separated on 7% polyacrylamide gel in TAE. The gel was dried and exposed on X-ray film overnight. (B) The ratio of MT/β-actin of SNU cell lines. Comparative sensitivity of the gastric cancer cells that express MT differentially to cisplatin analogs The resistance of the gastric cancer cells that express MT to cisplatin was compared. SNU-16 and SNU-601, which showed the lowest MT expression levels, were sensitive but SNU-638, which exhibited the highest MT expression levels, were resistant to cisplatin (Fig. 3 ). On the other hand, the gastric cancer cell lines expressing moderate MT expression levels (SNU-16, SNU-484, SNU-620, SNU-5 and SNU-668) showed a lower correlation between the sensitivity to cisplatin and MT expression (Fig. 3 ). In addition, 3 gastric cancer cell lines with very low (SNU-484) or moderate MT levels (SNU-1 and SNU-620) which are as resistant to cisplatin as SNU-638 (Fig. 2 and 3 ), suggesting no involvement at all of MT in cisplatin resistance or the involvement of other resistance mechanisms except MT. Figure 3 Cytotoxic effect of cisplatin in the gastric cancer cell lines. Cisplatin cytotoxicity was determined using an MTT assay. Correlation of MT mRNA expression and sensitivity to the cisplatin analogs The cytotoxicity of the cisplatin analogs in the SNU-638 and SNU-601 cells, which expressed different MT levels, was determined by an MTT assay. The SNU-638 cells were approximately 11 times, 4 times and 2 times more resistant to cisplatin, carboplatin and heptaplatin, respectively, than the SNU-601 cells (Fig. 4 and Table 1 ), suggesting a lower involvement of MT in the resistance to heptaplatin. Figure 4 Cytotoxic profiles of the cisplatin analogs in the SNU-601 and SNU-638 cell lines showing low and high MT expression levels, respectively. Table 1 Comparison of cytotoxicities of the cisplatin analogs in various gastric cancer cell lines Ratio of IC 50 a Cell line Cisplatin Carboplatin Heptaplatin 638/601 b 11.23 5.10 2.00* CIS/601 c 50.30 55.95 28.36* MT/Mock d 54.77 e 3.00 1.57* a , The 50% inhibitory concentration (IC 50 ) for a particular agent was defined as the drug concentration which results in a 50% reduction in cell number to the untreated control. IC 50 values were determined directly from semilogarithmic dose-response curves. The means were obtained from the experiments carried out at least in triplicate. b , The ratios of IC 50 values of SNU-638 to that of SNU-601 (Fig. 4) c , The ratios of IC 50 values of SNU-601/Cis to that of SNU-601 (Fig. 5) d , The ratios of IC 50 values of SNU-601/MT to that of SNU-601/Mock (Fig. 7) e , The ratio calculated from extrapolated data obtained in SNU-601/MT (Fig. 7) , P < 0.05 versus cisplatin or carboplatin group Comparison of resistance to cisplatin analogs in gastric cancer sublines resistant to cisplatin The cisplatin-resistant gastric cancer subline, SNU-601/Cis was selected in the presence of 2 μg/ml cisplatin. The SNU-601/Cis cells showed an approximately 49-fold, 56-fold and 29-fold increased resistance to cisplatin, carboplatin and heptaplatin, respectively (Fig. 5 and Table 1 ). Figure 5 Cytotoxic effects of cisplatin, carboplatin and heptaplatin in the SNU-601/WT cells and its cisplatin-resistant subline SNU-601/CIS. The cytotoxicity was determined using the MTT assay. Mean ± SE of triplicate determination is given. Sensitivity of MT-transfected SNU-601 cells to cisplatin analogs Sensitivity to cisplatin analogs was compared between SNU-601 transfected with the vector containing the full-length MT cDNA (pIRESneo2/MT) and with the empty vector (pIRESneo2/Mock). Although the SNU-601/MT showed a 66% increase in MT expression when compared to the SNU-601/Mock cells, the SNU-601/MT cells scavenged significantly ROS generated by hydrogen peroxide or paraquat as compared with those in SNU-601/Mock using a fluorescence probe (Fig. 6 ). This result suggests that SNU-601/MT expresses not only a higher quantity of MT than does SNU-601/Mock but also the MT functions to scavenge ROS. Figure 6 Comparison of ROS-scavenging activities between the SNU-601/Mock and the SNU-601/MT sublines. The reaction took place with 1 × 10 5 cells and 1 μM 2',7'-DCFH diacetate in 3 ml phosphate-buffered saline. The cells were exposed to H 2 O 2 and paraquat for 4 hours. The fluorescence intensity was determined using a fluorometer with excitation wavelength at 485 and emission wavelength at 530 nm. The results are expressed as means ± SE (n = 3). PQ, paraquat; , P < 0.05. The sensitivities of the SNU-601/MT cells to cisplatin analogs were tested using the MTT assay. The SNU-601/MT cells showed a lower degree of resistance to cisplatin and carboplatin, but was paradoxically sensitive to heptaplatin when compared to the SNU-601/Mock cells (Fig. 7 ). Figure 7 Cytotoxic effects of cisplatin, carboplatin and heptaplatin in the SNU-601/Mock and SNU-601/MT sublines. Cytotoxicity was determined using the MTT assay. Mean ± SE of triplicate determination is given. Comparison of MT mRNA expression in SNU-601 after treatment of zinc and cisplatin analogs To examine the effect of zinc and cisplatin analogs on MT mRNA expression, the maximum non-cytotoxic concentrations of each drug in the 3-day MTT assay were chosen and then treated for 1 day. After the treatment with 100 μM ZnCl 2 , 0.6 μg/ml cisplatin, 2 μg/ml carboplatin and 0.2 μg/ml heptaplatin for 24 hr, MT mRNA level was determined using the RT-PCR assay. The MT mRNA expression level was significantly increased by ZnCl 2 but was decreased by heptaplatin. In contrast, there were no changes in MT mRNA expression level by cisplatin and carboplatin (Fig. 8 ). Figure 8 MT expression in the SNU-601 cell line after a treatment with zinc and the cisplatin analogs. The SNU-601 cells were treated with 100 μM ZnCl 2 , 0.6 μg/ml cisplatin, 2 μg/ml carboplatin, 0.2 μg/ml heptaplatin for 24 hrs. The MT mRNA expression level was determined by the RT-PCR assay. Effect of heptaplatin on heavy metal-induced MT expression in SNU-601 cells Of the cisplatin analogs, heptaplatin is not only relatively less related to MT but also decreases the MT expression level. We tested whether or not heptaplatin could influence the heavy metal-induced MT expression. Each IC 50 concentration of CdCl 2 or ZnCl 2 obtained from the 3-day MTT that did not influence the growth of the SNU-601 cells for 24 hr were used in this study. The SNU-601 cells were treated with 32 μM CdCl 2 or 200 μM ZnCl 2 in the presence or absence of 200 ng/ml heptaplatin for 24 hr. After a 24-h treatment, MT expression level was determined by the RT-PCR assay and Western blot analysis. Fig. 9 shows that heptaplatin inhibits the MT gene expression induced by cadmium but not zinc, suggesting the differential modulation of metal-induced MT expression. Figure 9 Effect of heptaplatin on MT induction by heavy metals in the SNU-601 cell lines. The SNU-601 cells were treated with 32 μM CdCl 2 or 200 μM ZnCl 2 in the presence or absence of 200 ng/ml heptaplatin for 24 hr. The MT expression level was determined by (A) the RT-PCR assay and (B) the Western blotting method. Discussion The cisplatin analogs are among the most active and widely used cytotoxic anticancer drugs. However, the acquisition or presence of resistance significantly undermined the curative potential of these drugs against many malignancies. Alterations in the cellular pharmacology, including decreased drug accumulation, increased cellular thiol levels and increased repair of platinum-DNA damage, have been observed in the cisplatin-resistant cancer cell lines [ 19 ]. Since heptaplatin is effective against cisplatin-resistant cancer cells, its development has shed light on cisplatin-refractory cancer patients. However the mechanism by which heptaplatin is effective against cisplatin-resistant cancer cells is unclear. Since MT is involved in cisplatin resistance [ 26 ], the molecular mechanisms for the effect of heptaplatin against the cisplatin-resistant cancer cell lines was investigated with respect to the involvement of MT. In this study, the gastric cancer cell lines expressed different basal MT mRNA levels, whose mechanisms remain to be determined. It was suggested that DNA hypomethylation was responsible for the higher basal hMT-IIa mRNA levels in the cisplatin-resistant cells [ 20 ]. In this study, MT expression in the gastric cancer cell lines was not always related to the resistance to cisplatin, suggesting the involvement of other mechanisms except MT. But heptaplatin is more effective against the MT-overexpressing gastric cancer cell line selected for resistance to cisplatin than cisplatin and carboplatin were. In addition, the cytotoxicity by cisplatin and carboplatin but not heptaplatin was reduced by a pretreatment with zinc, which can induce MT remarkably (data not shown). Taken together, heptaplatin was more effective against both cisplatin-resistant gastric cancer cell subline SNU-601/CIS and MT-overexpressing SNU-638 than was cisplatin or carboplatin. These results suggest a possibility that the cytotoxicity of heptaplatin was less influenced by MT. To test the hypothesis, the function of MT involved in the cytotoxicity of the cisplatin analogs was confirmed by the transfection of MT. The cytotoxicity of heptaplatin was not influenced by the MT transfection, which indicates that the anticancer activity of heptaplatin to the cisplatin-resistant gastric cancer sublines could be less associated with MT. In addition, heptaplatin inhibited the MT expression caused by cadmiun but not zinc. This result suggests that metals differentially regulate MT, which is supported by the literature. The inhibition mechanism for H7, a protein kinase C inhibitor, may be different between the zinc- and cadmium-treated cells. H7 blocks zinc transport but not cadmium transport although rottlerin, a PKC inhibitor that can block both cadmium and zinc transport [ 21 , 22 ]. Hypoxia activates MT expression through the metal response elements and that this activation involves the metal transcription factor-1 (MTF-1) [ 23 ]. MTF-1 was found to be present in the cytoplasm of the cells. Upon zinc stimulation, MTF-1 was translocated to the nuclei and activated MT gene expression [ 24 ]. However, no MTF-1 translocation was observed in cells treated with cadmium [ 25 ]. Whether or not MTF-1 is involved in inhibiting the cadmium-induced MT induction by heptaplatin remains to be determined. In summary, these results indicate that molecular mechanisms responsible for the effect of heptaplatin against the cisplatin-resistant gastric cancer sublines is at least in part due to the lower involvement of MT as well as its attenuation of MT induction. Materials and Methods Cell culture and the selection of the gastric cancer cell subline for cisplatin resistance The human gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668) were obtained from the Cancer Research Center in Seoul National University (South Korea). The cells were cultured in RPMI 1640 (GibcoBRL Grand Island, NY, U.S.A.) supplemented with 10% FBS (Sigma Chemical Co. St. Louis, MO, U.S.A.). The cells were maintained as a monolayer culture and subcultured at confluence. The cisplatin-resistant gastric cancer cell subline was selected by chronic exposure to gradually increasing cisplatin concentrations ranging from 200 ng/ml (IC 50 value) to 2,000 ng/ml on an intermittent dosage schedule. Cytotoxicity assay The in vitro cytotoxicity of the drugs was determined using an MTT (Sigma Chemical Co. St. Louis, MO, USA) assay previously described by Pieters et al . [ 26 ]. The 50% inhibitory concentration (IC 50 ) for a particular agent was defined as the drug concentration that causes in a 50% reduction in the number of cells compared to the untreated control for 3 days. The IC 50 values were determined directly from the semilogarithmic dose-response curves. Cisplatin and carboplatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and heptaplatin (Sunpla ® ) was generously donated by SK Pharmaceutical (Seoul, Korea). Cisplatin analogs were dissolved in phosphate buffered saline and used immediately because of their instability in aqueous solution. RNA extraction RT-PCR assay The total RNA was extracted from the cells using the acid guanidium thiocyanate-phenol-chloroform method [ 27 ]. The MT and β-actin mRNA transcripts were detected using an RT-PCR assay. The RNA from each sample was reverse transcribed using 200 units of Moloney murine leukemia virus reverse transcriptase (GibcoBRL, Grand Island, NY, U.S.A) and oligo (dT) primer for 1 h at 37°C. The resulting cDNA was diluted 1:5 with distilled water and amplified with 2.5 units of Taq polymerase (Perkin-Elmer, Foster City, CA, USA) and 10 pmole of each primer using a GeneAmp PCR2400 (Perkin-Elmer, Foster City, CA, USA). The MT expression level was detected with the sense and antisense primers corresponding to the nucleotides (5'-ATGGACCCCAACTGCTCG) and (5'-TCAGGCGCAGCAGCTGCA), respectively, of the published cDNA sequence [ 28 ] and yielded a 220-bp PCR product. The β-actin expression level, as a control of the RNA quantity, was detected with the sense and antisense primers corresponding to the nucleotides 1912–1932 (5'-GACTATGACTTAGTTGCGTTA) and 2412–2392 (5'-GCCTTCATACATCTCAAGTTG), respectively, of the published cDNA sequence [ 29 ], yielding a 501-bp PCR product. Twenty one PCR cycles for MT was carried out as follows: denaturation at 94°C for 30 s, annealing at 57°C for 60 s, and extension at 72°C for 1 min was used. Sixteen cycles of PCR for β-actin of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s. After the last cycle, all the PCR products were subjected to a final extension for 5 min at 72°C. For quantitation, 5 mCi/ml of [α- 32 P] dCTP was added to each reaction mixture. Subsequently, the PCR products were combined and electrophoresed on 7.5% nondenaturing polyacrylamide gels. The bands were scanned using a densitometer (Pdi, Huntington Station, NY, USA). The quantity of each mRNA transcript was normalized with that of β-actin mRNA. Autoradiographic films of the RT-PCR assay were subjected to densitometric analysis using a densitometer. Protein extraction and Western blot analysis for MT The total cell lysates from 2 × 10 6 cells were prepared by lysing the harvested cells in an extraction buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in phosphate-buffered saline) supplemented with 2 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co. St. Louis, MO, USA) and 10 μg/ml leupeptin (Sigma Chemical Co. St. Louis, MO, USA). The DNA was sheared by sonication. The sample buffer for MT analysis was used without 2-mercaptoethanol [ 30 ]. Western blotting analysis was performed using a slight modification of the method reported by Towbin et al . [ 31 ]. The proteins were transferred onto a nitrocellulose membrane by electroblotting at a current of 60 V overnight using a transfer buffer containing 5% mercaptoethanol. The membrane was incubated in a blocking solution (5% skim milk) for 1 hr at room temperature, washed, and then incubated with a monoclonal mouse antibody (E9 clone, 1:500, Dako Corporation, Carpinteria. CA, USA) for MT-I and MT-II. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:1000) for 1 hr. The membrane was then stained using the ECL detection kit (Amersham Biosciences Corp., Piscataway, NJ, USA). Production of stable transfectant cell line Mammalian transfectants were produced as reported previously [ 18 ]. The mammalian expression vector, pIRESneo2 (Clonetech, Palo Alto, CA, USA), was used to clone the MT. A 220 base pair cDNA fragment containing the full open reading frame of human MT was amplified by RT-PCR from the RNA of AML-2/PQ400 [ 17 ], a paraquat-resistant AML-2 cell line that highly over-expresses MT. The PCR primer pairs were as follows: the forward primer 5'-CG GGATCC ATGGACCCCAACTGCTCG-3' to introduce a Bam H I site as underlined, and reverse primer: 5'ATAAGAAT GCGGCCGC TCAGGCGCAGCAGCTGCA-3' to introduce a Not I site as underlined. The resulting MT PCR product was cloned to a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). After confirmation of the sequencing, the MT was cut from the TOPO plasmid vector (pCR 2.1-TOPO), purified, subcloning to the multicloning site of the expression vector pIRESneo2 and subsequently sequenced to confirm the correct open reading frame. Transfection was performed using the LipofectAmine Plus reagents (GIBCO BRL, Grand Island, NY, USA). Approximately 1 × 10 6 of the SNU-601 cells were plated into a 60 mm tissue culture dish and cultured overnight. Prior to transfection, the growth medium was replaced with the serum free RPMI 1640 media and cultured. The LipofectAmine reagent containing 2 μg of pIRES/Mock and pIRES/MT was combined with the Plus reagent and applied to the cells. After culturing for five hours, the media was replaced with RPMI 1640 containing 10% FBS. After selection by culture in the medium containing 0.2 mg/ml of G-418 (GIBCO BRL, Grand Island, NY, USA), the emerging colonies were isolated with cloning rings. The stable transfectants, SNU-601/Mock and SNU-601/MT, were obtained. The selection medium was changed every 2–3 days. Determination of reactive oxygen species (ROS) using a fluorometric probe Dichlorofluorescin (DCFH) was used to measure the ROS concentration. After 2', 7'-dichlorofluorescin diacetate (DCFH-DA) crosses the membrane, it is de-esterified to DCFH which is oxidized to fluorescent DCF by the ROS [ 32 ]. Phosphate-buffered saline containing 1 × 10 5 /ml SNU-601/Mock or the SNU-601/MT cells was incubated with 1 μM DCFH-DA at 37°C for 4 hr. After incubation, the DCF fluorescence intensity was determined using a fluorometer at 485 nm for excitation and 530 nm for emission.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC533863.xml
545077	Keratocyte loss in corneal infection through apoptosis: a histologic study of 59 cases	Background Keratocyte loss by apoptosis following epithelial debridement is a well-recognized entity. In a study of corneal buttons obtained from patients of corneal ulcer undergoing therapeutic keratoplasty, we observed loss of keratocytes in the normal appearing corneal stroma, surrounding the zone of inflammation. Based on these observations, we hypothesized that the cell loss in the inflammatory free zone of corneal stroma is by apoptosis that could possibly be a non-specific host response, independent of the nature of infectious agent. Methods To test our hypothesis, in this study, we performed Terminal deoxyribonucleotidyl transferase-mediated d-Uridine 5" triphosphate Nick End Labelling (TUNEL) staining on 59 corneal buttons from patients diagnosed as bacterial, fungal, viral and Acanthamoeba keratitis. The corneal sections were reviewed for morphologic changes in the epithelium, stroma, type, degree and depth of inflammation, loss of keratocytes in the surrounding stroma (posterior or peripheral). TUNEL positivity was evaluated in the corneal sections, both in the zone of inflammation as well as the surrounding stroma. A correlation was attempted between the keratocyte loss, histologic, microbiologic and clinical features. Results The corneal tissues were from 59 patients aged between 16 years and 85 years (mean 46 years) and included fungal (22), viral (15), bacterial (14) and Acanthamoeba (8) keratitis. The morphological changes in corneal tissues noted were: epithelial ulceration (52, 88.1%), destruction of Bowman's layer (58, 99%), mild to moderate (28; 47.5%) to severe inflammation (31; 52.5%). Morphologic evidence of disappearance or reduced number of keratocytic nuclei in the corneal stroma was noted in 49 (83%) cases; while the TUNEL positive brown cells were identified in all cases 53/54 (98%), including cases of fungal (19), bacterial (14), viral (13), and Acanthamoeba keratitis. TUNEL staining was located mostly in the deeper stroma and in few cases the peripheral stroma. TUNEL positivity was also noted with the polymorphonuclear infiltrates and in few epithelial cells (10 of 59, 17%) cases, more with viral infections (6/10; 60%). Conclusions We report apoptotic cell death of keratocytes in the corneal stroma in infectious keratitis, a phenomenon independent of type of infectious agent. The inflammatory cells in the zone of inflammation also show evidence of apoptotic cell death. It could be speculated that the infective process possibly triggers keratocyte loss of the surrounding stroma by apoptosis, which could possibly be a protective phenomenon. It also suggests that necrotic cell death and apoptotic cell deaths could occur simultaneously in infective conditions of the cornea.	Background Infections of the cornea are potentially blinding diseases and may be caused by fungi, bacteria, viruses and a few protozoans. Despite best efforts with early diagnosis and specific antimicrobial treatment about one-third of cases require surgical intervention [ 1 ]. Those which respond to medical treatment result in scarring which may lead to varying degree of visual disability. The tissue destruction in infectious keratitis, irrespective of the etiologic agent, is the compound effect of cytokines and inflammatory mediators released by the microorganism, host inflammatory cells and the metalloproteases that act on the collagen [ 2 - 5 ]. The aim of treating corneal infections is not only to control the infection but also to restrict the tissue damage so that the corneal stroma maintains the transparency thus retaining its visual function. Though wound healing following surgical procedures including routine keratoplasty and refractive surgeries is well documented [ 6 - 11 ], healing after an infectious etiology is not well documented. We had observed the loss of keratocytes in deeper stroma in Acanthamoeba keratitis and had proposed that one of the mechanisms for this loss was apoptosis of keratocytes [ 12 ]. We have since then observed the paucity of stromal keratocytes in the zone surrounding the infected area in all types of corneal infections, prompting us to speculate that it is possibly a host mediated response to an infectious process. Apoptosis is an active process orchestrated by a series of events starting from activation of genes, release of enzymes, specific morphologic changes and ultimately leading to "an environmental-friendly cell death" whereby the dead cells are removed without affecting the bystander cells [ 13 - 15 ]. This is in contrast to necrotic cell death whereby the cell dies with release of various molecules that destroy the surrounding cells and stroma. Though tissue necrosis is an accepted fate of tissues in an infective process, it is associated with unwanted loss of visual function in infections of cornea. Apoptotic cell loss may be an attempt by the host stroma to escape necrosis, thereby minimizing tissue damage. The corneal buttons removed during therapeutic keratoplasty for uncontrolled infections could possibly throw some light on these events. This study was designed to look for the evidence of apoptotic cell death of keratocytes, along with the morphological changes in corneal tissues in infective conditions. Methods Patients and samples Corneal tissues from patients diagnosed as infectious keratitis undergoing therapeutic keratoplasty between 1998 and 2002 were reviewed. The corneal sections of those cases, in which the normal stroma surrounding the zone of inflammation and necrosis was visible, were included in the study. We included 59 patients diagnosed for fungal (22), viral (15), bacterial (14) and Acanthamoeba (8) keratitis in the study. The diagnosis of fungal, bacterial and Acanthamoeba keratitis was based on the microbiological investigations as described below. The etiologic diagnosis was consistent with histopathology of the corneal buttons excised during therapeutic penetrating keratoplasty. Clinical information was available in 43 cases from the ulcer database of our institute. The cases with missing clinical data on a specific parameter were excluded while calculating the statistical significance of that parameter. The clinical data was analyzed for duration of symptoms, size of ulcer, location, depth of infiltrates, presence of perforation, thinning, anterior chamber infiltration and duration of treatment. Microbiology All patients had undergone microbiological investigations by processing of their corneal scrapings for bacteria, fungus, Acanthamoeba . Corneal scrapings were subjected to Grams, Giemsa and Calcofluor white stain for direct smear examination and culture on blood agar, chocolate agar, brain heart infusion broth, thioglycollate broth, Sabouraud's dextrose agar and non-nutrient agar with Escherichia coli overlay. Immunofluorescent assay for HSV-1 was done on cold acetone fixed corneal scraping smears using anti-rabbit HSV-1 polyclonal antibody and FITC conjugated swine antirabbit immunoglobulin (DAKO, Denmark). In addition, polymerase chain reaction using primers specific for HSV-1 glycoprotein D gene was done on DNA extracted from corneal scraping with commercially available DNA zol solution (Helena Bio Sciences, UK) Histopathology The excised corneal buttons of the 59 patients were subjected to routine histopathology processing. The paraffin embedded sections were stained with hematoxylin eosin, periodic acid Schiff's and special stains like Gomori's methenamine silver stain and Grams stains. The sections were studied for histologic changes viz. ulceration, inflammation, vascular channels, necrosis, keratocyte loss and other details. Inflammation was graded in a semi-quantitative way as mild, moderate or severe based on the density of the inflammatory cells and the visibility of the stromal collagen in the background. We looked for the absence or the reduced number of the keratocytic nuclei in the corneal stroma surrounding the zone of inflammation. TUNEL staining T erminal deoxyribonucleotidyl transferase-mediated d- U ridine 5" triphosphate N ick E nd L abeling (TUNEL) staining was performed on all the 59 sections, as per the manufacture's instructions using In-Situ Cell Death Detection Kit, POD (Roche Diagnostics, Manheim, Germany). In brief, the sections were de-paraffinized, re-hydrated and incubated with proteinase K (20 ug/ml) for 10 minutes at room temperature. After rinsing with phosphate buffered saline (PBS) the sections were blocked with 3% hydrogen peroxide in methanol for 10 minutes. The sections were then incubated in the permeabilization solution consisting of 0.1% triton × 100 in 0.1% sodium citrate for 20 minutes. After washing the sections were incubated with TUNEL reaction mix at 37°C in a humidified chamber for 2 hours followed by washing and incubation with POD converter for 2 hours, the sections were stained with diaminobenzidine and counter stained with hematoxylin and observed under the microscope. Sections from the normal corneal tissues obtained from the eyebank corneas were used as control slides. Statistical analysis was done using Chi-Square test and Fisher Exact test. Results We studied the excised corneal buttons of 59 patients who underwent therapeutic penetrating keratoplasty for fungal, bacterial, viral and Acanthamoeba keratitis. The mean age of the patients was 46 years (range 15–85 years) with male to female ratio of 1 (22): 1.7 (37). Based on microbiologic and histologic findings, the buttons were diagnosed as fungal keratitis in 22; bacterial in 14, viral in 15 and Acanthamoeba keratitis in 8 cases. Clinical profile The clinical features of 43 patients at the time of presentation are given in Table 1 . The size and location of the ulcer with depth of infiltrates was noted. Based on the microbiology report, these patients were treated with specific antimicrobial therapy. The cases with advanced disease at presentation or not responding to treatment were subjected to penetrating keratoplasty. The patients were treated for a median duration of 8 days (1–164 days) with specific anti-microbial therapy respectively. Histopathology The histologic changes were observed in the sections of the excised corneal tissues are described in Table 2 . The common observations were ulceration (52, 88.1%) (figure 1 ), destruction of Bowman's layer (58,98%), mild to moderate (28; 47.5%) to severe inflammation (31; 52.5%). Vascular channels were seen in 13.6% of the cases, while necrosis was noted in 48 (81.4%) cases. The morphologic evidence of disappearance or reduced number of keratocytic nuclei (figure 2 ) in the corneal stroma was noted in 49 (83%) cases. Necrosis was more in cases of fungal keratitis as compared to other infections (22 of 22), while it was minimal in viral infections (7 of 15). Similarly severe inflammation was more in fungal keratitis (16 of 22 cases). TUNEL assay TUNEL staining could be assessed in 53 cases, rest 6 cases, it could not be evaluated due to either a total loss of keratocytic nuclei in the stroma, or absence of inflammation-free zone of corneal stroma. The TUNEL positive brown cells in the stromal region were noted in the region of inflammation with necrosis as well as in the surrounding stroma (figure 3 ), away from the zone of inflammation. In the zone of inflammation, the TUNEL positivity was seen as diffuse staining of the background as well as within the neurtrophils seen in that region. In the surrounding stroma, the TUNEL positivity was seen within the keratocytic nuclei as linear or granular nuclear staining (figure 4 ). This was seen in 53 (90%) cases, located mostly in the deep stroma (29; 49%) and in few cases the peripheral stroma (20; 34%). It was seen in 19 of 22 cases of fungal, 14/14 cases of bacterial, 14 of 15 cases viral and 6 of 8 cases of Acanthamoeba etiology. In addition, TUNEL positive cells were seen in the epithelium in 10 cases, (figure 5 ) located in the basal layer, middle layers as well as the superficial cells. Normal nuclei were also noted. The control slides showed no positive staining within the corneal stroma. Occasional positive cells were noted in the superficial cells of the corneal epithelium. The keratocyte changes and the TUNEL staining was compared to the size of ulcer, thinning, degree of inflammation, necrosis and etiologic agent, as shown in Table 3 . The correlation between apoptotic cell death (TUNEL positive cells) and the various types of infections could not be evaluated in view of high prevalence of positive staining in all types of infection. The epithelial cell apoptosis was seen in 10 cases, of which 6 were of viral etiology. Discussion Inflammation of the avascular transparent cornea is unique and different from other tissues in the body. Breakdown of the physiological barrier of epithelium and the tear film leads to the entry of microorganisms, which proliferate in the stroma. This is followed by a sequence of events including edema, inflammatory cell influx from tears, limbus, abscess formation and necrosis of the stroma [ 2 ]. When healing initiates there is epithelial migration of epithelial cells into the crater of the ulcer, along with growth of blood vessels into the stroma, proliferation and migration of stromal fibroblasts and influx of macrophages for scavenger activity, finally resulting in the scar formation. The degree of opacification and visual dysfunction appears to be related to the number and density of inflammatory cells and their location, and to the presence or absence of associated enzymatically induced stromal damage, vascularization, and other secondary changes. The visual recovery would therefore depend on the elimination of the inflammatory cell infiltrates, regression of edema, scarring and the newly developed vessels. Though the observation that the stromal keratocytes disappear after epithelial injury was published more than 30 years ago, the nature of cell death was attributed to apoptosis by Campos et al and subsequently by Wilson SE et al [ 16 - 18 ]. Wilson et al also reported keratocyte apoptosis in association with viral infection of the overlying corneal epithelium by the herpes simplex virus [ 19 ]. They had hypothesized that one of the primary functions of keratocyte apoptotic response is to inhibit the virus that infects the corneal epithelium from spreading into the stroma and to deeper areas by temporarily eliminating the cells that are needed for viral replication and extension. This is in accordance to the Samurai principle in biology " it is better to die than do a wrong", where the damaged cells commit suicide or the programmed cell death by apoptosis [ 20 , 21 ]. Apoptotic cell death can be evaluated by many tests like DNA laddering as documented by gel electrophoresis, detection of enzymes like caspases, expression of mRNA of the corresponding proteins and enzymes involved in the process and by TUNEL staining [ 22 - 26 ]. The latter involves labeling the nick ends of the DNA fragments by labeled digitoxin molecules mediated by tdt transferase enzyme. The advantage of this technique is it identifies the apoptotic cell death in the formalin-fixed, paraffin embedded tissues and thus can be interpreted in the light of other morphological changes that take place in the stromal tissues in infections of cornea. Ours is the first study demonstrating apoptosis in corneal tissue obtained from patients diagnosed as fungal, bacterial, viral and Acanthamoeba infections. A few animal experiments document the phenomenon of apoptosis in inflammatory conditions. In an animal experiment using rabbits, Moreau et al demonstrated that intrastromal injection of purified alpha-toxin mediates cell death of epithelial cells both by necrosis and apoptosis [ 27 ]. Similar observations have been made in our study where the infected corneal tissues showed both necrosis and apoptotic cell death. The diffuse TUNEL positive staining in the region of inflammation and necrosis also supports the hypothesis that the inflammatory cells also undergo apoptotic cell death. This process could be self-induced or due to the inflammatory cytokines which are capable of inducing the apoptosis of the host cells and also triggering apoptosis in themselves. Similar kind of observation has been made by Ozaki et al in artificially induced Arthus reaction, wherein they demonstrated that the inflammatory cells and the new blood vessels formed in the cornea regress by the process of apoptosis thereby reducing the haze and leading to corneal transparency [ 28 ]. A few other reports show that PRK produces more haze than lasik, not only because of the lack of the epithelial covering above but because of the polymorphonuclear infiltrates [ 29 ]. Therefore it could be speculated that apoptotic cell death is more of a protective phenomenon and reduces the degree of scarring that could result from necrosis alone. How this would affect the healing process and the scarring following the control of infection in early stages of corneal infection in humans in another aspect which requires further studies. It is not the scope of this study to look for the mediators of apoptosis, but evidence in literature points to the role of Fas-Fas ligand, interleukin 1, enzymes and free radicals in this process [ 30 , 31 ]. Loss of keratocytic nuclei was detected in 73% of cases on light microscopy, while TUNEL stain detected apoptotic cell death in 83% of cases included in the study. In 6 cases, it could not be assessed due to either the total loss of keratocytes, or due to presence of inflammatory infiltrates in all regions of corneal stromal, without an inflammatory-free zone for proper interpretation. This makes us suspect that probably it is seen in all cases of infectious keratitis. The keratocyte loss was noted mostly in the deep posterior stroma. This is similar to our study on Acanthamoeba keratitis where the remaining keratocytes in the posterior stroma showed TUNEL positivity [ 12 ]. The keratocyte loss following epithelial scraping is noted in the superificial stroma, which is the first region to be exposed following the insult to the epithelium [ 32 ]. In tissues from corneal ulcer as used in this study, the injurious stimulus is present both in the epithelium and the superficial stroma, the deeper keratocytes are possibly are continuously exposed to the insult, thus initiating the suicidal cell death. Since this study includes only those cases of infectious keratitis which underwent therapeutic keratoplasty and whose buttons histologically showed evidence of preserved stroma to comment upon, it is difficult to extrapolate the sequence of events in different phases of the infective process. Planned animal experiments could possibly throw more light on these events. But with the knowledge that the apoptotic bodies are rapidly removed form the tissues by the neighboring cells and the scavenger cells, it is possible for us to presume that apoptotic cell death does occur in late phases of the infectious process. Though TUNEL assay is an accepted method of morphological detection of apoptosis in cells and tissues, assessment of apoptosis by an additional method would have been more informative and could have overcome the limitation of this study. Conclusion In summary, the corneal tissues involved by various types of infection including fungal, viral and bacterial infections show evidence of inflammation, necrosis as well as apoptotic cell death of keratocytes and inflammatory cells. We speculate that the keratocyte loss adjacent to the infected tissue could be a protective phenomenon, which requires evaluation by further studies. Competing interests The author(s) declare that they have no competing interests. Authors' contribution This work was conceived and planned by Geeta K Vemuganti. Clinical and microbiological support was provided by Prashant Garg and Savitri Sharma respectively. The bench work and analysis was done by Kishore Reddy and Ghazala Iftekhar. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545077.xml
193605	From Gene Trees to Organismal Phylogeny in Prokaryotes:The Case of the γ-Proteobacteria	The rapid increase in published genomic sequences for bacteria presents the first opportunity to reconstruct evolutionary events on the scale of entire genomes. However, extensive lateral gene transfer (LGT) may thwart this goal by preventing the establishment of organismal relationships based on individual gene phylogenies. The group for which cases of LGT are most frequently documented and for which the greatest density of complete genome sequences is available is the γ-Proteobacteria, an ecologically diverse and ancient group including free-living species as well as pathogens and intracellular symbionts of plants and animals. We propose an approach to multigene phylogeny using complete genomes and apply it to the case of the γ-Proteobacteria. We first applied stringent criteria to identify a set of likely gene orthologs and then tested the compatibilities of the resulting protein alignments with several phylogenetic hypotheses. Our results demonstrate phylogenetic concordance among virtually all (203 of 205) of the selected gene families, with each of the exceptions consistent with a single LGT event. The concatenated sequences of the concordant families yield a fully resolved phylogeny. This topology also received strong support in analyses aimed at excluding effects of heterogeneity in nucleotide base composition across lineages. Our analysis indicates that single-copy orthologous genes are resistant to horizontal transfer, even in ancient bacterial groups subject to high rates of LGT. This gene set can be identified and used to yield robust hypotheses for organismal phylogenies, thus establishing a foundation for reconstructing the evolutionary transitions, such as gene transfer, that underlie diversity in genome content and organization.	Introduction The availability of complete sequences of genomes for clusters of related organisms presents the first opportunity to reconstruct events of genomic evolution. By comparing related genomes and inferring ancestral ones, we can identify events, such as specific chromosomal rearrangements, gene acquisitions, duplications, and deletions, that have produced the observed diversity in genome content and organization. The Bacteria offer the most immediate opportunities for such reconstruction, because many clusters of related genomes are now available and because the genomes are small and contain relatively little repetitive sequence, reducing computational complexity. Among bacterial groups, the γ-Proteobacteria presents the most intensively studied and sequenced cluster of genomes with varying degrees of relatedness. Intertwined with the problem of reconstructing genomic change is the problem of inferring phylogeny. Evading this issue is particularly difficult in the Bacteria. First, using complete genomes to obtain a robust phylogeny for all bacteria has presented problems due to the age of the group and the resulting loss of phylogenetic signal. Furthermore, lateral gene transfer (LGT) occurs in bacteria and has been claimed to be rampant for all classes of genes, potentially resulting in a diversity of phylogenetic histories across genes and complicating, or completely defeating, attempts to reconstruct bacterial evolution at both deep and more recent evolutionary depths ( Doolittle 1999 ; Nesbø et al. 2001 ; Gogarten et al. 2002 ; Wolf et al. 2002 ; Zhaxybayeva and Gogarten 2002 ). Although the existence of substantial levels of LGT in bacterial genomes is not disputed, the existence of a core of genes resistant to LGT has been proposed ( Jain et al. 1999 ) and has received some support from recent studies using relatively intensive taxon sampling ( Brochier et al. 2002 ; Daubin et al. 2002 ). For purposes of reconstructing genomic change, what we seek is the organismal phylogeny—that is, the topology that traces the history of the replicating cell lineages that transmit genes and genomes to successive generations. The organismal phylogeny provides the backdrop against which events of genomic change, including LGT, have occurred. High incidence of LGT may cause the organismal phylogeny to be elusive, because we do not know which genes represent the true history of the cell lineages. The gene most used for reconstructing organismal phylogeny is the small subunit ribosomal RNA (SSU rRNA), which has been argued to rarely undergo transfer among genomes ( Woese 1987 ; Jain et al. 1999 ). But even this gene may undergo occasional LGT or recombination ( Ueda et al. 1999 ; Yap et al. 1999 ). Furthermore, by itself, it provides insufficient information to resolve phylogenies, particularly for cases of heterogeneous rates and patterns of substitution. Thus, building conclusions about organismal phylogeny on the basis of SSU rRNA alone is unsatisfactory. The availability of complete genome sequences presents us with the potential to exploit the much greater set of genes that are expected to share the same history of transmission along the branches of the organismal phylogeny. A robust phylogeny based on more sequences could then be used to reconstruct genome-scale events, including LGT and rearrangements. But, while complete genome sequences have enormous potential for addressing phylogenetic issues, their utility for reconstructing bacterial phylogeny is initially quite limited due to the requirement of thorough taxon sampling within a clade for accurate reconstruction of phylogenies ( Zwickl and Hillis 2002 ; Hillis et al. 2003 ). Only now, with the continuing increase in numbers of fully sequenced bacterial species, is it becoming possible to obtain sufficiently dense taxon sampling to exploit the large amount of genomic sequence data for the purpose of phylogeny reconstruction. We have chosen one group of Bacteria, the γ-Proteobacteria, to address the problem of whether complete genome sequences can be used for robust reconstruction of the organismal phylogeny, despite high levels of LGT. The γ-Proteobacteria, distinguished on the basis of sequence signatures and structural differences in the SSU rRNA ( Woese 1987 ), is an ideal choice for this purpose. This group represents a model of bacterial diversification and includes free-living and commensal species, intracellular symbionts, and plant and animal pathogens. The sequence divergence of certain of its members ( Clark et al. 1999 ) suggests an age of at least 500 million years. At the same time, members are sufficiently closely related to enable us to reduce the problem of lack of phylogenetic signal and to identify a large set of unambiguous orthologs. Currently, the γ-Proteobacteria contains the highest density of fully sequenced genomes, including those of species ( Escherichia coli and Salmonella sp.) for which knowledge of gene function is more complete than for any other cellular organisms. The potential obstacles to phylogenetic inference that are found across the Bacteria are certainly present in the γ-Proteobacteria. In particular, LGT is known to be extensive in this group, based on studies of genome composition ( Lawrence and Ochman 1997 ; Parkhill et al. 2000 , 2001 ; Stover et al. 2000 ). Symbiotic lineages present particular issues for phylogeny reconstruction owing to huge losses of genes ( Shigenobu et al. 2000 ; Akman et al. 2002 ), accelerated sequence evolution, and shifts in base composition ( Moran 1996 ). These features create phylogenetic artifacts and make the use of additional data from genome sequences particularly desirable. Here we aim to use complete genome sequences to reconstruct the organismal phylogeny for the γ-Proteobacteria by first selecting a set of probable ortholog families and then determining whether most agree on a common topology. A major implication of our results is that the replacement of single-copy orthologous genes is extremely rare, even within phyla. Instead, LGT most often involves uptake of genes assuming functions that are not represented in the recipient and arriving from distantly related bacteria or from phage ( Daubin et al. 2003a ; Pedulla et al. 2003 ). A consequence is that most single-copy orthologous genes show broad phylogenetic agreement that reflects the organismal relationships and that provides a foundation for reconstructing events of genome evolution. Results Gene Families and Identification of Orthologous Genes The proteins of 13 complete γ-proteobacterial genomes were classified into an initial set of 14,158 homolog families, using the procedures described in Materials and Methods . Figure 1 A shows the distribution of the number of genes per family. A majority (7,655) of the families contain only one gene. As the criteria we applied for grouping genes into families are stringent, this number is expected to exceed the number of real orphan genes; indeed, annotations for many of these genes do claim homology with other genes in the included genomes. As a result, values for most of the genomes ( Figure 1 B) are higher than the number of genes annotated as orphans; for example, the number of this type of gene identified for Buchnera was 24, but the annotation indicates only four genes unique to this species ( Shigenobu et al. 2000 ). Moreover, our comparison was made only within this group of 13 bacteria, and some single-gene families may have homologs in other, more distant bacterial species. Pseudomonas aeruginosa yielded the highest number of unique genes, which represent nearly 41% of proteins of this genome. This is congruent with the result obtained during the original annotation of this genome: the authors were unable to identify relatives for 32% of the ORFs ( Stover et al. 2000 ). Figure 1 Number of Genes and Species within the Gene Families (A) Distribution of the number of genes contained in the homolog families. (B) Number of orphan genes in each species in parentheses. Abbreviations: Ba, Buchnera aphidicola ; Ec, Escherichia coli ; Hi, Haemophilus influenzae ; Pa, Pseudomonas aeruginosa ; Pm, Pasteurella multocida ; St, Salmonella typhimurium ; Vc, Vibrio cholerae ; Wb, Wigglesworthia brevipalpis ; Xa, Xanthomonas axonopodis ; Xc, Xanthomonas campestris ; Xf, Xylella fastidiosa ; Yp CO92, Yersinia pestis CO_92; Yp KIM, Yersinia pestis KIM. (C) Distribution of the number of species contained in the homolog families. At the other extreme, some families group large numbers of genes. The largest family contains 544 genes and corresponds to the ABC transporter family, known to be the largest protein family ( Tatusov et al. 1996 ; Tomii and Kanehisa 1998 ). The second-largest family, with 404 genes, corresponds to the histidine kinase response regulators ( Wolanin et al. 2002 ). Figure 1 C shows the distribution of number of species per gene family. Note that a large majority of families group only one or two species (8,035 and 2,693 families, respectively). In the families comprising only one species, Pseudomonas and Vibrio are heavily represented, with 2,397 and 1,474 families, respectively. The families containing two species often group two closely related genomes, such as the two Xanthomonas , the two Yersinia , Escherichia and Salmonella , and Haemophilus and Pasteurella . A total of 275 families are represented in all 13 species. Among these, 205 contain exactly one gene per species. We consider these 205 genes to represent likely orthologs and, consequently, to be good candidates for use in inferring the organismal phylogeny and the extent of LGT. The Extent of Conflict among Gene Families We constructed trees based on several combinations of data and methods (see Materials and Methods ), with the aim of generating a set of candidate topologies for the organismal phylogeny. These seven analyses produced a total of six topologies (numbered 1–6 in Figure 2 ). (The identical topology was obtained for the consensus tree and the tree based on the protein concatenation using the neighbor-joining [NJ] method and the γ-based method for correcting the rate heterogeneity among sites.) The trees differ, in particular, with regard to the positions of Wigglesworthia , Buchnera , and Vibrio . All topologies, except number 4 (that one resulting from the Galtier and Gouy distance method with the SSU rRNA), tend to place Wigglesworthia and Buchnera as sister taxa ( Figure 2 ). The sister relationship of Wigglesworthia and Buchnera was of particular interest because it would suggest a shared origin of symbiosis in an ancestor of these two species. Thus, we tested seven additional topologies (numbered 7–13 on Figure 2 ) that did not place these two species as sister taxa, but that otherwise resembled topologies 1–6. Figure 2 The 13 Candidate Topologies Topologies 1–4 correspond to tree reconstructions based on SSU rRNA. Topologies 5 and 6 correspond to the trees based on the concatenation of the proteins. Topologies 7–13 correspond to additional topologies constructed to test the sister relationship of the two symbiont species. Species abbreviations as in Figure 1 . Abbreviations: ML, maximum likelihood; NJ, neighbor joining; K, Kimura distance; G&G, Galtier and Gouy distance; γ, gamma-based method for correcting the rate heterogeneity among sites. The position of the root corresponds to the one obtained repeatedly using SSU rRNA. For each alignment, we tested the likelihood of the 13 topologies against the maximum-likelihood (ML) topology, using the Shimodaira–Hasegawa (SH) test, as recommended by Goldman et al. (2000 ). The question asked here was whether the tested topologies could be considered equally good explanations of the data. Figure 3 shows the result of this test. One topology (number 5) is in agreement with 203 of 205 alignments ( Figure 3 ). Three other slightly different topologies can be considered nearly as good on the basis of agreement with a large majority of alignments (for topologies 2, 3, and 6 agreement was with 197, 196, and 186 alignments, respectively; Figure 3 ). Figure 3 Result of the SH Test The graph shows the number of alignments accepting or rejecting each topology. The “Other Topologies” are those built to test the sister relationship of Wigglesworthia and Buchnera . The “Proteins” topologies are those obtained using both the protein concatenation and the consensus of trees from all 205 alignments. The “SSU rRNA” topologies were obtained using the SSU rRNA sequences with different methods. Cases of Lateral Transfer Of the 205 alignments, two were found to strongly reject the most accepted tree (topology 5) as well as all other topologies tested. We have investigated these two genes to determine whether the incongruence is likely to result from LGT. The proteins correspond to biotin synthase (BioB) and to the virulence factor MviN. The trees obtained using ML for each alignment are shown in Figure 4 A. In both cases, the position of Pseudomonas conflicts with all widely supported topologies; it is placed as sister-group to Vibrio (BioB) or as sister-group to the enterics Escherichia , Salmonella , and Yersinia (MviN). Although initial examination of the topologies obtained from these genes suggests more than a single LGT (comparing trees of Figure 4 A to topology 5 of Figure 2 ), the hypothesis of a single transfer in an ancestor of Pseudomonas could not be rejected for either gene based on the results of the SH test after removal of Pseudomonas from the alignments and from topology 5 and other widely supported phylogenies. The implication is that a single transfer event in an ancestor of Pseudomonas is sufficient to explain the conflict of bioB and mviN with trees derived from other genes. In addition, we searched GenBank for homologous genes in other species of Pseudomonas and built trees using NJ and the Poisson correction ( Figure 4 B). In each case, Pseudomonas species are grouped and display the same position as in the trees, based only on the 13 sequenced genomes ( Figure 4 A). Moreover, the bootstrap support was high for the grouping of Pseudomonas with Vibrio in the BioB tree and for the grouping of Pseudomonas with the enteric bacteria in the MviN tree. Thus, the phylogeny of each of these two genes can be explained as the result of a single LGT event, from different donors within the γ-Proteobacteria to a shared ancestor of these Pseudomonas species. Figure 4 Phylogenies for the Laterally Transferred Genes (A) ML trees obtained for BioB (left) and MviN (right). (B) NJ trees obtained for BioB (left) and MviN (right). Abbreviations: Pf, Pseudomonas fluorescens ; Pp, Pseudomonas putida ; Ps, Pseudomonas syringae . Other species abbreviations as in Figure 1 . In Escherichia , Vibrio , Salmonella , Yersinia , and Pseudomonas , bioB is flanked by bioF , also involved in biotin biosynthesis. To determine whether bioF could have been transferred with the bioB gene, we built a tree based on the protein translation of bioF using all species except Buchnera , Haemophilus , and Pasteurella , which lack this gene. The tree obtained did not show any unexpected position of Pseudomonas , indicating that only bioB has been horizontally transmitted. A possible explanation, consistent with the flanking position of bioB and bioF in the Pseudomonas genome, is that the original bioB gene was replaced through homologous recombination in a common ancestor of the included Pseudomonas species. Similar comparisons for mviN did not illuminate its history in Pseudomonas , as the flanking genes differed from those in other species. The observations for mviN are consistent with a transfer event from an enteric species to a new genomic position in a Pseudomonas ancestor. Robustness of the Inferred Organismal Phylogeny The general lack of conflict observed among the 203 remaining families was not due to the absence of phylogenetic signal in the gene alignments because most genes did conflict with several other topologies (see Figure 3 ). We interpreted this congruence as a reflection of shared history and a lack of LGT. Therefore, we chose these genes as the basis for inferring the true organismal phylogeny for these 13 species. The resulting tree was the same as that for the concatenation of all of the 205 genes and for the consensus of the trees obtained from all protein families (topology 5 in Figure 2 and tree presented in Figure 5 ). It differed only slightly from other tested topologies (see Figure 2 ) that also are not rejected by many individual alignments (see Figure 3 ). Finally, an SH test performed using the complete concatenated alignment shows that this topology is significantly more likely than all alternative hypotheses. Figure 5 Tree Based on the Concatenation of the 205 Proteins (NJ) The topology shown agrees with almost all individual gene alignments (topology 5 of Figure 2 ). The same tree is obtained after removing the two genes showing evidence for LGT. The position of the root corresponds to the one obtained repeatedly using SSU rRNA. All topologies separating Wigglesworthia and Buchnera were rejected by the majority of the alignments. In the best-supported topology (see Figure 5 ), Wigglesworthia and Buchnera are grouped and comprise the sister-group to the enteric bacteria Yersinia , Salmonella , and Escherichia . Previously published phylogenies, based on SSU rRNA, gave conflicting results for the positions of these symbionts, sometimes placing Buchnera as sister-group to Escherichia and Salmonella ( van Ham et al. 1997 ; Spaulding and von Dohlen 1998 ; Moya et al. 2002 ). Because the genomes of these endosymbionts present a strong bias toward A+T relative to other genomes in the set, their grouping could reflect convergence at some nucleotide sites. This convergence could affect both the SSU rRNA, which is enriched in A+T ( Moran 1996 ), and also the protein sequences, which are enriched in amino acids with A+T-rich codon families ( Clark et al. 1999 ). To test this hypothesis, we removed from the alignment of the protein concatenation all sites at which Buchnera and Wigglesworthia contain amino acids encoded by A+T-rich codons (phenylalanine, tyrosine, methione, isoleucine, asparagine, and lysine) ( Singer and Hickey 2000 ). Using the resulting alignment (about 30,000 residues), we have reconstructed two trees, one with the NJ method and the polyacid-modified (PAM) matrix; the other with the NJ method and the γ-based method for correcting the rate heterogeneity among sites and bootstrap. The trees obtained (data not shown) were identical and gave strong support to the grouping of Buchnera and Wigglesworthia and to their position as the sister-group of enteric bacteria ( Escherichia , Salmonella , and Yersinia ). Thus, this grouping is probably not an artifact of the biased composition of the endosymbiont genomes. Discussion The most striking result is the almost complete lack of conflict among the set of genes selected as likely orthologs. Only two of 205 ortholog families showed such disagreement, both involving the P. aeruginosa genome. Because the γ-Proteobacteria has been the bacterial group most often cited as showing high rates of LGT, this finding is unexpected. However, we note that the evidence for LGT from sequence features and comparisons of genome content ( Lawrence and Ochman 1997 ; Ochman and Jones 2000 ; Parkhill et al. 2001 ; Perna et al. 2001 ; Daubin et al. 2003a ) primarily implicate genes that are absent from related bacteria; such genes would not have been retained in our set of putative orthologs. Furthermore, such genes are not candidates for phylogeny reconstruction since they are missing from most taxa. We also eliminated the large set of homolog families present as more than one sequence within even one of the genomes. If families containing paralogs show relatively high susceptibility to LGT, the proportion of genes undergoing LGT would be underestimated by considering only the set with one sequence per genome. Our aim was to locate a set of genes giving strong and consistent signal regarding the organismal phylogeny, and our results do not imply a lack of LGT in genes other than the widespread, single-copy orthologs that we selected. By streamlining the dataset for our primary goal, we have excluded genes that undergo more frequent transfer. Phylogenetic evidence for LGT mostly involves transfer between distantly related organisms ( Nelson et al. 1999 ; Brown et al. 2001 ; Brown 2003 ; Xie et al. 2003 ), and most clear-cut cases involve genes that are sporadically distributed (e.g., Parkhill et al. 2000 ; Singer and Hickey 2000 ) and thus excluded from our selection of families. The selected set includes genes that are distributed across a wide set of bacteria and includes about 100 universally distributed genes, such as those encoding ribosomal proteins, DNA polymerase subunits, and transfer RNA synthetases ( Table S1 ). Thus, if LGT is affecting most categories of genes, it should be detectable in our set, resulting in discordance of phylogenies whether it occurs between related genomes (within the γ-Proteobacteria) or between very dissimilar genomes. Such discordance was extremely rare, affecting only two (1%) of our families. Several previous studies have provided evidence that a core of genes may resist LGT and give a consistent phylogenetic signal ( Jain et al. 1999 ; Brochier et al. 2002 ; Daubin et al. 2002 ). However, the same studies have noted high incidence of genes showing incongruence, and, because they involved deeper trees and incorporated a much less dense sampling of genes or of taxa, this incongruence has not been firmly identified as due to LGT or to phylogenetic artifacts. Furthermore, recent analyses based on other sets of taxa have led to the proposal that all sets of genes, including orthologous genes, are subject to high rates of LGT ( Nesbø et al. 2001 ; Gogarten et al. 2002 ; Zhaxybayeva and Gogarten 2002 ), thereby casting doubt on the idea that we can identify a core set of orthologs that reflect the organismal phylogeny. Our analysis indicates that LGT is unusual for single-copy orthologous genes; that is, a gene copy from one species usually does not replace its ortholog in another species. The apparent discrepancy is not due to a relative lack of LGT in this particular group of bacteria, which is known to acquire foreign genes frequently ( Lawrence and Ochman 1997 ; Parkhill et al. 2000 ; Perna et al. 2001 ). More likely explanations are that (1) our criterion for orthology was more stringent in ruling out undetected paralogy; (2) the use of quartet phylogenies ( Zhaxybayeva and Gogarten 2002 ) can be misleading owing to artifacts linked to taxon sampling ( Zwickl and Hillis 2002 ); and (3) our focus on a relatively closely related group of bacteria minimizes the problem of loss of phylogenetic signal and reconstruction artifacts in deep divergences. This result thus provides further evidence that, though bacterial genomes constantly acquire and lose significant amounts of DNA, the incidence of LGT for widespread orthologous genes is relatively low ( Daubin et al. 2003b ). Although we have likely excluded many actual orthologs, the set of retained genes provides a dataset that is sufficiently informative to give a highly resolved and well-supported phylogeny for these taxa. This study thus defines a minimal core of genes that show both wide representation and congruent phylogenetic signal in γ-Proteobacteria. We note that this core includes numerous genes in both “informational” and “operational” functional categories ( Table S1 ); thus, our results do not fit closely with the “complexity hypothesis,” that only informational genes avoid LGT ( Jain et al. 1999 ), although they do not exclude such a trend. Our set of 203 genes should not be considered as representative of all genes resisting LGT, since we did not explore the other gene families. The main functional feature distinguishing the set is likely to be essentiality, owing to the requirement of presence in all 13 genomes, including the reduced symbiont genomes. For the goal of selecting genes that reflect organismal phylogeny through vertical descent, our criteria (single copy and ubiquitous) appear to be more reliable than criteria based on functional information (informational genes, translational genes, etc.). Indeed, cases of LGT are known for informational genes (e.g., Brochier et al. 2000 ). One possible explanation for the lack of observed events of orthologous replacement might be that these are sufficiently rare that significant frequencies are encountered only when considering deeper phylogenetic levels. However, the group studied here, though recent enough to allow accurate phylogenetic reconstruction, is old. Indeed, the divergence of different Buchnera species has been dated to approximately 200 million years based on the host fossil record ( Clark et al. 1999 ), and the clade we have studied must be much more ancient. A conservative molecular clock estimate, based on rRNA and dating the divergence of Escherichia and Buchnera at 200 million years, places the origin of the group at more than 500 million years (calculations not shown). Thus, our finding that very few orthologs have been exchanged within the group and that none show evidence of having been imported from other bacterial lineages is relevant for the understanding of long-term bacterial evolution. It has been proposed that LGT may be more frequent within clusters of related bacteria and even that phylogenetic groupings, such as the γ-Proteobacteria, may reflect boundaries to LGT rather than recent shared ancestry of lineages ( Gogarten et al. 2002 ). Such a model, which is consistent with apparent concordance among ortholog families in studies with poor taxon sampling but predicts rampant discordance within a well-sampled bacterial cluster, is strongly rejected by our results. Our findings favor the view that the cohesion of major phylogenetic groups within the Bacteria is due to vertical transmission and common ancestry rather than to preferential lateral transfer of genes. However, the results presented here do not eliminate the possibility of nonrandom patterns of LGT for gene families that are more sporadically distributed. A robust phylogenetic framework for the organismal lineages provides the foundation for reconstructing the events of genome evolution. An example of the kind of biological inference that can be built upon a well-supported phylogeny is provided by the two endosymbionts included in our set. Wigglesworthia and Buchnera have sometimes been considered as closely related and sometimes not, based on relatively weak phylogenetic evidence provided by the SSU rRNA alone. Our confirmation of their close relationship raises the question of whether their common ancestor was an endosymbiont with a reduced genome or a free-living bacterium (perhaps one with a host-associated lifestyle that promoted formation of intimate symbiosis). Because Buchnera and Wigglesworthia do not share any genes absent from the other species, no particular genes can be implicated as conferring a predisposition to symbiosis, a result that eliminates some hypotheses about how symbiosis originates. Furthermore, although emphasis has previously been placed on the close relationship of Buchnera with E. coli , our results shows that the phylogenetic relationship is equally high with other enterics, such as Yersinia pestis , which indeed shares as many ortholog families with Buchnera as does E. coli . This knowledge of relationships to other genomes allows more accurate reconstruction of ancestral genome content and of the chromosomal deletions and rearrangements occurring during the evolution of reduced symbiotic genomes ( Moran and Mira 2001 ). One biological interpretation of our findings is that the immediate retention of an acquired gene within a lineage depends upon strong positive selection for its function ( Ochman et al. 2000 ) and that such selection is unlikely if a homologous gene is already present in the recipient genome. An implication, from the perspective of phylogeny reconstruction, is that single-copy homologs with widespread distribution are a source of reliable information for inferring organismal phylogeny. The existence of many other gene families with multiple members per genome or with erratic distributions across the set of genomes (see Figure 1 ) is consistent with a major role of LGT, gene loss, and gene duplication in the evolution of this bacterial clade. Combined with chromosomal rearrangements, these events are the major sources of genomic, and ultimately ecological, diversification of bacterial groups. By demonstrating the potential to establish robust organismal phylogenies using genome sequence data, our results provide a foundation for examining the rates and frequencies of LGT and other large-scale events in evolving genomes. Materials and Methods Data. The genomes chosen for this study correspond to 13 γ-Proteobacterial taxa that show different degrees of relatedness based on divergence of SSU rRNA and that include two symbionts having undergone large-scale genomic reduction ( Shigenobu et al. 2000 ; Akman et al. 2002 ). The protein sequences of the 13 complete genomes were retrieved from the GenBank database ( Benson et al. 2002 ). The species used were Escherichia coli K12 (accession number NC_000913; Blattner et al. 1997 ), Buchnera aphidicola APS (NC_002528; Shigenobu et al. 2000 ), Haemophilus influenzae Rd (NC_000907; Fleischmann et al. 1995 ), Pasteurella multocida Pm70 (NC_002663; May et al. 2001 ), Salmonella typhimurium LT2 (NC_003197; McClelland et al. 2001 ), Yersinia pestis CO_92 (NC_003143; Parkhill et al. 2000 ), Yersinia pestis KIM5 P12 (NC_004088; Deng et al. 2002 ), Vibrio cholerae (NC_002505 for chromosome 1 and NC_002506 for chromosome 2; Heidelberg et al. 2000 ), Xanthomonas axonopodis pv. citri 306 (NC_003919; da Silva et al. 2002 ), Xanthomonas campestris (NC_003902; da Silva et al. 2002 ), Xylella fastidiosa 9a5c (NC_002488; Simpson et al. 2000 ), Pseudomonas aeruginosa PA01 (NC_002516; Stover et al. 2000 ), and Wigglesworthia glossinidia brevipalpis (NC_004344; Akman et al. 2002 ). To identify genes likely to have been transmitted vertically through the history of the γ-Proteobacteria, we first eliminated proteins annotated as elements of insertion sequences or as bacteriophage sequences, since they are likely to be subject to lateral transfer. Such sequences were present in most genomes but lacking in a few ( B. aphidicola , W. brevipalpis , and P. multocida ). Table 1 shows the number of proteins that remain in each genome after such elimination. Table 1 Number of Protein-Coding Genes per Genome after Elimination of the Insertion and Bacteriophage Sequences a Chromosome 1 b Chromosome 2 Construction of the gene families. We applied a stringent criterion for eliminating nonhomologous sequences and paralogous sequences, since both are likely to lead to false conclusions regarding the organismal phylogeny and frequency of LGT. In particular, the criterion of “best reciprocal hits” between sequences for a genome pair can lead to false conclusions of orthology because the resulting gene pairs are not always closest relatives phylogenetically ( Koski and Golding 2001 ). Instead, we used a cutoff for the degree of similarity as reflected in the BLASTP bit scores ( Altschul et al. 1997 ). The bit score is dependent upon the scoring system (substitution matrix and gap costs) employed and takes into account both the degree of similarity and the length of the alignment between the query and the match sequences. We used it to detect homologous genes, described as follows. A bank of all annotated protein sequences of all included species was created. A BLASTP ( Altschul et al. 1997 ) search was performed for all the proteins in each genome against the protein bank. This implies that all proteins were searched against both their resident genome and those from the 12 other species. The match of a given protein against itself gives a maximal bit score. To determine a threshold to group genes into a family, we examined the distribution of the ratio of the bit score to the maximal (self) bit score based on the proteins of E. coli compared against proteins of the 12 genomes ( Figure 6 ). In each case, the distribution showed a clear bimodal pattern with a first peak of low similarity values, which is constant among comparisons and therefore probably represents random matches, and a second peak of higher similarity values, representing true homologous genes. For comparisons of E. coli proteins with those of the most distant species in our set, such as Vibrio , Xanthomonas , Xylella , and Pseudomonas , the separation of the two portions of the distribution occurs at about 30% of the maximal bit score. Thus, in order to apply a stringent criterion for homology, we inferred as homologous genes those presenting a bit score value higher or equal to 30% of the maximal bit score. A protein was included in a family if this criterion was satisfied for at least one member. Our cutoff was chosen to minimize inclusion of nonhomologous sequences within a family; consequently, it may exclude some homologs, especially fast-evolving ones. Figure 6 Similarity Levels for Pairwise Comparisons of Genes from Two Representative Genome Pairs Frequency distribution of the ratio (bit score/maximal bit score) in a BLASTP query of the proteins from E. coli on the proteins from the genomes of Salmonella enterica (solid line) and Vibrio cholerae (dashed line). The ratio of 0.3 allows identification of most homologs but excludes probable nonspecific matches (NS). After establishing homolog families, we selected the set that contained a single sequence in each represented genome and regarded these as likely orthologs that could give information about the organismal phylogeny and the frequency of LGT affecting orthologs in this bacterial group. Phylogenies. The alignments for each identified gene family were created using the CLUSTALW 1.8 program ( Thompson et al. 1994 ). We corrected the concatenated proteins alignment by removing ambiguous parts using the SEAVIEW sequence editor ( Galtier et al. 1996 ). The TREE-PUZZLE 5.1 program ( Schmidt et al. 2002 ) was used in order to determine the α parameter from the datasets for the γ-based method for correcting the rate heterogeneity among sites. We wished to generate a set of reasonable candidate topologies that could be tested against the alignments for individual genes. These topologies were generated based on the consensus of the 205 trees from individual protein families (one method, yielding topology 5 of Figure 2 ), on the concatenation of all the proteins (over 75,000 amino acids) (two methods, yielding topologies 5 and 6), and on the SSU rRNA (four methods, yielding topologies 1–4). In the case of the reconstruction of the trees based on the SSU rRNA, we used the DNAML module of the PHYLIP package version 3.6 ( Felsenstein 2002 ), which performs ML reconstruction using the γ-based method for correcting the rate heterogeneity among sites; the PHYLO_WIN program ( Galtier et al. 1996 ) using the NJ method with bootstrap and using two different distances, Kimura 2P distance and Galtier and Gouy distance, designed to reduce bias due to base composition ( Galtier et al. 1996 ); and the MEGA program ( Kumar et al. 1993 ) using the NJ method with bootstrap and with the γ-based method for correcting the rate heterogeneity among sites. We used the PROML module of the PHYLIP package version 3.6 ( Felsenstein 2002 ) to conduct a ML reconstruction using the Jones, Taylor, and Thornton (JTT) model of substitution ( Jones et al. 1992 ) and the γ-based method for correcting the rate heterogeneity among sites, on each of the 205 families of single-copy, orthologous proteins. The consensus of the trees of the 205 protein alignments was obtained using the CONSENSE module of the PHYLIP package version 3.6 ( Felsenstein 2002 ). As there are no missing data, we also concatenated all the proteins and used the PHYLO_WIN program ( Galtier et al. 1996 ), using the NJ method and the PAM distance matrix, and the MEGA program ( Kumar et al. 1993 ), using the NJ method with bootstrap and with the γ-based method for correcting the rate heterogeneity among sites, on the protein concatenation. For each of the 205 alignments, a comparison of the likelihood of the best topology with the likelihood of the candidate topologies shown in Figure 2 were performed with the SH test ( Shimodaira and Hasegawa 1999 ) implemented in TREE-PUZZLE 5.1 ( Schmidt et al. 2002 ). This test determines whether these potential organismal phylogenies are significantly rejected by the alignment and thus whether an event of LGT must be invoked. Finally, we used the SH test to determine whether more than one LGT event was required to explain the lack of congruence between the favored topology and two gene alignments that rejected that topology. For each case, we observed which taxon showed the most evident discordance in the topology derived from the exceptional gene alignment. We then removed the corresponding sequence from the alignment and the corresponding taxon from the widely favored topologies. Using an SH test, we determined whether the alignment continued to show significant conflict with the favored topologies. Supporting Information Table S1 Names and Functional Categories of the 205 Genes Used to Reconstruct the Phylogenetical Relationship of γ-Proteobacteria (123 KB DOC). Click here for additional data file. Accession Numbers The GenBank accession numbers discussed in this paper are NC_000907, NC_000913, NC_002488, NC_002505, NC_002506, NC_002516, NC_002528, NC_002663, NC_003143, NC_003197, NC_003902, NC_003919, NC_004088, and NC_004344.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC193605.xml
546218	Increased expression of integrin-linked kinase is associated with shorter survival in non-small cell lung cancer	Background Integrin-linked kinase (ILK) promotes tumor growth and invasion. Increased ILK expression is correlated with progression of several tumor types, but the expression of ILK has not been investigated in patients with non-small cell lung cancers (NSCLCs). Methods We investigated ILK expression in patients with NSCLC by means of immunohistochemistry. Results ILK expression was significantly associated with tumor grade, T status, lymph node metastasis and stage. (p = 0.0169 for tumor grade; p = 0.0006 for T status; p = 0.0002 for lymph node metastasis; p < 0.0001 for stage). The 5-year survival rates for patients with strong and weak or no ILK expression levels were 20% and 59%, respectively: the difference was statistically significant (p < 0.0001). A multivariate analysis of survival revealed that ILK expression, T status, N status and vascular invasion were statistically significant prognostic factors (p = 0.0218 for ILK; p = 0.0046 for T status; p < 0.0001 for N status; p < 0.0001 for vascular invasion). Conclusions Our study demonstrates that increased expression of ILK is a poor prognostic factor in patients with NSCLC.	Background Interaction of cells with the extracellular matrix regulates many physiological and pathological processes. These interactions are mediated by a large family of cell surface receptors known as integrins, which recognize several extracellular matrix proteins, including fibronectin, collagens, and vitronectin [ 1 ]. Integrins act as the bridge between extracellular matrix components and the cytoskeleton and other proteins, regulating cell survival, proliferation, differentiation, and migration [ 1 ]. Integrins are important mediators of tumor invasion and metastasis through interaction with extracellular matrix. All integrins are heterodimers composed of one copy each of two subunits, α and β. Many studies examined the association between integrins and clinicalpathology or prognosis in lung cancer. Reduced integrin α3β1 expression was reported to be a factor of poor prognosis in pulmonary adenocarcinoma [ 2 ]. Increased expression of integrin β1 was reported to be a poor prognosis in small cell lung cancer [ 3 ]. Integrin α5β1 was reported to be associated with lymph node metastasis of non-small cell lung cancer (NSCLC) [ 4 ], or to be a prognostic factor in node-negative NSCLC [ 5 ]. Integrin-linked kinase (ILK) interacts with cytoplasmic domain of both β1 and β3 integrins and is activated by cell- extracellular matrix interactions [ 6 ]. Overexpression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression [ 7 ]. Furthermore, increased ILK expression is correlated with progression of several tumor types, including prostate [ 8 ], gastric [ 9 ], and colon carcinomas [ 10 ]. However, to our knowledge, the expression of ILK has not been investigated in patients with NSCLC. Thus, we investigated ILK expression in series of 134 cases of curatively resected NSCLC by means of immunohistochemical assays to evaluate its clinical significance. Methods Patients A total of 134 patients (88 men and 46 women; mean age, 65 years; age range, 28 to 80 years) with NSCLC were studied (consecutive cases). All patients in this study had undergone curative tumor resection in our department between 1995 and 1998. Patients who died within 30 days after surgery and those who underwent exploratory thoracotomy were excluded from the study. Patients with a past history of another type of cancer were also excluded. With regard to histological type, 75 were adenocarcinomas, 54 were squamous cell carcinomas and 5 were large cell lung carcinomas. The lesions of these 134 patients were staged on both operative and pathologic findings according to the UICC TNM classification [ 11 ] (1997). There were 36 patients (27%) with stage Ia, 37 (28%) with stage Ib, 3 (2%) with stage IIa, 18 (13%) with stage IIb, and 40 (30%) with stage IIIa. None of the study subjects received pre- or postoperative chemotherapy and median follow-up of the patients was 75.3 months (range 60–96 months) and their outcomes were known. Immunohistochemical staining Resected tissue specimens were fixed in formalin, embedded in paraffin, and cut into 3-μm serial sections. The sections were subjected to hematoxylin-eosin staining and immunohistochemical analysis with ILK. A mouse anti-ILK monoclonal antibody (Santa Cruz Biotechnology, CA, USA) was used, and immunohistochemical staining was based on the avidin-biotin-peroxidase complex method and was performed with a Vestastatin kit (Vector, Burlingame, CA). Negative control sections were treated using nonspecific IgG in the primary antibody. Determination of expression of ILK Immunoreactivity was graded as from - to +++, according to the number of cells stained. Grades was defined as follow as: -, no positive cells; ±, less than 5% of tumor cells showed immunoreactivity; +, 5–50% of tumor cells showed immunoreactivity; ++, 50–80% of tumor cells showed immunoreactivity; +++, over 80% of tumor cells showed immunoreactivity. Grades ++ and +++ were regarded as strong expression, and grades ± and + were regarded as weak expression. The results of immunohistochemical staining were evaluated independently by three observers with no prior knowledge of patients' clinical data. The evaluation was suitable for 96% of the samples. The other specimens (4%) were re-evaluated independently, then classified according to the classification given most frequently by the observers. In this study, we compared the group of tumors of strong ILK expression with the group of tumors of weak or no ILK expression. Statistical analysis The association between ILK expression and clinical data was statistically evaluated by using Mann-Whitney U test or the chi-square test. Survival curves were calculated by the Kaplan-Meier method and were compared using the log-rank test. The correlation of variables with survival was analyzed by multivariate analysis using a Cox proportional hazards model. All statistical analyses were performed using the StatView software package (Abaracus Concepts, Berkeley CA). A P value of <0.05 was considered statistically significant. Results Expression of ILK and clinicopathologic parameters In non-neoplastic lung tissue, ILK was not detected in epithelial cells, while ILK expression was found in stromal cells including fibroblasts and infiltrating lymphocytes (Figure 1 ). In the cancer cells of many patients, ILK expression was detected in both cytoplasm and nuclei (Figure 2 , and 3 ). Of the 134 patients, 6 (4%) were classified as -, 34 (25%) as ±, 53 (40%) as +, 25 (19%) as ++, and 16 (12%) as +++. Patients with ILK ++ and +++ were placed together in a strong ILK group and were compared with weak or no ILK group. Tumor cells expressed ILK protein in most NSCLC cases, and strong expression was detected 31% (41 of 134) of the cases. Stromal cells in cancer lesion were also positive to ILK, and rate of positive cells and intensity of the staining were not different from those of stromal cells in non-neoplastic lesion. As shown in Table 1 , there were no significant differences between ILK expression status and clinical factors of age, gender, histology, lymphatic invasion and vascular invasion. However, the expression of ILK protein was significantly associated with tumor grade, T status, lymph node metastasis and stage (p = 0.0169 for tumor grade; p = 0.0006 for T status; p = 0.0002 for lymph node metastasis; p < 0.0001 for stage). Relationship between ILK expression and overall survival The 5-year survival rates for the groups with strong, and weak or no for ILK expression in their tumors were 20% and 59% respectively: the difference was statistically significant (p < 0.0001) (Figure 4 ). The univariate survival analysis revealed that tumor grade, T status, N status, stage, lymphatic invasion, vascular invasion and ILK expression all were statistically significant prognostic factors (p = 0.0003 for tumor grade; p < 0.0001 for T status; p < 0.0001 for N status; p < 0.0001 for stage; p < 0.0001 for lymphatic invasion; p < 0.0001 for vascular invasion; p < 0.0001 for ILk expression) (Table 2 ). However, age, gender, and histology were not significant factors. The multivariate survival analysis revealed that T status, N status, vascular invasion and ILK expression were statistically significant prognostic factors (p = 0.0046 for T status; p < 0.0001 for N status; p < 0.0001 for vascular invasion; p = 0.0218 for ILK expression) (Table 3 ). Discussion Recent studies have indicated that ILK facilitated the phosphorylation of AKT, which is required for AKT activation [ 12 ]. Activation of AKT upregulates vascular endothelial growth factor expression, and AKT is known to induce angiogenesis and suppress apoptosis [ 12 ]. By regulating, the activity of AKT as well as glycogen synthase kinase 3, ILK facilitates the assembly and activity of the β-catenin/LEF-1 transcription complex, and suppresses expression of the invasion suppressor E-cadherin [ 13 , 14 ]. Overexpression of ILK in epithelial cells results in anchorage-independent cell growth with increased cell cycle progression, and constitutive up-regulation of cyclin D and cyclin A expression [ 7 ]. Inhibition of ILK elicits cell cycle arrest and induces apoptosis [ 15 ]. Overexpression of ILK in epithelial cells induces tumorigenicity in nude mice, indicating that ILK can act as a proto-oncogene [ 16 ]. ILK is associated with a highly invasive phenotype of certain tumors [ 7 ]. In the current study, we detected ILK expression using immunohistochemistry on tumors from patients with NSCLC. ILK was strongly expressed in 31% of tumor samples, whereas there was no ILK expression in noncancerous pulmonary tissue samples from the same patients, except for fibroblasts and infiltrating lymphocytes. These finding suggest that ILK expression may serve as a useful marker of NSCLC. ILK is very low in healthy cells. In cancer cells, however, ILK activity is often increased, possibly as a result of a malfunctioning of upstream components in the integrin and growth factor signaling pathways. We found a significant correlation between strong expression and advanced T status, N status and stage. No correlation was found age, gender or histology. These results suggest that ILK expression is correlated with tumor progression in NSCLC. Cases with strong ILK expression were reported to be significantly more frequent in advanced gastric carcinoma [ 9 ]and advanced melanoma [ 17 ]. ILK-mediated pathway that may enhance tumor progression is its regulation on MMP expression [ 18 ]. During tumor progression, MMPs facilitate the pathological processes of tumor invasion, angiogenesis, and metastasis by breaking down the extracellular matrix. The overexpression of ILK has been shown to be result of increased MMP-9 expression [ 18 ]. We also demonstrated that the expression of ILK correlated with tumor grade. Recent studies have also linked ILK expression to tumor grade of prostate [ 7 ], gastric [ 9 ] or ovarian carcinomas [ 19 ]. The overall prognosis of patients with strong ILK expression was significantly poorer than that of patients with weak or no expression. For stage I or stage II/IIIa patients, prognosis was poorer for those with strong ILK expression than for those with weak or no ILK expression, although the differences were not significant (data not shown). Strong expression of ILK protein was reported to be significantly associated with presence of nodal metastasis [ 9 , 17 ]. The univariate survival analysis revealed ILK expression was a significant prognostic factor as well as T status, N status, stage, lymphatic invasion and vascular invasion. The status of ILK expression might be dependent on the status of lymph node metastasis or other variables. So the multivariate analysis for survival was performed. In the current study, the multivariate analysis revealed that ILK expression was picked up for its independent level of prognostic significance. Our results also showed that the tumors with a strong expression of ILK were associated with an increased recurrence, which suggests that patients with strong ILK expression may be prone to metastasis, or may already have occult systemic diseases. ILK expression level plays one of the key roles in the biology of NSCLC and defines a more aggressive tumor phenotype of NSCLC. Preoperative adjuvant therapy in NSCLC is designed to improve survival and reduce local recurrence. Recent reports have shown that preoperative adjuvant therapy has led to an increase in overall survival of NSCLC patients [ 20 , 21 ]. It is possible that this preoperative adjuvant treatment modality may be important for patients whose tumors have strong ILK expression. In the future, small molecule antagonists of ILK may be used to interfere with recurrence in tumor patients. Conclusion Our study demonstrates that ILK expression is a poor prognostic factor in patients with NSCLC. Thus, the utility of the expression of ILK could open up a new window for the molecular marker and the treatment of NSCLC. Competing interests The author(s) declare that they have no competing interests. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546218.xml
529422	Using Modified Antigenic Sequences to Develop Cancer Vaccines: Are We Losing the Focus?	Are short synthetic peptides the key to developing cancer vaccines? And what are the obstacles in the way?	Tumor Antigen Recognition by Cytolytic T Lymphocytes CD8 + cytolytic T lymphocytes (CTLs) are the primary effector cells of the adaptive immune system and have a major role in protecting us from a vast array of diseases including cancer. CTLs specifically recognize and lyse targets through the interaction of T cell receptors (TCRs) on the surface of the T lymphocyte with protein fragments (peptides) presented on the surface of target cells, in association with major histocompatibility complex (MHC) class I molecules. When a particular CTL interacts with a target cell, it rapidly divides to form a clonal population of T cells with the identical TCR. Townsend and colleagues first elucidated the molecular basis of target cell recognition by CTLs in 1985 [ 1 ]. They showed that antigens are processed inside the target cell into nine- or ten-amino-acid-long peptides, which are then presented at the surface in association with MHC class I molecules. This discovery suggested the possibility of using short synthetic peptides mimicking naturally processed antigens as immunotherapeutic drugs and vaccines. Short synthetic peptides are ideal for drug development because of the relatively low cost of production, easy storage, and high safety. However, not all peptides and MHC alleles work well together to stimulate CTLs. So for clinical use, either patients would have to be selected for treatment based on their MHC I type or it would be necessary to make multiple peptides to cover the majority of MHC class I alleles in a given population. Furthermore, before Boon and colleagues cloned the first antigen recognized by tumor-reactive CTLs in 1991 [ 2 ], it was not clear which antigens were recognized by tumor-reactive CTLs in humans; so, it was not possible to rationally design cancer vaccines. Now, however, a long list of more or less tumor-specific antigens has been generated [ 3 ]. Most of the peptides identified so far are either normal self proteins aberrantly expressed in cancer but not in most other adult normal tissues or tissue-specific antigens also expressed in certain types of cancer. Some patients show a spontaneous CD8 + T cell response (occasionally at high levels) that is specific for several of these antigens. The development of such responses, however, requires a large tumor load, occurs late in the disease, and probably does not cause the efficient destruction of the tumor cells [ 4 ]. Thus, a central objective in cancer immunotherapy is to efficiently produce tumor-reactive CTLs at an earlier phase of the disease. Heteroclitic Tumor Antigen Peptides Unfortunately, some synthetic peptides, including some corresponding to immunodominant epitopes (those which cause the biggest part of the immune response) from tumor antigens, only seem to bind MHC class I molecules with medium to low affinity and/or are recognized by specific T cells with relatively low avidity. These characteristics are the likely cause of the poor immune reaction generated by these peptides [ 5 ]. One strategy to improve the immune reaction is to make what are called heteroclitic antigen variants. By improving either peptide binding to MHC, recognition by TCRs, or both, these variants have increased peptide antigenicity and immunogenicity. Solinger and colleagues were the first to describe antigen variants producing T cell responses that were stronger than those elicited by the parental sequences [ 6 ]. Some heteroclitic tumor antigen peptides that showed highly improved antigenicity and immunogenicity in preclinical studies, and which also cross-reacted well with CTLs generated against the parental sequence, were tested in clinical trials. The peptides selected for trials mostly contained substitutions of anchoring amino acids that were designed to increase peptide binding to the MHC molecule while minimally changing the shape of the epitope [ 7 , 8 ]. In a study by Lee and colleagues in this issue of PLoS Medicine [ 9 ], despite the careful study design, vaccination with these peptides resulted in the recruitment of T cells that bound antigens less efficiently and had lower tumor reactivity than those from the endogenous response to the tumor. The authors propose that the cause for the decreased affinity of vaccine-elicited CTLs could be the high antigen density of these synthetic peptides on antigen-presenting cells. An alternative explanation, however, is that the synthetic peptides used for vaccination simply fail to faithfully mimic the naturally processed antigens ( Figure 1 ). The use of peptides that differ from those resulting from natural intracellular processing has previously given rise to similar problems [ 10 , 11 ]. In any case, the enormous diversity in the normal TCR repertoire provides a molecular explanation of the observed phenomenon. These results emphasize how difficult it is to translate findings, such as the spectacular results obtained by the vaccination of TCR transgenic mice with heteroclitic peptides [ 12 ], into an application for normal animals and humans. Figure 1 Synthetic Peptides Used for Vaccination May Fail to Faithfully Mimic the Naturally Processed Antigens The TCR repertoire specific for the natural tumor antigen (N) contains a group of CD8 + T cells that recognize N with high functional avidity and display high tumor reactivity (TCR-A, focused). TCR-A is stimulated by the natural ligand and expands during spontaneous responses to the tumor in some patients with antigen-expressing tumors. A larger group of CD8 + T cells able to recognize N also exists in the naïve T cell repertoire (TCR-B). TCR-B recognizes N with decreased avidity and shows low to undetectable tumor reactivity (unfocused). Heteroclitic peptides (H) are analogs of N that contain modifications that increase their immunogenicity. They are selected on the basis of their increased recognition by T cells from the TCR-A group. When used as immunogens, they elicit a group of CD8 + T cells specific for H (TCR-C). The reactivity of TCR-C will be variable and will depend on the extent of overlapping between the TCR-A, -B, and -C groups. In Lee and colleagues' study [ 9 ], most of the elicited CD8 + T cells belong to the TCR-B group (unfocused), suggesting a large overlap between TCR-C and TCR-B and a more limited overlap of TCR-C with TCR-A. This phenomenon can be explained by the structural difference between N and H. Other factors that may differ between natural and peptide-induced immune responses, including the density of the peptide on antigen-presenting cells and the mode of presentation (i.e., the nature of antigen-presenting cells), could also contribute to the outcome. Conclusion It is increasingly clear that even the smallest alteration in the structure of the MHC peptide complex can result in significant changes in which TCRs are selected after vaccination. Thus, manipulating the immune T cell repertoire in vivo through the use of heteroclitic tumor antigen peptide variants could be harder than anticipated. As the field moves rapidly towards the use of new vaccine adjuvants with high immunogenic potential [ 13 ], reassessment of the immunogenicity of natural sequences could be worthwhile in some cases. In addition, the careful analysis of antigen-specific T cell clones, such as that reported here by Lee and colleagues, will be crucial to ascertain the quality of the elicited immune response.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529422.xml
509241	A molecular 'signature' of primary breast cancer cultures; patterns resembling tumor tissue	Background To identify the spectrum of malignant attributes maintained outside the host environment, we have compared global gene expression in primary breast tumors and matched short-term epithelial cultures. Results In contrast to immortal cell lines, a characteristic 'limited proliferation' phenotype was observed, which included over expressed genes associated with the TGFβ signal transduction pathway, such as SPARC , LOXL1 , RUNX1 , and DAPK1 . Underlying this profile was the conspicuous absence of hTERT expression and telomerase activity, a significant increase in TβRII , its cognate ligand, and the CDK inhibitor, p21 CIP1/WAF1 . Concurrently, tumor tissue and primary cultures displayed low transcript levels of proliferation-related genes, such as, TOP2A , ANKT , RAD51 , UBE2C , CENPA , RRM2 , and PLK . Conclusions Our data demonstrate that commonly used immortal cell lines do not reflect some aspects of tumor biology as closely as primary tumor cell cultures. The gene expression profile of malignant tissue, which is uniquely retained by cells cultured on solid substrates, could facilitate the development and testing of novel molecular targets for breast cancer.	Background In breast cancer, cell based experiments are largely conducted with a few spontaneously arising cell lines from late stages of disease that demonstrate unlimited proliferation (immortalization). However, the breadth of tumor heterogeneity and early stages of breast tumorigenesis remain under represented in such assays. We, and others have adapted tissue processing and culture conditions to enable the selective isolation and expansion of tumor cell populations from primary breast cancer [ 1 - 9 ]. Closely reflecting the biological heterogeneity and relatively slower growth rate of malignant cells in primary tumor tissue [ 10 ], in vitro tumor cell populations are highly variable at the microscopic level and generally require long intervals between passages. The vast majority of such cultures (>90%) display a finite mitotic lifespan. While molecular changes underlying growth cessation in non-malignant breast epithelial cultures are well studied [reviewed in [ 11 ]], barriers to the continued proliferation of tumor-derived cultures remain undetermined. In contradistinction to previous reports on the characteristics of rare immortal cell lines developed from primary tumors [ 4 , 5 , 7 , 8 , 12 ], we have focused on early passage tumor cultures (that may not necessarily culminate as immortal cell lines). This approach encompasses a broader range of disease since <10% of primary breast tumors spontaneously develop into immortal cell lines. Although limited direct comparison of primary breast tumor tissue and corresponding short-term cultures has assisted in authenticating the malignant origin of tumor-derived cultures [ 1 - 3 , 9 ], relatively little is known regarding the degree of phenotypic and functional concordance between malignant epithelial populations in human breast tissue and their counterparts in vitro . In depth comparative analysis of such isogenic malignant cell populations could provide important insights regarding cellular pathways that function independently of environmental constraints, as well as the genome wide consequences of constitutive growth arrest of breast tumor cells widely encountered in laboratory dishes. Here we describe a comprehensive comparison of matched samples of primary breast carcinoma tissue, and early passage tumor cultures. We have used cDNA microarray-based global gene expression profiling to determine common molecular phenotypes of tumor tissue and tumor-derived epithelial cultures, which distinguish them from cancerous and non-cancerous immortal cell lines. Results In order to ensure the propagation of tumor epithelium without apparent contamination with fibroblasts and other cellular components, previously described methods including the selective release of nests of malignant cells from the connective tissue, were used [ 2 , 3 ]. As illustrated in Figure 1A , the characteristic epithelial morphology, and microscopic heterogeneity between tumor cultures from independent cases was observed. To confirm that these cultures represented pure populations of epithelial cells, indirect cytokeratin immunostaining was performed (Figure 1A ). Clinical information and additional cell culture details are listed in Table 1 . Figure 1 A. Microscopic phenotype of primary tumor tissue and corresponding tumor-derived epithelial and stromal cell cultures. ( 1–3 ) – H & E-stained frozen sections showing histology of representative cases processed for RNA isolation and cell culture. Case number 061T – 1; 066T – 2; 068T – 3. Note abundant tumor cells in the samples. ( 4–6 ) – Early passage epithelial cultures; brightfield, 100× magnification. Note morphological variation between cultures in the context of an epithelial phenotype. ( 7–9 ) – abundant cytokeratin expression in the cytoplasm of cultured primary tumor cells analyzed by indirect immunofluorescence. Magnification – 400×. ( 10–12 ) – tumor-derived fibroblast cultures; brightfield, 100× magnification. Note morphological distinction between epithelial and stromal cells isolated from the same tissue sample. B – E. cDNA-based gene expression profiles of tumor tissue and tumor derived epithelial cultures. Rows represent genes, and columns represent samples. B, C – unsupervised two-dimensional hierarchical clustering of 17 samples. B – cluster shows genes under expressed in primary tumor tissue and tumor cultures compared to immortal cell lines. C – cluster shows genes over expressed in primary tumor cultures and tumor tissue compared to immortal cell lines. D, E – Gene expression patterns, which distinguish between group 1, consisting of immortal cell lines, and group 2, consisting of primary tumor tissue and tumor cultures. D – Results of SAM analysis showing a thumbnail of 681 differentially expressed genes. E – SAM-identified genes which are in common with the cluster in panel B are shown by red vertical bar, and with the cluster in panel C are shown by green vertical bars. Color scale indicates expression level. Table 1 Clinical characteristics of Primary Tumors used for Gene Expression Analysis Primary Tumor Cultures Sample # Sample ID Age TNM Stage Tumor Grade Tumor histology ER status Cell culture passage at RNA isolation 1 022T NA NA 1 IDC NA 3 2 044T 50 NA 3 IDC Neg 2 3 047T 79 II 2 IDC Pos 5 4 054T 59 IV 3 IDC Pos 8, 12 5 061T* 62 III 2 ILC Pos 5, 6 6 066T* 47 II 2 IDC NA 6, 7 7 068T* 52 II 1 IDC Pos 5, 6 8 071T NA II 3 IDC NA 3 9 076T 51 III 2 IDC Pos 2 10 257T 41 IIB 3 IDC Neg 8 11 672T 60 II 3 IDC Neg 5 12 701T 59 I 1 IDC Pos 7 13 713T 77 II 3 IDC+ILC Pos 5, 7 14 1555T 38 NA 2 ILC NA 3, 4 15 1569T NA NA NA IDC NA 3, 4 16 1570T 66 IIA 2 IDC+ILC Pos 2 17 1599T* 64 I 2 IDC NA 3 18 1607T 58 I 1 IDC NA 5 19 1617T 64 III 2 IDC Pos 2 20 1620T 70 III 3 IDC Pos 2 21 1625T 80 IIB 3 IDC Pos 2 * Matched tissue samples analyzed by cDNA microarrays NA – not available; IDC – invasive ductal carcinoma; ILC – invasive lobular carcinoma ER – estrogen receptor; Neg – negative; Pos – positive Concordant aspects of global gene expression in breast cancer tissue and tumor-derived cultures Seventeen RNA samples were analyzed by cDNA microarrays. These were comprised of 4 cases of primary breast tumor tissue and 1–2 matched tumor cultures, 2 additional unmatched tumor cultures, and 4 immortal breast cell lines. Cell lines were selected to represent estrogen receptor (ER) positive (T47D) and ER negative (SKBR3, BT20) tumors, and non-cancerous breast epithelium (ENUt7, ref. [ 13 ]). Unsupervised clustering analysis of 7362 clones (4743 unique genes/ESTs) grouped the samples into three separate clusters representing immortal cell lines, tumor tissue, and tumor cultures. Figure 1B displays a cluster of genes, which were over expressed in immortal cell lines but under expressed in tumor tissue and tumor cultures, while Figure 1C displays a 'mirror image' gene expression pattern (under expressed genes in immortal cell lines, which were over expressed in tumor tissue and tumor cultures). The clusters selected for illustration represent ~90% gene correlation. The entire data set displayed additional clusters [Figure S1 – see http://genome-www.stanford.edu/breast_cancer/PTCC/]. Comparisons of large data sets as described above frequently result in "significant" patterns of gene expression by chance alone. We employed the Significance Analysis of Microarrays (SAM) for independently verifying genes that are differentially expressed between classes or groups of samples. SAM analysis identified 930 clones, representing 681 unique genes/ESTs, whose expression was significantly (>2-fold) different between group 1 comprised of immortal cell lines, and group 2 comprised of tumor tissue and tumor cultures (0.05% false discovery rate). A full list of the differentially expressed genes is provided in [Table S2 – see http://genome-www.stanford.edu/breast_cancer/PTCC/]. As expected on the basis of the similarity in gene expression observed between tumor tissue and tumor cultures in the unsupervised array data (Figure 1B,1C ), these clusters were also present in the SAM profile (Figure 1D ). As shown in Figure 1E , genes upregulated in immortal cell lines (indicated by red vertical bar) reflecting significantly shorter doubling times (for example, RFC4 , CENPA , TOP2A , CCNA , MCM7 , PCNA , CDC2 ) were primarily those associated with the 'proliferation' cluster described by Ross et al [ 14 ]. In contrast, upregulated transcripts in tumor tissue and tumor cultures (Figure 1E , indicated by green vertical bar) included genes involved in epithelial differentiation ( MAL, MAFB, RUNX1, KRT5 ), in the induction of apoptosis ( DAPK1 ), and in tumor angiogenesis, and extravasation ( SPARC ) (Gene Ontology – GO annotations, ref [ 15 ]). Primary epithelial cell cultures, in contrast to fibroblasts and rapidly growing cell lines, undergo rapid growth arrest in 10% fetal calf serum (FCS). This is why we, and others have propagated cultures of primary tumor epithelium in 0–5% FCS (1–9). Immortal cell lines, however, grow optimally in 10% FCS; lower concentrations retard growth. Therefore, to optimize growth conditions for both, 2% FCS was chosen for primary tumor cultures and 10% FCS for cell lines. It is conceivable that an increased concentration of FCS may account for differences in gene expression between primary tumor cultures and immortalized cell lines. In this case, one would expect increased proliferation in the primary tumor cultures at 10% FCS, when in fact, growth is severely inhibited by this approach. As summarized in Table 2 , based on microarray expression data of 38,999 cDNA clones, the average correlation between matched tumor tissue and short-term tumor cultures (7 sets) was 0.41 (sd = 0.03) in contrast to 0.10 (sd = 0.09) between tumor tissue and immortal cell lines (16 pairs). These correlation coefficients differed significantly in a Mann-Whitney rank sum test (p = 0.007) demonstrating that the gene expression profile of tumor tissue was more consistent with that of the corresponding tumor culture than it was with any of the immortal cell lines tested. Table 2 Pair wise correlations between primary tumors, matched epithelial cultures, and immortal breast epithelial cell lines Matched Primary Tumor Pair Immortal Breast Epithelial Cell Lines Tissue Cell Culture ENUt7 SKBR3 BT20 T47D R correlation 1599T 1599TC 0.31 0.05 0.07 0.01 0.23 061T 061TC1 0.44 0.10 0.03 0.03 0.24 061TC2 0.43 066T 066TC1 0.44 0.16 0.02 0.03 0.20 066TC2 0.39 068T 068TC1 0.50 0.12 0.05 0.00 0.27 068TC2 0.54 Determinants of replicative arrest in primary breast tumor-derived cultures Towards the determination of specific molecular changes underlying the finite proliferative lifespan in tumor cultures, gene expression analysis, by QRT-PCR, was conducted on an expanded set of 39 samples comprised of multiple early passage epithelial cultures obtained from 16 primary breast cancers, 8 cases of normal breast epithelial organoids and, 12 immortal breast epithelial cell lines. As noted above, since genes in the proliferation cluster displayed minimal expression in primary tumor cultures, first we considered the possibility of growth arrest due to a lack of telomerase activity and subsequent telomeric attrition. The relative expression of hTERC and hTERT subunits of telomerase, encoding the structural RNA component, and the component with reverse transcriptase activity, respectively, was measured. While commonly used immortal cell lines (T47D, MDA231) displayed several-fold higher transcript levels for hTERT , undetectable to minimal levels were observed in 6/8 independent primary tumor cultures (1599T, 713T, 1569T, 1570T, 1617T, 1620T). The primary tumor culture with the highest relative expression of hTERT (257T) has developed into an immortal cell line (Figure 2A ). Figure 2 A. QRT-PCR analysis of hTERT and hTERC in primary tumor cultures compared to levels in normal breast organoids, matched fibroblasts, and immortal breast epithelial cell lines ( T47D , MDA231 , and ENUt7 ). The Y-axis is minimized to display the range of relative gene expression. The highest hTERT expression level, denoted by the asterisk, was 22-fold (sample 257T). B . TRAP assay measurement of telomerase activity shown with, and without heat inactivation of cell lysates. Extracts equivalent to 1000 cells are displayed. IC – internal PCR control. For sample 054T, 2 independent fractions of the tumor are shown; SP – mechanically dissociated spillage, DIG – enzymatically digested tissue. To confirm the functional impact of hTERC and hTERT down regulation, telomerase activity was measured directly by the TRAP assay. As expected, primary cultures with detectable transcript levels, and immortal cell lines of cancerous and non-cancerous origin displayed significant telomerase activity, while those tumor cultures that did not display gene expression showed no activity. In the primary tumor sample, 257T, robust telomerase activity was found as early as passage 8. Similarly, telomerase activity was detectable in early passage epithelial cultures propagated from cells isolated by mechanical dissociation (SP – spillage), or enzymatic digestion (DIG) of the tumor sample 054T (Figure 2B ). In the next step towards identifying the determinants of replicative arrest, primary tumor cultures were compared with immortal cell lines for relative expression of genes associated with the negative regulation of the cell cycle in general, and with epithelial cell proliferation in particular. This analysis included 9 candidate genes in 3 signaling pathways: (1) members of the CIP/KIP family of cyclin-dependent kinase inhibitors (CDKIs), p21 CIP1/WAF1 , p27 KIP1 , and p57 KIP2 (2) members of the INK family of CDKIs, p15 INK4B , and p16 INK4A (3) members of the TGF-β family, TGFβI , TGFβII and the signaling receptors, TβRI , and TβRII . As illustrated in Figure 3A , we observed that in 10/12 breast cancer cell lines, p21 CIP1/WAF1 levels were 2 to138 fold lower than steady state levels in normal breast epithelium. In contrast, 4/20 primary tumor culture samples showed this range of p21 CIP1/WAF1 expression (p = 0.0003). For the CDKIs, p27 KIP1 , and p57 KIP2 , several fold decrease in gene expression was observed in all primary tumor cultures and most immortal cell lines as well (p = 0.04 and 0.69, respectively). Similarly, no significant differences were apparent for p15 INK4B , and p16 INK4A gene expression in the two groups (p = 0.07 and 0.39, respectively). For members of the TGF-β family, while significant differences were not observed in the expression of TGFβI and TβRI , a median increase of 2 fold and 4 fold were found in the expression of TGFβII and TβRII respectvely. In contrast, immortal cell lines, showed a median decrease of 7-fold for TGFβII and 4-fold for TβRII (p = 0.0035 and 0.0011, respectively). All p values were derived by the Mann-Whitney test. Figure 3 Comparative QRT-PCR analysis of genes encoding negative regulators of cell proliferation in primary tumor cultures and immortal cell lines. A – Expression levels of individual genes represented as fold increase or decrease over gene expression in normal breast organoids (average of 8 independent reduction mammoplasty cases). Data shown are averages of triplicate measurements. Dots represent immortal cell lines (red) and primary tumor cultures (black) shown in panels B and C . B – multivariate analysis of data in A displayed as a hierarchical clustering dendrogram. The scale shows the distance between groups comprised of normal breast, primary tumor cultures, and immortal cell lines. For samples 1569T, 1555T, 713T, and 054T, gene expression was analyzed in epithelial cells at 2 different passages in culture (passage number indicated as 3', 4', 5' etc.) C – a plot of 3 principal components (U1, U2, and U3) of 9-dimensional space, representing the 9 genes evaluated in samples in panels A and B . Based on its level of gene expression, a place is assigned to each cell sample in this space. Data points representing epithelial organoids from the normal breast (pink) appear to be tightly clustered, whereas those representing primary breast tumor cultures (green) and immortal cancer cell lines (blue) show considerable scatter. We used MANOVA to compare expression of the 9 above-mentioned genes in immortal cell lines, primary tumor cultures, and normal breast epithelium. It was apparent that the multivariate means for the 9 genes differed for each of the 3 groups above and that both immortal lines and primary tumor cultures differed from normal breast epithelium (p = 0.0001) as also depicted in the hierarchical clustering dendrogram of these samples (Figure 3B ). A 3-D plot of the first three principal components, which together account for 83% of the total variation in expression in the 9-dimensional gene space, is shown in Figure 3C . This display format demonstrates that the three types of cell samples cluster in different parts of the three dimensional space and that primary tumor cultures and immortal cell lines are significantly different from normal breast and from each other in the expression of the negative growth regulators evaluated here (p = 0.0002). The relatively large Wilks' lamda (0.271) for immortal cell lines vs. primary tumor cultures is most likely due to the greater spread of these groups in the 9-dimensional gene space, while normal samples are tightly grouped together. This finding may be related to the heterogeneity between individual tumors. Overall, the genes in the array-based clusters, shown in Figure 1B and 1C , could be categorized as positive or negative regulators of proliferation respectively according to GO annotations. Genes such as, PCNA , CKS1B , TPX2 , UBE2C , CDC6 , confirmed by the statistically significant analysis of microarray data, SAM, were among the positive proliferation genes differentially expressed by immortal cell lines, and matched tumor tissue/cell culture samples (Figure 1E , also see Table S2 – http://genome-www.stanford.edu/breast_cancer/PTCC/, for full list). Expression levels of negative proliferation genes identified in the SAM data, such as, TβRII , and CDKN1A ( p21 CIP1/WAF1 ) were confirmed by QRT-PCR (Figure 3A ) Discussion The cDNA microarray analysis of primary breast tumors and their epithelial counterparts propagated in cell culture has revealed close similarities in the expression of several hundred genes. Notably, increased expression of genes deemed to be 'growth limiting' by virtue of their consistent down regulation in immortal cell lines, was observed. The patterns of gene up regulation or down regulation were remarkably consistent in independent immortal lines (including those derived from non cancerous breast epithelial cells) as was the contrasting gene 'signature' in cultures derived from independent cases of breast carcinoma. Together, these observations have led us to conclude that the continuous selection of rapidly proliferating cells culminating in the immortalized phenotype favors the loss of several aspects of gene expression retained by early passage primary tumor cultures. Our data suggests that the limited growth potential of primary tumor cultures results from two major proliferative barriers, (a) telomerase inactivation, potentially leading to telomere attrition, and (b) negative growth signaling by upregulated TGFβ resulting in a significant increase in transcription of the CDKI, p21 CIP1/WAF1 , consistent with the findings of induction or stabilization of this gene in a variety of immortalized cells exposed to exogenous TGFβ [ 16 , 17 ]. Elevated levels of p21 CIP1/WAF1 in turn appear to have a direct impact on telomerase regulation [ 18 ]. As expected of slow growing primary breast tumors, in vivo , p21 CIP1/WAF1 positive cells are detected in the majority of cases [ 19 ]. Also pertinent in this regard is the fact that substrate induced changes in cell shape upregulate TGFβ transcription [ 20 ]. Since the promoter region of the TGFβ gene contains a shear stress response element [ 21 , 22 ], it seems likely that TGFβ induction occurs in response to unknown stresses in the in vitro environment and initiates a cascade of growth inhibitory events, including telomerase inactivation. While epithelial cells derived from most primary breast tumors rapidly revert to regulated growth dictated by environmental signals, immortal breast epithelial cells are insensitive to such cues, which may be the underlying basis for their continued selection in vitro . Notably, loss or functional inactivation of the cognate receptor often enables tumor cells in vivo to overcome the growth inhibitory effect of TGFβ early in tumorigenesis, however, during metastatic progression, autocrine TGFβ appears to play a tumor-promoting role, possibly enabling tumor cells to survive significant changes in microenvironment [ 23 ]. In this light, induction of TGFβ in response to the in vitro environment in primary breast tumor cultures portrays a critical phase of tumor progression. Additionally, towards the full manifestation of differentiated phenotypes in primary tumor cultures, cell propagation in a three-dimensional growth matrix, pioneered by Bissell and colleagues [ 24 ], is imperative. Such studies are currently ongoing in our laboratory. Tumor-derived immortal cell lines generally display robust proliferation and have thus filled an important need for functional cancer cell model systems. While immortal cell lines continue to provide molecular and biological insights regarding proliferation-related hallmarks of malignancy, their functional application as indicators of efficacy in cancer drug development is relevant mostly to rapidly proliferating high-grade tumors. Many breast tumors do not fall into this category, and are not necessarily indolent. Thus, additional targets in diverse gene clusters must be identified for novel drug designing. In fact, such targets could be applicable to tumors at early or late stages as recent studies suggest that genes conferring invasive and/or metastatic characteristics late in progression often become dysfunctional at earlier stages of tumorigenesis [ 25 ]. Moreover, since our data demonstrate that primary tumor cultures routinely derived from surgical discard tissue display phenotypic, and most likely functional aspects of breast cancer, they provide a strong rationale for the experimental manipulation of such cells towards revealing the causative role of genetic polymorphisms in cancer susceptibility, an important goal which is unlikely to be fulfilled with currently used model systems. Conclusions This microarray-based analysis demonstrates that epithelial cultures isolated from primary breast tumors retain phenotypes of the malignant tissue, which are eliminated during the selection of rapidly proliferating cell populations that comprise commonly used in vitro model systems. Thus the opportunity for basic and clinical application of functional cells derived from the full range of pathological breast tissue, instead of a few immortal cell lines should not be missed. Methods Clinical specimens and cell culture Pathologically confirmed tumor tissue and non malignant reduction mammoplasty samples were collected as fresh specimens under IRB approved guidelines at the California Pacific Medical Center, San Francisco, Stanford University, and the University of California, San Francisco between 1997 – 2000. Additional tumor samples were obtained from the NCI Cooperative Human Tissue Network. A portion of the tissue was snap frozen and cryopreserved for histological confirmation and nucleic acid isolation prior to cell culture. Tumor samples were processed as previously described [ 2 , 3 ]. A total of 21 independent cultured specimens were used for gene expression analysis. Primary tumor cultures and the non-tumorigenic, ENUt7 cell line were propagated in low calcium MCDB170 medium supplemented with 2% FCS. Breast cancer cell lines were cultured in the recommended growth media supplemented with 10%FCS; MCF7 , BT20 , MDA231 , MDA157 in DME-H21; BT474, CAMA1 , T47D , ZR75 in RPMI 1740; MDA435 , MDA134 , MDA361 in L15. To confirm the epithelial phenotype of primary tumor derived cells, cultures fixed with 50% ethanol: acetone were immunostained with anti pan-cytokeratin, as previously described [ 1 ]. RNA isolation, microarray methods and data analysis Frozen tissue was trimmed to yield >90% tumor cell enrichment. Total RNA was extracted with the RNAeasy Mini kit (Qiagen), amplified using an optimized T7 based protocol [ 26 ], labeled with Cy5, and hybridized to ~47,000 feature cDNA microarrays as previously described [ 26 , 27 ]. Hybridized arrays were scanned (Axon) and images were analyzed using GenePix ® Pro 4.0 software (Axon Instruments). Spots were selected for analysis if signal intensities in both Cy3 and Cy5 channels were 2.5 times higher than background. The arrays were clustered using a hierarchical clustering algorithm [ 28 ], which groups genes and samples on the basis of their expression similarities. The results were visualized using TreeView software [ 29 ]. The clustering was performed on clones whose expression varied at least 3-fold from the mean in one or more samples and was measurable in over 80% of the samples. Significance Analysis of Microarrays [ 30 , 31 ], used for identifying genes whose expression differed significantly between groups, was performed on data from 38,999 cDNA clones. After the selection of spots 2.5 times over background, and the elimination of clone redundancy, data was retrieved on 930 clones, representing 681 unique genes. Quantitative Real Time PCR (QRT-PCR) RNA samples were treated with RNAse-free DNAse (Roche) to remove genomic DNA, and reverse transcribed. Fifty ng of cDNA was used as template for PCR amplification with specific oligonucleotide primers, (designed using Primer Express 1.5 software). Following denaturation and cycling reactions (40 cycles) the products were analyzed (Applied Biosystems 5700 Sequence Detection System). The cycle number at which the amount of amplified target reached a fixed threshold was designated as the threshold cycle (C T ). The higher the initial amount of transcript template, the lower the C T value. For quantitation of gene expression, the target gene value normalized to the expression of an endogenous reference ( β actin ) was designated as ΔC T . Relative gene expression was calculated by the formula 2 -ΔΔCT . ΔΔC T was obtained by subtracting the ΔC T of test sample from the ΔC T of normal breast epithelial fractions, called 'organoids', isolated from mechanically and enzymatically dissociated reduction mammoplasty tissue (average of 8 independent specimens). Telomerase activity Cell lysates prepared from primary tumor cultures, immortal cell lines, and normal breast organoids were analyzed by the telomerase repeat amplification protocol (TRAP) as per manufacturer recommendation (Intergen). Briefly, trypsinized cell pellets were resuspended in lysis buffer at 500 cells/microliter, incubated on ice for 30 minutes, centrifuged at 12,000 × g for 20 mins, and the telomerase containing supernatant used for PCR amplification. Reaction products were resolved in 10% PAGE gels and visualized with SYBR Green-1 (Molecular Probes). Heat inactivated controls were included for each sample. Linearity of the assay was determined with cell dilutions ranging from 100 to 10, 000 cells. Statistical methods We calculated Pearson correlations from the microarray data generated from 38,999 clones to measure similarities among 7 matched tumor tissue: cell culture sample pairs and among 16 tumor tissue: immortal cell line pairs. For QRT-PCR data, multivariate analysis of variance (MANOVA) was used to compare the expression levels of genes involved in negative growth regulation between immortal cell lines, primary tumor cultures, and non malignant breast epithelial cells. Tests for statistical significance were based on Wilks lamda criterion, a multivariate analog of the F-test for univariate analysis of variance (ANOVA), which tests the equality of means. Calculations were made in Data Desk, version 6.2. Principal components for all genes over all the data were computed and S-Plus was used to plot the data for the first 3 principal components. Authors' contributions SHD conceived the study, provided overall direction and coordination to the research, and drafted the manuscript. YJ carried out the microarray analysis. YB carried out QRT-PCR, and other molecular analyses. DHM performed the statistical analysis of data. ZM performed the pathology review and tissue dissection. SSJ participated in the design and analysis of microarray-based experiments and in editing portions of the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC509241.xml
545063	Accuracy of parents in measuring body temperature with a tympanic thermometer	Background It is now common for parents to measure tympanic temperatures in children. The objective of this study was to assess the diagnostic accuracy of these measurements. Methods Parents and then nurses measured the temperature of 60 children with a tympanic thermometer designed for home use (home thermometer). The reference standard was a temperature measured by a nurse with a model of tympanic thermometer commonly used in hospitals (hospital thermometer). A difference of ≥ 0.5 °C was considered clinically significant. A fever was defined as a temperature ≥ 38.5 °C. Results The mean absolute difference between the readings done by the parent and the nurse with the home thermometer was 0.44 ± 0.61 °C, and 33% of the readings differed by ≥ 0.5 °C. The mean absolute difference between the readings done by the parent with the home thermometer and the nurse with the hospital thermometer was 0.51 ± 0.63 °C, and 72 % of the readings differed by ≥ 0.5 °C. Using the home thermometer, parents detected fever with a sensitivity of 76% (95% CI 50–93%), a specificity of 95% (95% CI 84–99%), a positive predictive value of 87% (95% CI 60–98%), and a negative predictive value of 91% (95% CI 79–98 %). In comparing the readings the nurse obtained from the two different tympanic thermometers, the mean absolute difference was 0.24 ± 0.22 °C. Nurses detected fever with a sensitivity of 94% (95 % CI 71–100 %), a specificity of 88% (95% CI 75–96 %), a positive predictive value of 76% (95% CI 53–92%), and a negative predictive value of 97% (95%CI 87–100 %) using the home thermometer. The intraclass correlation coefficient for the three sets of readings was 0.80, and the consistency of readings was not affected by the body temperature. Conclusions The readings done by parents with a tympanic thermometer designed for home use differed a clinically significant amount from the reference standard (readings done by nurses with a model of tympanic thermometer commonly used in hospitals) the majority of the time, and parents failed to detect fever about one-quarter of the time. Tympanic readings reported by parents should be interpreted with great caution.	Background Parents and health care workers use temperatures measured by parents at home to determine if a child requires medical assessment. Health care workers sometimes make decisions about the need for investigations and hospital admission based on the temperature that parents report. This is especially true in the newborn or in the immunocompromised patient, where any temperature above the normal range can be indicative of bacterial infection. Health care workers have used commercial tympanic thermometers in hospitals for over 15 years. These thermometers are quick and easy to use and minimize the risk of nosocomial infection. Tympanic thermometers are now available for home use at a cost of about $50 US. There are no published studies comparing measurements obtained with tympanic thermometers designed for home use to measurements obtained with other types of thermometers, or comparing readings obtained by parents with these instruments to those obtained by health care workers. The primary objective of this study was to determine the diagnostic accuracy of readings obtained by parents with a tympanic thermometer designed for home use. Methods The Health Research Ethics Board of the University of Alberta approved this study and parents and older children signed a consent form. Reference standard Traditionally, rectal temperatures have been used as the reference standard in pediatrics. However, readings done by a nurse using a model of tympanic thermometer commonly used in hospitals were chosen as the reference standard for this study as they are easier to obtain than are rectal temperatures and tend to be closer to PA temperatures when the patient is febrile [ 1 ] or when the temperature is changing [ 2 , 3 ], although the opposite may be true in the steady state [ 4 ]. Subjects Parents of patients from the General Pediatric Clinic, the Emergency Department, or on the inpatient units of the Stollery Children's Hospital were eligible for the study. Parents of febrile or afebrile children aged 6 months to 16 years were approached on the days when the study nurse was available, but preference was given to patients who were being assessed because of fever. Parents were not approached if it would be inconvenient for them to participate in the study, or if they did not communicate in English, but there were no other exclusion criteria. Study procedures After informed consent was obtained from the parent (and the child when practical), the parent was given the instruction sheet from the Braun Thermoscan (Thermoscan Inc., San Diego, CA) tympanic thermometer (home thermometer) to read. They were then asked to measure their child's temperature when they felt they understood the instructions. They then recorded the temperatures they measured on a sheet of paper. Without looking at this sheet of paper, one of three trained research nurses then measured the child's temperature using the same thermometer. The nurse then also measured the child's temperature with CORECHECK (ALARIS Inc., San Diego, CA) tympanic thermometer (hospital thermometer). All readings were done in the same ear with a new probe cover. Outcome measures The mean absolute difference between the parents' reading and the reading by the nurse using the same thermometer was calculated. Readings were then analyzed separately for the febrile children. A temperature of ≥ 38.5 °C on the reading taken by the nurse using the hospital thermometer was defined as a fever. The percentage of times that the readings differed by 0.5 °C or more was calculated as this was considered a clinically significant difference. Even if the parent and the nurse obtained similar readings from the home thermometer, it would still be possible that these readings are inaccurate, as there are no published studies demonstrating the accuracy of this thermometer. Therefore, the readings obtained by the nurse and the parent from the home thermometer were compared to those obtained by the nurse using the hospital thermometer (the reference standard for the study). If the readings obtained by the parent differed considerably from those obtained by the nurse, but the nurse then obtained very consistent readings with the two different thermometers, one could conclude that the parents were inaccurate because of human error. On the other hand, if the nurse obtained inconsistent readings with the two different thermometers, this would suggest that either instrument or human error could be the problem. The measures of central tendency were determined, and the intraclass correlation coefficient determined for the three sets of readings. The three sets of measurements were also compared using the Bland-Altman method [ 5 ] where the differences between two measurements are plotted against the average of the measurements. Sample size calculation It was not possible to calculate a sample size, as the expected standard deviation between the three sets of readings could not be predicted. We estimated that a sample size of 60 children would be adequate. Results Characteristics of study subjects Sixty parents were approached and all agreed to participate in the study. Their children were 35 males and 25 females aged 5 months to 15 years (median 3 years). Although the majority of patients were being assessed because of fever, only 17 children were febrile at the time of the study. All 3 measurements were obtained within 5 minutes for 55 of the 60 cases. Main results Table 1 shows the results of the study. The mean absolute difference between the temperature recorded by the parent and the temperature recorded by the nurse using the home thermometer was 0.44 ± 0.61 °C (Figure 1 ). The difference was similar in febrile and afebrile children. The higher reading was from the parent in 20 of the 60 cases. Parents detected fever with a sensitivity of 76% (95% CI 50–93%), a specificity of 95% (95% CI 84–99%), a positive predictive value of 87% (95% CI 60–98%), and a negative predictive value of 91% (95% CI 79–98 %) (Table 2 ). Table 1 Comparison of results of temperatures measured by parents using a tympanic thermometer designed for home use, a nurse using the same thermometer, and the same nurse using a tympanic thermometer designed for hospital use Mean absolute difference (°C) Range of absolute differences (°C) % of time where absolute difference ≥ 0.5 °C Parent versus nurse using home tympanic thermometer All children n = 60 0.44 ± 0.61 0.0 – 3.1 33% Parent versus nurse using home tympanic thermometer Afebrile children n = 43 0.44 ± 0.65 0.0–3.1 35% Parent versus nurse using home tympanic thermometer Febrile children n = 17 0.45 ± 0.51 0.10 – 1.9 29% Nurse using home thermometer versus same nurse using hospital thermometer n = 60 0.24 ± 0.22 0.0 – 1.0 13% Parent using home thermometer versus nurse using hospital thermometer N = 60 0.51± 0.63 0.0 – 3.4 72% Figure 1 Comparison of readings done by a parent to readings done by a nurse using a home tympanic thermometer The mean absolute difference between the temperatures measured by the nurse using the home thermometer versus the hospital thermometer was 0.24 ± 0.22 °C. The higher reading was from the hospital thermometer in 25 of the 60 cases. There was agreement between the readings taken by the nurse from the two thermometers with regard to the presence of fever, except in one case where one reading was 38.4 °C and the other was 38.6 °C (Table 2 ). Nurses detected fever with a sensitivity of 94 % (95 % CI 71–100 %), a specificity of 88 % (95% CI 75–96 %), a positive predictive value of 76% (95% CI 53–92 %), and a negative predictive value of 97% (95% CI 87–100 %) (Table 2 ). In comparing the readings obtained by the parent using the home thermometer to those obtained by the nurse using the hospital thermometer, the mean absolute difference was 0.51 ± 0.63 °C, with 72% of the readings differing by ≥ 0.5 °C. Table 2 Ability of parents and nurses to detect fever using a home tympanic thermometer, with a hospital tympanic thermometer being the reference standard Nurse reported fever on hospital thermometer Nurse reported no fever on hospital thermometer Parent reported fever on home thermometer 13 2 Parent reported no fever on home thermometer 4* 41 Nurse reported fever on home thermometer 16 5 Nurse reported no fever on home thermometer 1** 38 Readings by parents were 37.8 °C, 37.9 °C, and 38.4 °C twice with corresponding hospital thermometer readings being 38.6 °C, 38.5 °C, 38.6 °C, and 39.6 °C *Reading was 38.4 °C on home thermometer and 38.6 °C on hospital thermometer The intraclass correlation coefficient for the three sets of readings was 0.80. Figures 2 , 3 , and 4 show that the variation between the readings was consistent at different temperatures. Figure 2 Scatter plot of the difference between temperature measured by a parent and that measured by a nurse using a thermometer designed for home use Figure 3 Scatter plot of the difference between temperature measured by a parent using a thermometer designed for home use and a nurse using a thermometer designed for hospital use Figure 4 Scatter plot of the difference between temperature measured by a nurse using a thermometer designed for home use and a a thermometer designed for hospital use Discussion It is now common for parents to report a child's body temperature as measured by a tympanic thermometer designed for home use. The only published study looking at the reliability of parents at measuring tympanic temperatures reported on the consistency of readings, rather than comparing the temperatures measured by parents to those measured by health care workers or by another method [ 6 ]. The current study showed that readings obtained by parents differ from those obtained by a nurse using the same instrument by a mean of 0.44°C, with 33% of the readings differing by a clinically significant amount (0.5°C or more). The absolute differences were similar for febrile and afebrile children. Using the reference standard of a tympanic temperature measured by a nurse with a model of thermometer commonly used in hospitals, the parents did not detect a fever in four of 13 cases (although in two of these cases, the reading by the parent was just 0.1 °C below our definition of fever). This suggests that the readings obtained by a parent with a tympanic thermometer are sometimes not reliable. In fact, previous studies showed that parents' subjective assessment of whether their child had a fever had a sensitivity of 81.8% [ 7 ] and 88.9% [ 8 ], which is similar to the 76 % sensitivity in the current study when parents used a tympanic thermometer. In comparing the readings taken by a nurse with a home tympanic thermometer to a hospital tympanic thermometer, the mean absolute difference was 0.24 °C, with 13 % of the readings differing by a clinically significant amount. It is not clear if this degree of discrepancy is to be expected when a skilled operator takes two tympanic readings, or if it was a true difference between the two types of tympanic thermometer. The nurse would have identified all but one of the 17 children with fever as being febrile with either thermometer, and that child had a temperature of 38.4 °C on one thermometer and 38.6 °C on the other thermometer. Therefore, it appears that with a skilled operator, the tympanic thermometer designed for home use is reasonably reliable, and is likely to detect fever even if the actual reading is not always accurate. The chief limitation of this study is that readings obtained by a nurse from the hospital tympanic thermometer must be reliable for the results of this study to be accurate. Some studies have concluded that tympanic thermometers do not give an accurate measurement of body temperature [ 7 , 9 , 10 ]. However, the reference standard in these studies (axillary or rectal temperature) may not be ideal. Studies have shown that when used by a skilled operator on a cooperative patient with rapidly changing body temperature, the readings from models of tympanic thermometers commonly used in hospitals more closely approximate pulmonary artery or esophageal temperature than do rectal or axillary temperatures [ 2 , 3 ]. Therefore, a tympanic temperature measured with a hospital tympanic thermometer was considered the best available reference standard for this study. However, if the nurses used incorrect technique, this would affect the results of the study. Falsely high readings with a properly calibrated tympanic thermometer should only occur if the thermometer itself has been stored above room temperature, so the fact that in 25 of the 60 cases the parent obtained a higher reading than did the nurse suggests the possibility that the nurses were not using ideal technique. Another limitation is that we could not blind the nurses for comparing their own two temperature readings. There is some evidence that falsely low readings can be obtained if tympanic readings are obtained in rapid succession as placement of the thermometer results in local cooling, so it is possible that readings would have been more comparable had we waited at least 2 minutes between readings [ 11 ] It is possible that parents would have performed better had they leisurely read the instructions in their own home rather than doing so in a hospital-based setting, or had they had time to practice. However, it is also possible that they performed better than they would have in the home, as they knew evaluation of their performance was occurring. It is also possible that parents who would choose to buy a tympanic thermometer will differ from our study population. It is not clear what advice to give to parents about purchasing a thermometer. The ability of parents from the inner city to correctly use and read a mercury glass thermometer was poor in previous studies [ 12 , 13 ]. An electronic thermometer is easier to read and is now relatively inexpensive, but may not be as accurate as a mercury thermometer [ 14 ]. The results of the current study suggest that readings obtained by parents with a tympanic thermometer often differ by a clinically significant amount from readings obtained by nurse. Despite their ease of use, there are reports of parents inserting tympanic thermometers in the rectum [ 15 ]. It is not clear if parents could more accurately use an infrared temporal artery thermometer. A study showed that using the highest of three temporal artery temperature obtained by a parent or nurse, a reading of ≥ 37.8 °C was 97% sensitive and 84% specific for detecting a rectal temperature of ≥ 38.5 °C, and that the limits of agreement for temperatures obtained by parents versus nurses using this thermometer were -0.6 °C to 0.7 °C [ 16 ]. This is lower than the limits of agreement for the current study, but it is possible that the parents would have performed better had we recorded the highest of three readings. Conclusions With the recent outbreak of Severe Acute Respiratory Syndrome, rapid measurement of body temperature (sometimes by personnel who are not health care workers) has become an important tool to indicate if people can board airplanes and if staff can work. It is therefore important to determine the accuracy of non-health care workers at measuring body temperature. This study showed that the temperatures measured by parents with a tympanic thermometer often differ by a clinically significant amount from those measured by a nurse with a tympanic thermometer. There is not a consistent gradient or direction of the gradient between the two temperatures, which makes it difficult to interpret the temperatures reported by parents. There was a smaller gradient between the readings taken by a nurse with a tympanic thermometer designed for home use compared with those taken with a model of tympanic thermometer commonly used in hospitals, but there were still potentially clinically significant discrepancies 14% of the time. This makes it difficult to conclude if the poor performance of the parents relates entirely to human error, or if there is also an element of instrument error. The fact that in comparison with our reference standard (tympanic temperature measured by the nurse with a hospital thermometer) the readings taken with the home thermometer by the nurse correlated much better than those taken by the parent suggests that the parents did not use ideal technique. In any case, one should interpret temperatures taken by parents with a tympanic thermometer with great caution. Although parents will detect the majority of fevers with these instruments, the absolute numbers obtained may not be accurate. Again, we find that in pediatrics, the clinical picture is a more useful piece of information than are "the numbers"! Abbreviations °C – degrees Celsius PA: pulmonary artery Competing interests The author(s) declare that they have no competing interests. Authors' contributions JLR wrote the protocol and the manuscript. HJ helped with patient recruitment and reviewed the manuscript. DWS did the statistical analysis and reviewed the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC545063.xml
544194	Nondestructive analysis of urinary calculi using micro computed tomography	Background Micro computed tomography (micro CT) has been shown to provide exceptionally high quality imaging of the fine structural detail within urinary calculi. We tested the idea that micro CT might also be used to identify the mineral composition of urinary stones non-destructively. Methods Micro CT x-ray attenuation values were measured for mineral that was positively identified by infrared microspectroscopy (FT-IR). To do this, human urinary stones were sectioned with a diamond wire saw. The cut surface was explored by FT-IR and regions of pure mineral were evaluated by micro CT to correlate x-ray attenuation values with mineral content. Additionally, intact stones were imaged with micro CT to visualize internal morphology and map the distribution of specific mineral components in 3-D. Results Micro CT images taken just beneath the cut surface of urinary stones showed excellent resolution of structural detail that could be correlated with structure visible in the optical image mode of FT-IR. Regions of pure mineral were not difficult to find by FT-IR for most stones and such regions could be localized on micro CT images of the cut surface. This was not true, however, for two brushite stones tested; in these, brushite was closely intermixed with calcium oxalate. Micro CT x-ray attenuation values were collected for six minerals that could be found in regions that appeared to be pure, including uric acid (3515 – 4995 micro CT attenuation units, AU), struvite (7242 – 7969 AU), cystine (8619 – 9921 AU), calcium oxalate dihydrate (13815 – 15797 AU), calcium oxalate monohydrate (16297 – 18449 AU), and hydroxyapatite (21144 – 23121 AU). These AU values did not overlap. Analysis of intact stones showed excellent resolution of structural detail and could discriminate multiple mineral types within heterogeneous stones. Conclusions Micro CT gives excellent structural detail of urinary stones, and these results demonstrate the feasibility of identifying and localizing most of the common mineral types found in urinary calculi using laboratory CT.	Background Clinical laboratory assessment of urinary stones is typically conducted using destructive methods of analysis [ 1 - 5 ] and is usually geared to identify stones only by their primary mineral content. It is rare that stones get classified as having multiple components, and even less likely that notation is given to describe the pattern or distribution of different minerals in heterogeneous stones. Thus, stones are commonly classed as being calcium oxalate monohydrate (COM), uric acid, cystine, etc. When this information makes its way to the patient's chart, the individual may be classified simply, for example, as a COM stone former. This tends to underestimate the complexity of an individual's stone history as, indeed, it has been determined that the vast majority of stones actually contain more than one type of mineral [ 6 ]. Knowing the mineral composition of a patient's stones has obvious value in determining a treatment plan, but stone structure may be important as well. It has long been appreciated that there is variability in stone fragility to shock waves in lithotripsy, that stones of a given mineral type do not all break the same [ 7 ]. Stone fragility, and the factors that may influence variability in fragility, are not well known. There are some clues, but the story is incomplete and deserves attention. As an example, consider the case of cystine stones. Rough surface cystine stones tend to break readily, but smooth surfaced cystine can be very difficult to break [ 8 ]. Rough cystine typically contains internal radio-lucent regions (possible voids). Numerical modeling, and studies with artificial stones made to contain voids, suggests that such defects could be sites of structural weakness [ 9 ]. Thus, the internal structure of a stone may very well contribute to its fragility. The purpose of this report is to present micro computed tomography (micro CT) as a potential method for the analysis of urinary stone composition and morphology in a nondestructive manner at very high resolution. Micro CT, which has seen considerable use as a research tool in bone biology [ 10 ], has the ability to reconstruct 2-D and 3-D images of urinary stones that allow the 3-D image of the stone to be cut and viewed in multiple planes with voxel sizes of 8–34 μm. In the present study, we observed that in addition to providing exceptional detail of the fine structure of human urinary stones, micro CT was able to differentiate six common mineral constituents using x-ray absorption (attenuation) values alone. Methods Human urinary stones were obtained following percutaneous nephrolithotomy procedures or from a stone analysis laboratory (Beck Analytical Services, Indianapolis, IN). A representative collection of urinary stones of pure and heterogeneous mineral content was selected for use in this study. Calibrating micro CT attenuation to mineral content Stones for this work had already been analyzed using conventional methods (microscopic, chemical and IR spectroscopic), with portion used here typically the half of the stone remaining intact after that analysis. For some stones, pre-CT analysis was completed on a cohort stone taken during the same surgical procedure. Compositions of the 11 stones used for combined micro CT and IR microspectroscopy included pure COM, pure calcium oxalate dihydrate (COD), 91% COD/9% COM, 99% COM/1% hydroxyapatite, 52% COM/48% uricite, pure uricite, pure struvite, two pure cystine stones, and two pure brushite stones. Stones were embedded within methyl methacrylate and sliced into 1–2 mm slabs using a diamond wire saw (Well Saw, Delaware Diamond Knives, DE). A Perkin-Elmer AutoImage infrared microscope interfaced to a Perkin-Elmer Spectrum 2000 fourier transform infrared (FT-IR) spectrometer (Perkin Elmer, Shelton, CT) was used to collect infrared spectra on the flat surface of the stone slices. The spectrometer directs infrared light onto the specimen through a microscope lens, and collects reflected light onto a cooled mercury-cadmium-telluride detector. Spectra were collected in grids of 200 × 200 μm intervals, averaging 16 individual scans on each spot, yielding spectral resolution of 4 cm -1 . Spectra for COM, COD, apatite, uric acid, struvite, cystine, and brushite were all easily identified and distinguishable from one another. Uricite and uric acid dihydrate could not be distinguished above the background noise of the spectra, and so regions are reported as uric acid. The same stone slices analyzed by FT-IR microspectroscopy were also scanned by micro CT, using a Scanco MicroCT 20 instrument (Scanco Medical AG, Bassersdorf, Switzerland) (Figure 1 ). The system utilizes a 7 μm spot-size microfocus x-ray source (0.16 mA, 50 kVp) that is detected by a charge coupled device array. The scans on stone slices were completed using standard resolution (512 × 512 pixels) and a 17.4 mm specimen holder which produced image slice thicknesses and pixel widths of 34 μm. For each stone slice, a CT image slice was obtained within ~ 50 μm of the cut surface. Figure 1 Micro CT system (Scanco MicroCT 20) . The micro CT unit itself is on the right, a cube approximately 0.5 m across. The computer workstation to the left of the micro CT unit controls the collection of scan data, storage and archival of data, and is used for image reconstruction. The FT-IR analysis yielded a 2-D map of mineral composition for a defined region of each stone slice. Regions of the 2-D map showing pure mineral were compared to the identical region on the micro CT image slice taken just below the cut surface (Figure 2 ). Micro CT attenuation values from these pure mineral regions were recorded. Figure 2 Calibrating micro CT attenuation to pure mineral . On stone slice shown ( top ) FT-IR spectra were collected on each of 126 regions indicated by the grid area. Examples of spectra are shown for three regions, which represent the three classes of spectra collected from cut surface of this stone. Spectra indicated that the mineral was either purely calcium oxalate monohydrate (COM), purely apatite (HA), or a mixture of these two minerals. Corresponding regions-of-interest on micro CT image slice ( bottom ) taken just beneath the cut surface are shown with blue squares, and micro CT attenuation values are given below spectra. Using this method, regions of pure mineral were identified on stone slices and corresponding regions-of-interest measured in micro CT images to determine CT attenuation of different minerals. Imaging of intact stones: nondestructive stone analysis In scanning intact stones with micro CT, actual slice thickness and pixel widths were dependent on the diameter of the specimen holder and resolution parameters, and ranged from 25–34 μm for images shown in this study. Examples of scans of intact stones are presented to show the potential for performing analyses of stone composition and structure using micro CT alone. Results The imaging capability of micro CT is simply remarkable. Additional Data File 1 shows a stack of micro CT images of a single stone, archived as a movie. Refer to this file to view the reconstruction ability of micro CT, as well as to appreciate the power of micro CT to clearly display very high resolution images of stone structure. Nine sectioned stones, analyzed as shown in Figure 2 , were found to contain six different mineral types by IR spectrum. Micro CT attenuation values were taken from regions of compositional homogeneity, as shown by FT-IR microspectroscopy, and Figure 3 displays the results. Values for micro CT attenuation were non-overlapping for the six minerals. Means ± standard deviations for attenuation values were 22,207 ± 709 for hydroxyapatite, 17,771 ± 837 for COM, 14,767 ± 680 for COD, 9434 ± 439 for cystine, 7633 ± 248 for struvite, and 4201 ± 564 for uric acid. Two brushite stones were also analyzed by this method, but no regions on the cut stone surface larger than about 0.5 mm across were found that were pure; spectra frequently showed intermixing of brushite with COD. Micro CT attenuation for 11 ROI's on these two stones averaged 19,145 ± 486, with a tight range of 18,540 to 19,944. Figure 3 Micro CT attenuation values taken from pure mineral regions identified by FT-IR microspectroscopy . Regions-of-interest representing pure mineral, confirmed by FT-IR mapping, were drawn on micro CT images collected just beneath the cut surface of stone slice, and average value for CT attenuation (in machine-specific units) was recorded. Horizontal lines indicate minimum and maximum values, and number of regions-of-interest indicated in parentheses. Note that each mineral composition is associated with non-overlapping attenuation values (uric acid 3515 – 4995, struvite 7242 – 7969, cystine 8619 – 9921, calcium oxalate dihydrate 13815 – 15797, calcium oxalate monohydrate 16297 – 18449, and hydroxyapatite 21144 – 23121). Since micro CT attenuation values for six of these minerals did not overlap, mineral composition of pure mineral regions could be inferred using micro CT alone. For example, Figure 4 displays a simple, homogeneous stone and a complex, heterogeneous stone for which micro CT attenuation values were used to identify mineral composition. The homogeneous stone (left panel) showed uniform CT attenuation values in regions-of-interest of various sizes and shapes, placed at various positions across the image, all consistent with this stone being purely COM. The image of the heterogeneous stone (right panel) displays intricate structure in which the grayscale color difference in selected regions of interest yielded different (i.e., non-overlapping) x-ray attenuation values – values that when compared to FT-IR calibration data (Figure 3 ) could be identified as uric acid, COM, and hydroxyapatite. Figure 4 Example of micro CT identification of mineral composition . Visually, there appears to be only one mineral contained in the stone in the left panel and three different minerals that comprise the stone in the right panel. Micro CT attenuation identified the left stone as pure COM, and the right stone as a mixture of hydroxyapatite (bright white, highest attenuation), COM (gray) and uric acid (close to black). Speckled nature of colors – particularly apparent in image at left – is due to image noise as a result of increased magnification. Over the past few years, we have imaged hundreds of urinary stones using micro CT, and we have begun to learn some of the capabilities of this method that can enhance the value of the analytical information presented above. Figure 5 shows some of the imaging capabilities of micro CT software. Panel A is a photograph of a COM stone and panel B is a representative micro CT image slice in which the concentrically lamellar nature of the stone is easily seen. The dark lines separating lamellae indicate regions that absorb x-rays poorly, possibly representing regions of organic matrix. The 3-D surface rendering capability of micro CT software is shown in Figure 5C , where surface topography is shown. Further, the software permits the image to be cut and rotated in multiple planes, as shown by the wedge cut that exhibits the 3-D lamellar distribution of voids in two planes of view (Figure 5D ). Figure 5 Imaging and software power of micro CT . Pure COM stone viewed by conventional photography ( A ) and micro CT ( B-D ). B shows a typical micro CT image slice, C a 3-D reconstruction of the stone surface, and D a wedge cut that displays the internal 3-D morphology of the stone. Figure 6 shows features of a stone revealed using micro CT data in a different way. Panel A is a photograph of a mulberry-type COM stone and panel B is a single image slice of this stone by micro CT. The micro CT image shows a COM outer shell deep to which is a thin ring of hydroxyapatite (white) surrounding a core of low attenuation. The core consists of void spaces, which are defined to be regions of extremely low attenuation, again perhaps representing organic matter. Panel C shows a 3-D surface rendering of the stone from the micro CT image stack. Panel D displays a surface rendering of the apatite regions, superimposed within a translucent version of panel C so that the position of the apatite within the stone is easily visible. Rendering for panels C and D were obtained using MacVol, a freeware program. Figure 6 Visualization of components of a mixed COM/HA stone . A. Digital photograph of stone, on 1 mm grid. B. Micro CT slice through middle portion of stone; arrow indicates ring of high attenuation (hydroxyapatite). Gray material outside this ring had attenuation value in the range for COM. C. 3-D surface rendering from micro CT displaying surface topology. D. 3-D surface rendering with transparent shell to show 3-D localization of hydroxyapatite within stone. Total stone volume and volume of internal voids can also be calculated using images obtained by micro CT. Figure 7 shows a transparent 3-D reconstruction of a cystine stone with void spaces appearing as internal granular objects. The stone volume is 257.7 mm 3 and the volume of internal voids is 0.3 mm 3 . This method can also be used to display and quantitate the 3-D distribution of mineral components within a complex, heterogeneous stone. Figure 7 3-D reconstruction of a cystine stone with regions of low attenuation (voids) . Outer shell of stone was made transparent to display distribution of inner void regions depicted as small bodies. Total stone volume was 257.7 mm 3 and total volume of internal voids was 0.3 mm 3 . Discussion Micro CT yields excellent high resolution analysis of stone structure. It is a relatively fast method, taking approximately 2 hours for a complete 30 μm slice scan of a 1 cm diameter urinary stone. Micro CT allows nondestructive mapping of the internal and surface structure of urinary stones and permits identification of mineral composition based on x-ray attenuation values. Micro CT cannot differentiate mineral types when the stone is highly complex and micro-heterogeneous with significant mixing of different mineral types at a scale below the spatial resolution of the instrument. With this limitation, complete analysis for most urinary stones seems feasible using micro CT alone. Establishing micro CT standards for complete stone analysis will require a larger data set than was obtained in the present study; for example, the measurements obtained for apatite were from only one stone that was composed mostly of COM. The x-ray attenuation of apatite was the highest of all the minerals tested, which would be expected from other CT studies [ 12 ], but the attenuation value in pure apatite stones might well be different. It will also be important to study more brushite stones to see how this clinically important mineral can be distinguished from other minerals. Brushite stones are rare, making up only about 1% of total stones [ 6 ], but when present they are a special clinical problem, as brushite stone formers tend to form stones rapidly, and the stones are difficult to break with shock wave lithotripsy [ 13 ]. A recent study showed that less than 2% of brushite stones are pure brushite [ 6 ], but the incidence of close intermixing of mineral within brushite stones, as seen in the present study, has not been studied. The source of variation of micro CT attenuation in stone regions that are pure by IR analysis is not apparent. Note in Figure 3 that the range of attenuation values measured for COM is greater than that seen for other minerals. This range of values could be due to varying amounts of matrix included among the COM crystals [ 14 ], or it could be due to mixing of small amounts of COD or other mineral, amounts small enough not to be detected by IR. The vast majority of stones contain more than one component [ 6 ], but many of these mixed stones show obvious spatial separation of the different materials, as observed in the stones shown in Figures 4b and 6 . If a significant number of stones show close intermixing of minerals, the use of micro CT for stone analysis may be more limited than is suggested in the present study. One application of stone analysis using micro CT will be the study of stone fragility in shock wave lithotripsy. It has been known from the earliest days of shock wave lithotripsy that some kinds of urinary stones are broken by shock waves more easily than others [ 11 ]. However, only recently has it been better appreciated that stone behavior in lithotripsy is highly variable, even among stones composed of the same major mineral type [ 7 ]. Some of this variable behavior in lithotripsy may be accounted for by the structural arrangement of stone components, which can be viewed by CT [ 15 ]. Further testing of hypotheses concerning stone structure and stone fragility can be done in vitro , using micro CT to analyze stones before breakage in the lithotripter. Another logical application of micro CT is for materials testing of stones. Several studies have reported materials properties of stones [ 16 - 18 ], but stone heterogeneity complicates this effort. One study by Zhong et al. demonstrated that depending on the site of hardness testing within the same stone, different measurements could be obtained [ 19 ]. Thus, micro CT could help identify regions of homogeneous mineral content within a stone and also show any structural internal weakness, such as a crack or void space, prior to testing. This process would enable researchers to identify regions suitable for materials testing and, therefore, provide more reliable data. Our findings with micro CT suggest that similar stone analysis may one day be possible as a preoperative diagnostic tool. Clinical helical CT is evolving and enhancements continue to be made to increase overall imaging functionality. For instance, newer multi-detector helical CT (MDCT) affords much greater spatial resolution than conventional single-detector helical CT. Williams et al. used four-row MDCT to show that some degree of internal structure can already be seen in urinary stones [ 15 ]. Zarse and colleagues also used four-row MDCT and demonstrated that CT can identify mineral composition in vitro when suggested scanning parameters were used [ 20 ]. Eight-row MDCT has been found to improve z-axis resolution and scan time, while reducing artifact streaking for an overall improvement in diagnostic imaging [ 21 ]. Sixteen-row MDCT instruments are now available that use isotropic imaging, the same technology used in micro CT. This essentially translates to equal resolution and voxel size in any plane (sagittal, coronal, and axial) [ 22 ]. Moreover, like micro CT, MDCT has the option to reconstruct 3-D images of the entire viewing area. This is advantageous since mapping the 3-D spatial distribution of mineral content in stones could yield important information useful in determining proper treatment for the patient. Overall, the continued development of helical CT technology points to imaging capability in the future that could provide stone analysis equivalent to what we describe for micro CT. Conclusions Micro CT provides high resolution in vitro imaging of urinary calculi for nondestructive stone analysis. Fine resolution coupled with the 2-D and 3-D reconstruction capabilities of micro CT yields an imaging diagnostic that offers excellent images of surface and internal stone structure. Mineral deposition pattern and regions of potential structural weakness, such as voids, were easily visible. Six common stone minerals were found to occupy non-overlapping ranges of attenuation value, which allows the identification of mineral types using micro CT alone. This technology carries the potential for immediate application to non-destructive analysis of stone structure and composition in clinical stone analysis laboratories. The demonstration that stone composition can be determined by micro CT is proof of concept and an important step toward the use of helical CT to provide similar analysis in patients. Competing interests The author(s) declare that they have no competing interests. Authors' contributions CZ did the experimental work for calibrating the attenuation values, put together several of the figures, including the image stack movie, and drafted the manuscript. AJ performed the FT-IR analyses. EH did many of the micro CT scans and managed the library from which stones were chosen. SK and JL provided some of the stones from patients, and SK performed the 3-D imaging of cystine stone. JM, JL, and AE initiated much of the intellectual direction for the study. JM and AE also played important roles in the FT-IR analysis part of the study. JW performed the 3-D image construction in MacVol, drafted several of the figures, and performed the primary editing of the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here: Supplementary Material Additional File 1 Micro CT image stack through a urinary stone.MOV 6.61 MB Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544194.xml
521730	Language Evolution	How did language develop and evolve? Here, linguists, cognitive scientists, behavioural ecologists, and theoretical biologists all offer their disparate views on this emerging field	A ban in the 1866s by the French Academy of Sciences on publications about the origin of human language must have been one of the strangest bans in the history of sciences. Yet it was highly effective. After the ban, scientists and interested laymen had to wait for more than a century to hold a textbook on language evolution in their hands. Language Evolution, a compilation of essays by a diverse group of respected researchers, is amongst the first books that try to tackle what is arguably one of the hardest scientific problems. The editors set themselves the ambitious target of creating an up-to-date book about this emerging field, and they have to be congratulated for their efforts. Linguists, cognitive scientists, behavioural ecologists, and theoretical biologists all offer their view on the origin of human language and, refreshingly, do not shy from pointing out the real or assumed weaknesses of the other approaches. One of the main themes of the book is the evolutionary approach and the importance of biological structures and properties that were co-opted in the development of language (pre-adaptations). In one essay, Michael Studdert-Kenedy and Louis Goldstein propose that speech, as a motor function, draws on phylogenetically ancient mammalian oral capacities for sucking, licking, swallowing, and chewing. Thus, our hominid ancestors adopted an apparatus already divided neuroanatomically into discrete components. Complementing this evidence, Marc Hauser and Tecumseh Fitch compare human speech production and perception with that of nonhuman species. They conclude that many traits that were formerly thought to have evolved specifically for speech (such as having a descended larynx or categorical perception) are also present in other species. But perhaps the most interesting idea about pre-adaptation comes from the work of neuroscientist Michael Arbib on ‘mirror’ neurons in monkeys. These neurons are a subset of the grasp-related premotor neurons that discharge not only, as other premotor neurons do, when the monkey executes a certain class of actions, but also when the monkey observes more or less similarly meaningful hand movements made by the experimenter (or by another monkey). The area in which these grasp-related neurons are found is analogous with the Broca's area in human brains, which is involved in assessing the syntax of words. This observation serves as the basis for the mirror-system hypothesis, which postulates that Broca's area in humans evolved from a basic mechanism not originally related to communication but rather from the mirror system for grasping in the common ancestor of monkey and human. As a result, the mirror system provides a possible ‘neural link’ in the evolution of human language. There is still much debate about the selection pressures that led to the evolution of language. Observing the overabundance of potential selective scenarios for why language evolved, the linguist Derek Bickerton voices his scepticism: ‘The fact that these and similar explanations flourish side by side tells one immediately not enough constraints are being used to limit possible explanations.’ One frequent source of confusion, he notes, is equating language with speech by not distinguishing between modality, lexicon, and structure. Hauser and Fitch share Bickerton's scepticism and urge scientists to rely more on the traditional comparative approach, which was always the strength of Darwinian evolutionary theory. Primatologist Robin Dunbar, who originally proposed that grooming (group bonding) could have provided the stimulus for language, dismisses two other possible scenarios—hunting and tool-making—as potential ecological contexts for the evolution of human language. Gestural origins are also dismissed in his theory, because gestural languages do not seem to develop spontaneously and also require a line-of-sight contact making them useless at night. Interestingly, Steven Pinker rules out both Dunbar's theory of grooming and Geoffrey Miller's theory of sexual selection, whereas Bickerton rules out grooming, gossip, mating contract, and Machiavellian intelligence as likely contexts for the origin of human language. Also under fire in the book is the idea that the human brain is somehow equipped at birth with a ‘universal grammar’ out of which all human languages later develop. Several authors try to provide alternatives to innate predispositions, such as the importance of function to categorization (Michael Tomasello) and the importance of cultural transmission to the structure of language (Simon Kirby and Morton Christiansen). Arbib explicitly questions the traditional Chomskyan theory of innate linguistic predispositions and argues that what humans have and had in the past is ‘language readiness’ rather than a fixed universal grammar. Neuroscientist Terrence Deacon also puts an alternative theory forward. According to Deacon, many of the language universals reflect semiotic constraints inherent in the requirements for producing symbolic reference rather than innate predispositions. Thus, neither evolved innate predispositions nor culturally evolved and transmitted regularities can be considered as the ultimate source of language universals. He draws a parallel with mathematical operations (addition, subtraction, etc.) and with prime numbers. Symbolic reference, he argues, is constrained by the structure it refers to. The editors claim, in the light of this diversity, that ‘this book is intended to bring together, for the first time, all the major perspectives on language evolution’. We have two concerns with this aim. First, two books of the same organization and scope have been published in the past six years based on the material from language evolution conferences ( Hurford et al. 1998 ; Knight et al. 2000 ). Although this first concern might be just splitting hairs, the second is more substantial: several crucial aspects of language evolution are not represented at all or are just touched superficially. One of these missing themes is the selective advantage of early language. As discussed, many of the contributors express their scepticism towards the selective scenarios found in the literature—and indeed towards such constructions in general—but there is no review and no balanced evaluation of these selective scenarios. Since one of the key questions of language evolution is the selective advantage of early language, the lack of such a review is a major weakness. A balanced account could have been presented even if the editors and most of the contributors are frustrated by the plethora of selective scenarios. Related to the possible selective advantage of language is the issue of genetic background. Although there is mention of the so-called FOX genes—some mutations of which are associated with language disorders—there is no detailed discussion of our current knowledge of genetics related to language. Another lightly treated theme is the neural basis of language and language evolution. Understandably it is one of the most difficult issues concerning human language, and no one expects the editors or any of the contributors to come up with an answer to all the questions. What is missing again is a good survey outlining the problems and the current findings of the field. The weaknesses of the book come from its structure and organization. The editors, instead of outlining a structure and asking specialists to contribute to that structure, appear to have let every contributor write freely about their current ideas and current research without regard to the bigger picture. This definitely shows the interests of the contributors and outlines the current state of the art; it leaves gaps, however, in the coverage of crucial topics related to the evolution of human language.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521730.xml
551599	Increasing stability of water-soluble PQQ glucose dehydrogenase by increasing hydrophobic interaction at dimeric interface	Background Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has a great potential for application as a glucose sensor constituent. Because this enzyme shows no activity in its monomeric form, correct quaternary structure is essential for the formation of active enzyme. We have previously reported on the increasing of the stability of PQQGDH-B by preventing the subunit dissociation. Previous studies were based on decreasing the entropy of quaternary structure dissociation but not on increasing the interaction between the two subunits. We therefore attempted to introduce a hydrophobic interaction in the dimeric interface to increase the stability of PQQGDH-B. Results Amino acid residues Asn340 and Tyr418 face each other at the dimer interface of PQQGDH-B, however no interaction exists between their side chains. We simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile, to create the two mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile. Furthermore, residues Leu280, Val282 and Val342 form a hydrophobic region that faces, on the other subunit, residues Thr416 and Thr417, again without any specific interaction. We simultaneously substituted Thr416 and Thr417 to Val, to create the mutant Thr416Val/Thr417Val. The temperatures resulting in lose of half of the initial activity of the constructed mutants were increased by 3–4°C higher over wild type. All mutants showed 2-fold higher thermal stability at 55°C than the wild-type enzyme, without decreasing their catalytic activities. From the 3D models of all the mutant enzymes, the predicted binding energies were found to be significantly greater that in the wild-type enzyme, consistent with the increases in thermal stabilities. Conclusions We have achieved via site-directed mutagenesis the improvement of the thermal stability of PQQGDH-B by increasing the dimer interface interaction. Through rational design based on the quaternary structure of the enzyme, we selected residues located at the dimer interface that do not contribute to the intersubunit interaction. By substituting these residues to hydrophobic ones, the thermal stability of PQQGDH-B was increased without decreasing its catalytic activity.	Background Water-soluble quinoprotein glucose dehydrogenase (PQQGDH-B) from Acinetobacter calcoaceticus has great potential for application as a constituent of an electron mediator-type glucose sensor. The conventionally utilized enzyme for glucose measurement, glucose oxidase (GOD), inherently utilizes oxygen as its electron acceptor during the oxidation of glucose. In contrast, PQQGDH-B is completely independent of oxygen, resulting in improved accuracy and rapidity during glucose measurement. However, because the substrate specificity and stability of PQQGDH-B remain somewhat inferior to those of GOD, we have been engaged in the improvement of these enzymatic properties through protein engineering to further increase the application of PQQGDH-B in glucose monitoring systems [ 1 - 17 ]. The subunit structure of PQQGDH-B was determined to be homodimeric with no activity observed in its monomeric form [ 17 ]. Correct quaternary structure is essential for the formation of active enzyme and dissociation of the dimer conformation triggers inactivation of the enzyme. We have previously reported on the increasing of the stability of PQQGDH-B against the dissociation of quaternary structure by chemical cross-linking [ 12 ], by constructing tethered enzyme [ 13 ], and by introducing Cys residues at the dimer interface to form a novel intersubunit disulfide bond [ 14 ]. All of these attempts were based on decreasing the entropy by decreasing the possibility of dimer dissociation, but no attempts had been made to increase the interaction between the two subunits. In this paper, we report on our rational designing of hydrophobic interaction in the dimer interface to increase the stability of PQQGDH-B. We identified protein regions at the dimer interface where potential novel hydrophobic interactions could be introduced by amino acid substitution, thereby increasing the stability. Results Modeling novel dimer hydrophobic core Among 19 amino acid residues located at the dimer interface, 8 residues were predicted not to be involved in the formation of hydrogen bonds, electrostatic interactions, or hydrophobic interactions (Fig. 1 ). We focused on the residues Asn340, Tyr418, Thr416, and Thr417, as they are not involved in the formation of the active site cavity and are therefore suitable candidates for amino acid substitution. Although residues Asn340 and Tyr418 face each other on the surface of the dimer interface (Fig. 2 ), no interaction exists between their side chains. We therefore simultaneously substituted Asn340 to Phe and Tyr418 to Phe or Ile to create the mutants Asn340Phe/Tyr418Phe and Asn340Phe/Tyr418Ile, respectively. Furthermore, the hydrophobic region composed of residues Leu280, Val282, and Val342 faces residues Thr416 and Thr417, again with no specific interaction. We therefore substituted Thr416 to Val and Thr417 to Val to create the double mutant Thr416Val/Thr417Val. Figure 1 The amino acid residues located at the dimer interface. The two subunits are represented in red and blue, respectively, using the RasMol molecular visualization software 26. The 19 amino acid residues at the interface are shown in space filling format, of which 8 residues (orange and light blue) are predicted not be involve in hydrogen bond formation, electrostatic interaction, or hydrophobic interaction at the interface. Figure 2 Hydrophobicity of PQQGDH-B dimer interface. Hydrophobic regions are shown in green and hydrophilic regions are shown in blue. Residues that have been substituted in this study are indicated. The structural images were generated using Molecular Operating Environment. Characterization of mutant enzymes The activity and stability of each of the three constructed double mutant enzymes were then analyzed. All three mutant enzymes showed slightly higher thermal stability than wild-type PQQGDH-B upon incubation for 10 min at various temperatures (Fig. 3 ). The temperatures resulting in lose of half of the initial activity were shifted by approximately 3°C higher in the mutants compared to the wild type (Table 1 ). As the time course of thermal inactivation at 55°C follows first-order kinetics (Fig. 4 ), half-lives were calculated using logarithmic regression of residual activity. The wild-type enzyme inactivates at 55°C with a half-life of 9.5 ± 1.4 min, while all three double mutants showed greater thermal stability, with half-lives of 5–6°C greater (Table 1 ). Figure 3 Thermal stability of wild-type and mutant PQQGDH-Bs. Residual activity was measured at 25°C after 10-min incubations at different temperatures of the following protein samples (0.075 μg/mL): Wild-type ◆, Asn340Phe/Tyr418Phe □, Asn340Phe/Tyr418Ile △, and Thr416Val/Thr417Val ○. Table 1 Kinetic parameters and thermal stability of PQQGDH-Bs. V max(U/mg) K m(mM) T h (°C) a t 1/2 (min) b Wild type 3030 20 53.9 ± 0.9 9.5 ± 1.4 Asn340Phe/Tyr418Phe 3100 20 57.7 ± 0.4 14.9 ± 1.1 Asn340Phe/Tyr418Ile 2500 20 57.5 ± 0.3 15.5 ± 1.5 Thr416Val/Thr417Val 2800 16 56.5 ± 0.9 14.8 ± 1.4 a T h represents the temperature at which half the initial activity is lost in 10 min. b t 1/2 represents the half-life at 55°C. Figure 4 Time course of thermal inactivation of wild-type and mutant PQQGDH-Bs. Protein samples (0.075 μg/mL) were incubated at 55°C and aliquots were taken at different times to measure residual activity. The analyzed samples contained the following PQQGDH-Bs: Wild-type ◆, Asn340Phe/Tyr418Phe □, Asn340Phe/Tyr418Ile △, Thr416Val/Thr417Val ○. Investigation of the kinetic parameters of the wild-type and mutant enzymes shows that the mutations did not significantly affect the overall kinetic properties of PQQGDH-B (Table 1 ). The specific activities of Asn340Phe/Tyr418Phe (3100 U/mg), Asn340Phe/Tyr418Ile (2500 U/mg), and Thr416Val/Thr417Val (2800 U/mg) were close to that of the wild-type enzyme (3030 U/mg). Except for Thr416Val/Thr417Val, which had a Km value of 16 mM, the mutants had Km values identical to 20 mM Km value of the wild-type enzyme. Discussion PQQGDH-B is a 6-blade β-propeller protein, with each blade consisting of a 4-stranded anti-parallel β-sheet (W-motif) [ 18 ]. The strands in each W-motif are labeled A-D from the inside to the outside of the molecule [ 18 , 19 ]. All strands are connected by loops, which are named according to the strands they connect. Based on PQQGDH-B structural information, these loop regions have been associated with a number of important functions, such as substrate binding, co-factor binding, and formation of the enzyme active site. As with other β-propeller proteins, the catalytic site and substrate-binding pocket of PQQGDH-B is made up of the cleft formed by loops DA and BC [ 20 ]. The enzyme surface composed of loops AB and CD is therefore located opposite the functional region. In the present study, we have introduced mutations at Asn340, Tyr418, Thr416, and Thr417, which are all located in the loop 5CD region. Considering that these residues are located far from the functional region and do not contribute in the structure of functional region, it is not surprising that their substitutions did not significantly alter the enzyme's kinetic parameters, particularly its catalytic activity. Based on the predicted structure of the mutant enzymes shown in Figure 5 , the binding energy of each subunit was calculated, using both the AMBER89 and CHARMM22 force fields, and compared with those of the wild-type enzyme (Table 2 ). As expected, the increases in hydrophobicity at the subunit interface resulted in increases in their binding energies calculated by both methods. Estimation of the number of hydrophobic interactions based on these same predicted models revealed 4 to 5 novel hydrophobic interactions at the interface of all the mutants, while none were found in the wild-type one. These results based on structural predictions are consistent with the observed improvements in thermal stability. Table 2 Predicted binding energy of each mutant subunit calculated using AMBER89 and CHARMM22 force fields. Binding energy (kcal/mol) AMBER89 CHARMM22 Wild type -249 -118 Asn340Phe/Tyr418Phe -291 -134 Asn340Phe/Tyr418Ile -273 -126 Thr416Val/Thr417Val -278 -131 Figure 5 Hydrophobicity of mutant PQQGDH-B dimer interface. Hydrophobic regions are shown in green while hydrophilic regions are shown in blue. The interfaces shown are those of the Asn340Phe/Tyr418Phe (left), Asn340Phe/Tyr418Ile (middle), and Thr416Val/Thr417Val (right) mutants of PQQGDH-B, with their respective mutation sites circled. The structural images were generated using Molecular Operating Environment. The addition of hydrophobic interactions in other enzymes has been reported to result in thermostability increases of 2 to 10°C [ 21 - 24 ], comparable to the results of the current study. Recent observations of enzymes from thermophilic organisms indicate that their extraordinary thermal stability is due to hydrophobic interactions. Oligomeric enzyme stability was also reported to be improved through engineering to increase the hydrophobic interaction at the oligomer interface [ 25 ]. However, these studies were based on homology analyses between mesophilic and thermophilic bacteria together with random mutagenesis library analyses [ 21 - 24 ]. Although some sequences have been found to be homologous to PQQGDH-B, these are all putative ORFs with no functional information reported. Therefore, there exists no reliable template to help improve the stability of this enzyme by increasing the dimer interface interaction. Conclusions We improved PQQGDH-B's thermal stability by increasing the dimer interface interaction through rational design based on its quaternary structure. We demonstrated that this can be achieved by selecting residues located at the dimer interface that do not contribute to the intersubunit interaction and substituting them to hydrophobic ones via site-directed mutagenesis. In each case tested, the enzyme's thermal stability was increased without decreasing its catalytic activity. This rational design approach will provide relevant information for future designs by combining with other mutant PQQGDH-Bs with narrowed substrate specificity and improved catalytic efficiency. Methods Chemicals Glucose, phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), and magnesium chloride were obtained from Kanto Kagaku (Tokyo, Japan), 3-(N-morpholino) propane sulfonate (MOPS) from Dojin (Kumamoto, Japan), and pyrroloquinoline quinone from Mitsubishi Gas Chemical Company (Tokyo, Japan). All other regents were of analytical grade. Kpn I was obtained from TOYOBO (Osaka, Japan) and Hin dIII from New England BioLabs (Beverly, USA). Site-directed mutagenesis The structural gene for wild-type PQQGDH-B was previously amplified by polymerase chain reaction (PCR) and inserted into the expression vector pTrc99A (Pharmacia) to create pGB [ 15 ]. A 1.2-kbp Kpn I- Hin dIII fragment containing the PQQGDH-B gene was transferred from pGB to pKF18k and mutagenesis was carried out with the Mutan-Express Km kit (Takara) according to the manufacturer's instructions with the oligonucleotides Asn340Phe (5'-GGTGGGACAAA GAA TTTACCAGTCC-3'), Tyr418Phe (5'-CGGTACAGCGTCATC AAA AGTAGTGC-3'), Tyr418Ile (5'-CGGTACAGCGTCATC AAT AGTAGTGC-3'), and Thr416Val/Thr417Val (5'-CAGCGTCATCATA AACAAC GCTATAAGTTGGATC-3'). The mutations (underlined) were confirmed by automated DNA sequencing (ABI PRISM Genetic analyzer 310, Applied Biosystems). The mutated genes were digested with Kpn I and Hin dIII and were replaced into pGB to construct expression vectors containing mutated PQQGDH-B. Numbering of the amino acid positions starts from the first residue of the signal peptide (24 residues). Enzyme preparation and assay The PQQGDH-B genes were expressed in Escherichia coli and the enzymes purified as previously reported [ 15 , 16 ]. Following a 30-min preincubation in 10 mM MOPS-NaOH (pH 7.0) containing 1 μM PQQ and 1 mM CaCl 2 at room temperature (25°C) to produce the holoenzyme, GDH activity was measured by using 0.6 mM PMS and 0.06 mM DCIP. The enzyme activity was determined by measuring the decrease in absorbance of DCIP at 600 nm. Analysis of PQQGDH-B stability The thermal stability of wild-type and mutant PQQGDH-B was determined with 0.075 μg/mL protein, as previously reported [ 15 ]. Thermal inactivation experiments were carried out by incubating each holoenzyme in 200 μL of 10 mM MOPS-NaOH, pH 7.0, at 55°C. Aliquots were taken every 5 min and kept at 4°C for 2 min, followed by incubation at room temperature for 30 min. The residual enzyme activity was determined as described above. Since the initial time course for thermal inactivation at 55°C followed first-order kinetics, the thermal stability of each mutant enzyme was expressed as a half-life. The thermal stability of Asn340Phe/Tyr418Phe, Asn340Phe/Tyr418Ile and Thr416Val/Thr417Val were also determined by incubating purified enzyme at various temperatures for 10 min. The residual activities were determined as described above, and were compared with the initial activities. Prediction of three-dimensional structure and quaternary-dimensional structures Three-dimensional and quaternary structures were predicted using Molecular Operating Environment (MOE) (Chemical Computing Group Inc., Quebec, Canada). By using the available PDB data of the wild-type PQQGDH-B, 1QBI [ 18 ], we made the appropriate substitutions with all possible side-chain orientations to predict the structures of the Asn340Phe/Tyr418Phe, Asn340Phe/Tyr418Ile and Thr416Val/Thr417Val mutants. After addition of hydrogen atoms to the PQQGDH-B structure and optimization of orientation of some hydrogen atoms by MOE, the structures were subjected to energy minimization using the AMBER89 or CHARMM22 force field within the MOE program until the final energy gradient was < 0.01 kcal/mol·Å. Authors' contributions ST carried out the site-directed mutagenesis of PQQGDH-B as well as the preparation and characterization of the resulting proteins. SI carried out the 3D modeling and participated in the design of the study. SF participated in interpretation of the results and in drafting the manuscript. KS conceived of the study, participated in its design and coordination, as well as in drafting the manuscript. All authors read and approved the final manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC551599.xml
368180	Microstimulation of Neurons Distinguishes Neural Contribution to Perception	null	The brain is an overwhelmingly complex organ packed with billions of nerve cells, performing a myriad of different functions. To decipher the roles of individual neurons in processing sensation or actions, scientists can measure the neural activity of animals that are shown particular objects or perform simple tasks. In this way, neurons are categorized as having preferences, also known as selective responses. These techniques have been particularly helpful in determining, or mapping, preferences of visual areas in the cerebral cortex. For example, some neurons respond to the color of an object, while others respond to the direction that object is moving. What is less well understood, however, is how the brain integrates information from individual neurons for complex processes such as perception and behavior. That is, how does neural activity affect what we see and do? The neural contribution to mokeys' perception of motion Microstimulation, a technique that activates a cluster of nerve cells by zapping them with a weak electrical current, has helped make causal links between neurons and behavior. For instance, when neurons in an area of the visual cortex that are “tuned” to a particular direction of motion are microstimulated, the way monkeys perceive moving dots on a video screen changes. Microstimulation seems to change what they see. Similar work has also been done for neurons that respond to binocular disparity—the depth-of-field information you gain because each eye has a slightly different view of the world. But many neurons respond, or are tuned, to more than one dimension, leading scientists to wonder how information from these multidimensional neurons contributes to perception—especially when some of that information is irrelevant to a given task. As they report in this issue, Gregory DeAngelis and William Newsome find that neurons tuned to both direction and binocular disparity contribute little to monkeys' perception of motion. The researchers asked three rhesus monkeys to determine the direction a group of dots was moving on a TV screen—a task that can be done regardless of the perceived depth of the dots. The authors had already located two different types of neurons in each of the monkey's brains: sites tuned strongly to direction and multidimensional sites tuned to both direction and binocular disparity. They then determined each site's exact preference: the direction of motion and degree of binocular disparity (if present) that triggered maximum neural activity. The researchers then showed the monkeys several sets of video displays, some with the dots moving in the “preferred” direction and some not. The microstimulation acts somewhat like adding dots in the preferred direction—which confuses monkeys when the real dots are moving against preference and aids them in trials of preferred moving dots. If the behavioral effect of microstimulation—whether it be a help or a hindrance—was significant, it meant that the monkeys were monitoring the activated neurons to perform the task at hand; if there was no change, the stimulated neurons were not being recruited. DeAngelis and Newsome hypothesized that multidimensional neurons (which are also tuned to the irrelevant dimension of binocular disparity) might be ignored during pure motion perception tasks. For two of the three monkeys, this was true. Microstimulation of multidimensional sites had no effect on their behavior, compared to the significant effect of microstimulation of direction-only sites. But for the third monkey, called monkey R, microstimulation of both types of sites had significant effects on his performance. He didn't seem to be ignoring anything. The authors proposed that the monkeys could be using different neural strategies to complete the same task. This conclusion is supported by the fact that monkey R performed better on the task than the other monkeys; he appeared to be recruiting any neuron with applicable information, unlike the others, who seemed to rely on neurons tuned solely to direction of motion. Furthermore, for the few multidimensional sites that affected behavior, their contribution was tempered by how well the depth, or disparity, of the video matched the preference of the stimulated neurons. The results of this paper show that even if neurons carry information that can aid in perceptual decision making, they may not participate, depending on how they are tuned along other (irrelevant) stimulus dimensions. All directional neurons are not created equal—some are more useful than others for a particular task. Whether neurons that respond to a particular stimulus contribute to the task at hand depends on how closely that stimulus hews to the neurons' preference as well as on the subject's learned strategy for performing the task. This neural flexibility, the authors point out, suggests that the brain uses complex, variable strategies to respond to changing environmental stimuli. Techniques like microstimulation will be helpful in drawing the connections between neural activity and behavior.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC368180.xml
538275	Public sector reform and demand for human resources for health (HRH)	This article considers some of the effects of health sector reform on human resources for health (HRH) in developing countries and countries in transition by examining the effect of fiscal reform and the introduction of decentralisation and market mechanisms to the health sector. Fiscal reform results in pressure to measure the staff outputs of the health sector. Financial decentralisation often leads to hospitals becoming "corporatised" institutions, operating with business principles but remaining in the public sector. The introduction of market mechanisms often involves the formation of an internal market within the health sector and market testing of different functions with the private sector. This has immediate implications for the employment of health workers in the public sector, because the public sector may reduce its workforce if services are purchased from other sectors or may introduce more short-term and temporary employment contracts. Decentralisation of budgets and administrative functions can affect the health sector, often in negative ways, by reducing resources available and confusing lines of accountability for health workers. Governance and regulation of health care, when delivered by both public and private providers, require new systems of regulation. The increase in private sector provision has led health workers to move to the private sector. For those remaining in the public sector, there are often worsening working conditions, a lack of employment security and dismantling of collective bargaining agreements. Human resource development is gradually being recognised as crucial to future reforms and the formulation of health policy. New information systems at local and regional level will be needed to collect data on human resources. New employment arrangements, strengthening organisational culture, training and continuing education will also be needed.	Introduction This paper considers health sector reform and its impact on human resources for health (HRH) in developing countries and countries in transition. Health sector reform has been defined as the "sustained purposeful change to improve the efficiency, equity and effectiveness of the health sector" [ 1 ]. Health sector reform involves many fundamental changes to the way in which public services are financed, organised and delivered in both developing and developed countries, and often operates as part of a wider programme of public sector reform. Fiscal reform, the introduction of market mechanisms and decentralisation are three key elements of health sector reform. This paper will show the impact of these elements on human resources for health and attempt to assess the changing demand for health workers. A series of recommendations will seek to address some of the issues that have emerged for HRH demand during the process of health sector reform. Impact of health sector reform on human resources for health (HRH) Fiscal reform The introduction of new budget management systems, designed to maintain financial control throughout government, is one of the most important elements of fiscal reform. These incorporate new financial planning and control systems that emphasise what outputs a department or agency will be expected to deliver. Overall, there is a focus on the performance of public services [ 2 ]. Mechanisms for monitoring and enforcement of targets are designed for all government departments. In the health sector, over 50% of costs are labour costs, so that demonstrating effectiveness depends largely on attempting to measure the work of health staff. Measuring outputs in health care is often difficult because of having to capture both the quality of care and patient outcomes. Fiscal reform introduces new ways of allocating resources in line with government objectives [ 3 ]. There may not be a precise match with individual sectoral objectives. Fiscal reform also tries to encourage improved use of resources, which may inform the reorganisation and management of central agencies and the downsizing of the civil service. Health sector employees are often part of the civil service, and so civil service reform has an impact on the employment and deployment of health workers. Civil service downsizing results from policies to cut the costs of the public sector and transfer the delivery of services to the private or non-profit sectors. These changes lead to a reduction in the size of the public health care workforce. Compensation schemes may include retraining and lump-sum severances to ease the transition of workers into the private sector. This may be accompanied by wage policy reform to limit and contain wage expenditures, again with the potential to affect health workers [ 4 ]. Trying to improve the performance of the health sector, one of the objectives of health sector reform, has been a slow process because the savings from reducing the size of the workforce are often not enough to raise salaries for the remaining staff. Several countries, including Zambia, have set up a separate health service agency, operating as a semi-autonomous government agency, that employs staff directly. Some writers argue that agencies need to be well-managed on a limited budget rather than be seen as an escape from civil service restrictions [ 5 ]. In addition, "the importance of political and institutional context in which reforms have to be implemented has been undervalued" [ 5 ]. Many ministries have a poor record of human resource management and planning. New information systems can provide a more accurate picture of the current number, type and distribution of staff, but civil service systems rarely provide incentives to reduce staff budgets. There may also be attempts to strengthen linkages between government departments, which are relevant for the health sector. The decentralisation of budget management is another element of fiscal reform. In the health sector, this has been reflected in the decentralisation of service provision to semi-autonomous hospitals, because hospitals often consume the largest part of the health sector budget [ 6 ]. Set up as institutions run on business principles, "corporatised" hospitals bring the results of fiscal reform to local level. With limited available resources, there may be pressure to generate income through the introduction of user fees such as for health services, as well as trying to achieve outputs and outcomes at the lowest cost. Delegation of financial authority can also provide managers with the scope to use existing resources differently or consider different ways of delivering services, such as by using a range of local providers [ 2 ]. The motivations for introducing corporatisation may also vary from wanting to increase efficiency and achieve cost saving and quality improvements to just wanting to "free up a public function from constraints of ... red tape" [ 6 ]. Cassels argues that decentralisation has provided "major contradictions for health care" between accountability, competing priorities and equity and tensions between small-scale participation and managerial effectiveness required at a large scale [ 5 ]. In Mexico, new systems of financial management affected public health institutions by restricting the maintenance and upgrading of equipment and imposing cuts in the wages of health workers. This has led to the deterioration of working conditions and the quality of care provided by the public health sector [ 7 ]. Financial management Part of a programme of fiscal reform involves the development of new systems and structures of financial management, which have organisational implications [ 3 ]. The role and functions of the finance ministry in central government are strengthened and it develops a dominant role over other government departments. This affects government health ministries, because the priorities of the finance ministry are often different from those of the health ministry and priority setting and resource allocation issues become sources of conflict. The nature of the relationship between the finance and health ministries has been exposed in the development of poverty reduction strategy papers [ 8 ]. In health systems, new forms of financing for the health sector may involve moving from a tax-based system to an insurance system. This, in turn, introduces new forms of budgetary management and control between insurance funds and health service providers as well as new systems of payment collection. Financial management is often accompanied by new information technology systems, which have the potential to change the ways in which public services are monitored [ 2 ]. Market mechanisms The introduction of market mechanisms is often driven by the goal of fiscal stability. Stronger systems of budget and management control are introduced that focus on results. They affect sectoral priorities and available human resources. Market mechanisms may be introduced by making health care institutions operate within an internal health care market and subjecting some health services to wider market testing. As part of developing a managed market in the public sector, the health sector is often reorganised into two separate purchasing and provider functions. The purchasing entity, typically a national or regional health authority, buys services from provider units within the government sector and is also encouraged to buy services from a range of providers in the private and NGO sectors [ 9 ]. This has immediate implications for the employment of health workers in the public sector, because the public sector may reduce its workforce if services are purchased from other sectors. The private sector may start to expand. Health workers often move from the public to the private sector because of better prospects and higher pay. Market testing has often led to changes in the size of the public sector workforce, increasing short-term and temporary employment contracts, and changes in wage levels [ 10 , 11 ]. User fees User charges have been introduced as a way of generating income for the health sector. User fees in many countries have affected access to services and equity [ 12 , 13 ]. In Nicaragua, the introduction of user fees and separate services for private, paying patients started as a national initiative but is now incorporated into local health systems. User fees have become the main source of decentralised revenue. At hospital level, 30% goes towards salary supplements [ 14 ]. In Honduras, the revenues collected from user fees have contributed only 2% to the Ministry of Health expenditures but the administrative costs are 67% of the revenues collected [ 15 ]. In most countries, however, the preparation of staff and supporting systems for implementing user charges has been minimal. The introduction of user fees places new pressures on health workers, especially when user fees contribute to the actual wages and salaries of health workers. A recent World Bank report (2002) presents informal payments as a hindrance to health sector reform. Payments made to health workers are considered to draw resources away from the health care system because they are given to individuals rather than institutions. Such payments operate as a private, unregulated system and the practice is often illegal. Poor people often avoid using health care facilities because of the need to make informal payments [ 16 ]. Stronger management capacity is needed to support and coordinate public, private and NGO providers and provide accountability so that revenue from user fees goes directly for service improvements [ 17 ]. This would also depend on health workers' being paid an adequate salary and the introduction of transparent systems to support the collection of user fees within the health care sector. Performance management Public sector and health sector reform often introduce new approaches to managing staff. Perhaps the most important innovation is "thinking differently about staff", which effectively underpins other changes. The three most innovative dimensions are "flexible staffing and recruitment practices, recognising achievement and developing performance contracts" [ 2 ]. The element of fiscal reform that emphasises outputs and outcomes of government services informs the development of performance management. It aims to address management problems relating to poor employee performance management, wage and non-wage incentives, job classification systems and ineffective payroll and personnel systems. Performance management may also be introduced as a way of improving standards within public services and making services more responsive to citizens. Wider programmes of training and capacity building for staff can accompany this. Some developing countries have experimented with performance management systems, with limited success [ 9 ]. Often the new "corporatised" hospitals have only limited management autonomy, and governments lack the capacity to manage performance in the health system [ 9 ]. Decentralisation The delegation and decentralisation of administrative and management processes often accompany budgetary reforms. In Nicaragua, decentralisation was used to introduce market reforms. Budget cuts, loss of resources from primary health care, user fees and privatisation were introduced at the same time [ 14 ]. In 1991 Local Integrated Health Care Systems (SILAIS) were introduced, which are made up of a hospital and a network of primary care units. Each SILAIS has a separate Board of Directors consisting of local officials, church officials, health sector representatives, community members and the SILAIS director. This group monitors services and approves the local health plan and budget, but accountability remains unclear. The Ministry of Health controls funding through "performance agreements, and controls 80% of the health budget including staff levels and composition". Only recently have Local Health Systems been given the power to sack staff [ 14 ]. In Uganda at the time of decentralisation, salaries for staff on the payroll were a central responsibility, although this has now been decentralised through a special conditional grant. In the past, professional staff were put on the national payroll and nursing aides were hired locally for work in rural health centres and health posts and paid for by the Ministry of Local Government. After the decentralisation reforms, nursing aides were supposed to be paid by local committees, but in practice this often did not happen and they were not paid for long periods [ 18 ]. Botswana and Tanzania have had long experience of decentralisation. As a result of health sector reform, health staff were transferred to local government contracts although senior staff remained employed by the Ministry of Health. This has led to confused loyalties and management responsibilities. In some districts the "personality factor" has meant that individuals working together have managed to overcome some of these problems, in spite of the systems introduced. Senior staff who have subsequently been transferred to local government complain that that there is "little relationship between promotions/disciplinary actions and performance". In both countries there is some scope for local decision-making in relation to personnel management, but there is still resistance to distributing staff according to local needs. More incentives and other measures are considered necessary if regional imbalances of staff are to be addressed [ 17 ]. Decentralisation may lead to a loss of resources for the health sector. In Uganda, after decentralisation, once central government stopped a block grant, primary heath care was not given the allocation at local level that had been expected by the Ministry of Finance. There were also considerable district variations in the allocation of health resources. Although some districts did increase their health allocation, in many cases decentralisation led to fewer resources for health. One of the reasons cited for the decline in allocation of resources to the health sector was that a large part of the health budget goes on salaries and wages, which do not show any dramatic change in the sector. Decentralisation in this context led to problems of financial management and corruption at local level, new problems of governance with a lack of accountability and concerns over quality of services [ 18 ]. Some changes have run contrary to the main aims of reform, such as increased centralisation of controls over pay. Much health sector reform was to strengthen and rationalise budgeting, financial control and staff classification, but in some cases control over health sector staffing has remained at national level [ 19 ]. Even when transfer of budgets has taken place, there is confusion between local government and health sector responsibilities. Changes in provision The use of the private sector as a health service provider has had implications for the recruitment and retention of staff in the public sector. Some services have been privatised and are now run by local, national or international private companies. Other services have been contracted out to both private and non-profit service providers. This has resulted in movement of health workers from public to private or non-profit sectors [ 17 ]. In the public institutions that remain, market conditions have been introduced and services are contracted out, which has resulted in a widespread decrease in job security in many countries. Health workers have moved from collective-bargaining arrangements to individual contracts. Decentralisation and privatisation have contributed to the breakdown of national collective bargaining. In Eastern and Central Europe, new organisations and professional associations and reorganised trade unions have led to a breakdown in labour relations expertise [ 20 ]. Changes of responsibility for managing health services, from national to local level and from public to private sectors, have led to some confused accountabilities for health workers [ 11 ]. Health workers have moved from being accountable to both a public service and to their profession, to being accountable to a commercial employer with performance-related pay and conditions. This often causes tension between professional standards and pressure from the commercial employer. The process of health sector reform has had an impact on human resources for health through new systems of financial and performance management, decentralisation and the introduction of market mechanisms. This has led to changes in the demand for health workers and in some cases the types of skills and expertise required from health workers. At the same time, the capacity of the new management systems is unable to create conditions in which a new health workforce can be developed. This can be seen particularly clearly in the process of budget decentralisation, which leads to a focus on local decision-making but where the capacity of local institutions to recruit, train and manage local health service workers is limited. This has an influence on the quality of health care delivered. Implications for HRH demand Demand for human resources in health systems that have experienced health sector reform must be considered in terms of the numbers of health workers and the skills and expertise needed currently and what will be anticipated in the future. There is a growing awareness that human resource issues need to be prioritised more effectively within reforms in order to secure an adequate health care workforce to deliver services now and in the future. Public sector culture Although health sector reform has included elements of human resources strategies such as improved education and training, restructured salary scales and a closer link between performance and reward, it has also had a fundamental impact on organisational culture and public sector ethos, which in turn influence demand for human resources. A study of four countries in Eastern and Southern Africa concluded that "human resource development, personnel management and staff motivation are critical issues" [ 17 ]. Tanzania, although it has invested in human resources development, found that low salaries, delayed promotion opportunities and poor working conditions led to dissatisfaction in the workforce. Staff performance has been found to be unsatisfactory. Although monetary and non-monetary allowances were supposed to compensate for low wages, they have led to poor teamwork and lack of continuity in health service operations. The regional health team was found to spend 40% of its time out of the region on training and meetings. Burkina Faso introduced health sector reforms in 1991 but they have not been fully implemented. It has recently introduced civil service reform, which "aims at a more flexible management and better performances of personnel". In a country where services are centralised, with an imbalance in personnel and low staff motivation and poor standards of care, there is resistance to the new reform. There has been a decline in standards of service between 1986 and 1997 [ 21 ]. Poor financial and human resources policies and management are resulting in high cost and poor quality of care. A recent study concluded: "Human resources should become the central focus for reform" [ 21 ]. Matheson (2002) points out that "the least systemically orientated area of recent public management reforms has been human resource management.... There is a danger that the constitutional, legal, cultural and leadership factors, which together create what is important and distinctive about public services, are not reflected on, or are dismissed as the bureaucratic problem which must be 'reformed' " [ 22 ]. Demand for health workers Some national health sector reforms reduced the numbers employed in the health sector, as in Chile and Latvia. Others, as in Mexico and Zambia, led to a rise in employment [ 11 ]. In almost all countries, health worker employment was restructured. Health sector reform has often aimed to restructure organisations to reduce costs and the power of the workforce. Pressure of work and hours worked have in many cases increased since health sector reform. There is also an increased workload due to lack of staff, pressure for results and staff reductions [ 10 , 11 ]. This affects the future demand for health workers. Health services have usually been seen as "essential services" and so health workers had the legal status of public servants. They had to account to both employers and professional bodies subject to strict regulation and registration rules. The effect of privatisation has been to change the pay and terms of employment and the legal status of health workers [ 11 ]. The public health sector has changed from being a public service to one with a greater commercial focus. This may have an effect on recruitment. The Nicaraguan government has continued to cut the number of doctors, changing to an hourly rate of reimbursement rather than salaries, and ending the commitment of government to employ graduating medical students [ 14 ]. This process is effectively influencing the demand for doctors in the public sector. The introduction of flexible contracts and fall in full-time permanent contracts has been a characteristic of most reforms, leading to a reduction in long-term employment security. Some of these changes in terms and conditions have led to health workers' taking on second jobs. This may be caused by the increase in part-time employment and low and erratically paid wages [ 20 ]. In Eastern and Central Europe, women have been most affected by the reduction in jobs in the heath sector. Their prospects for redeployment are often limited due to a lack of mobility [ 11 ]. The growth of part-time work in the public sector is a sign of the changing demand for health workers. Low pay levels have led to staff leaving the public sector and moving to the private sector, NGOs and aid agencies [ 9 ]. Low pay also contributes to low administrative capacity, as well as poor organisational discipline. In an analysis of health worker motivation, health sector reform was found to influence health worker motivation through changing organisational structures and community-client roles [ 23 ]. Organisational factors influence worker motivation through management structures and processes, communication processes, organisational support structures and processes, and ways of providing feedback about organisational and individual performance. These changes in organisational culture have often had a negative impact on workers' motivation. Important informal factors – for example, staff commitment – have "become the prime means of direction, motivation, coordination and control" [ 22 ]. When staff commitment deteriorates over time, health workers may migrate, not only from the public sector to the private sector, but internationally. This results in a shortage of skilled health workers within the public sector, precipitating a growing demand for skilled health workers. The aim of introducing market mechanisms to the public sector has been to improve economic efficiency. New skills are needed to implement commissioning and contracting of services. For example, contracts can be a powerful form of regulation if drawn up and monitored effectively, but increased expertise is required to establish this form of regulation. Process of reform Understanding the process of reform is important for understanding how changes have taken place but also what the critical factors are for successful policy implementation in future. "The process of reform offers numerous opportunities to alter the political equations that impede change" [ 19 ]. This is also significant for understanding the potential role that health workers can play within reforms. In Latin America, health sector reform has been characterised by various forms of privatisation, competition among providers, new insurance systems, management autonomy for hospitals and increased community participation. The goals were efficiency, accountability and improved quality of services. A recent study looked at how groups, such as unions, play different roles in relation to reforms with some opposing and others supporting reform. Some public sector health workers have played an important role in supporting change [ 24 ]. The importance of "principled agents" in public sector organisations has also been noted [ 25 ]. Public servants can be motivated by managerial and incentive schemes to lead and support change. Linking popular and unpopular reforms has often led to reformers' changing their attitudes to reform. Networks of reformers can also play a role in supporting reformers in environments hostile to change [ 19 ]. Communities also influence health worker motivation through their expectations of services. As health sector reform also aims to empower service users, this focus will have a significant impact on the individual health worker in future [ 23 ]. The evidence showing the extent to which users of health services have been empowered by health sector reform is limited in developing countries [ 13 ]. However, health workers do play an important role in the implementation of health sector reform policies. The lack of consideration of the value of human resources in health sector reform programmes has meant that this has often been ignored. Gilson et al. (2003) recommended that: "Technical analysts might consider working with middle managers and health workers to ensure adequate consideration is given to implementation realities in proposal development" [ 12 ]. Knowledge gaps The process of health sector reform is not complete. More research is needed to monitor changes still taking place as well as the outcomes of the reforms. One of the major changes is the role that health workers play in both the public and private sectors. How health workers perceive their roles in the different sectors and what the implications are for motivation, particularly in the public sector, will need further exploration. The role of the public sector is changing and this is reflected in public sector institutions and the public sector ethos. How this changed public sector can demonstrate a commitment to health workers as well as harnessing their own commitment, still needs to be explored. A further area of research needs to improve the understanding of human resources in health sector appraisal studies "by incorporating functional, institutional and policy dimensions. Only then will human resources become in practice the most valuable resource within any national system" [ 26 ]. Conclusion There is a growing awareness that human resources for health (HRH) must be addressed more effectively within public sector reform. Stein thinks that HRH strategies need to be a "primary objective for public organisations" [ 27 ]. Public sector reforms have sometimes been characterised as containing the paradox of aiming to reward performance and empower staff whilst at the same time implementing downsizing and redundancy – the "human costs of reform" [ 28 ]. Issues resulting from these changes include loss of institutional memory and the use of downsizing as a way of making financial savings rather than administrative reform. Changing rules and processes do not always lead to changes in organisational culture. "Multi-faced interventions sustained long enough to achieve change" will be needed to change public sector culture [ 13 ]. To address some of these issues, action at strategic, regional and local levels will be needed to strengthen skills, expertise and analysis of HRH and to strengthen the integration of HRH issues into health policy making and with relevant agencies [ 26 ]. New human resources systems are needed at regional and local level. Additional data on existing employment, retention and deployment issues must be made available to decision makers so as to relate them to health equity issues [ 29 ]. An increased awareness of the importance of improved coordination of facility planning and human resource planning at national and local level is needed. A better understanding of the role of organisational culture and public sector ethos in health worker motivation is needed [ 23 ]. This might be achieved by developing case studies of health workers as "drivers of change" and more process research to look at emerging practice. The working conditions of health workers need to be improved. This might be achieved through developing more flexible employment arrangements that are employee-focused. The public sector needs to be encouraged to establish a "living wage" and other forms of worker security so that terms and conditions of public sector workers are better than those of private sector workers [ 26 ]. Health workers need to have access to continuous professional development that includes skills for performance management, management of contracts and other new ways of operating in reformed systems [ 29 ]. The role of central government in setting standards for professional practice and legal requirements for registration needs to be strengthened so that human resources policies, registration and regulation are mutually supportive. Registration requirements that include experience in rural or remote areas would help to address uneven distribution of health workers. Declaration of competing interests The author(s) declare that they have no competing interests.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC538275.xml
406402	Niche Markets	Is evolutionary theory is incomplete and are we failing to understand phenomena as disparate as ecosystem development and the interplay of genes and culture in shaping human evolution?	This book, the latest in the excellent Monographs in Population Biology series from Princeton University Press, is a work of advocacy in which the authors argue that evolutionary theory is incomplete and that, in consequence, we are failing fully to understand phenomena as disparate as ecosystem development and the interplay of genes and culture in shaping human evolution. What we are missing, they argue, is an appreciation of niche construction, the process by which an organism modifies the abiotic and biotic environment in which it is subject to natural selection. The authors' major assertion is that the importance of niche construction is so great that it should be regarded “after natural selection, as a second major participant in evolution” and that it is “not just an important addition to evolutionary theory” but “requires a reformulation of evolutionary theory”. Bold claims indeed. After introducing the conceptual framework Odling-Smee et al. set out a series of arguments to support this position, the first of these being the empirical case for the existence of niche construction. Niche construction, as broadly interpreted here, is everywhere. Animals build nests, burrows, and protective cases and so alter the environment they experience in a way that may select for further adaptations. The changes caused by some animal species, such as beavers and earthworms, are of a sufficient magnitude that the environment experienced by a host of other species is affected. Many plant species also modify the environment they experience by generating organic litter; influencing hydrological and biogeochemical cycles; affecting temperature, humidity, and light regimes; and, over the longer term, determining the make up of the atmosphere. Decomposer and chemoautotrophic microorganisms similarly influence biogeochemical transformations, while parasitic species can manipulate the behaviour and internal environment of the hosts they infect. Perhaps less obvious examples of niche construction are the many types of migration and cultural evolution that, like physical transformations, cause the organism's descendants to experience a different selective environment. After this broad, accessible survey, the authors change key rather abruptly and explore two-locus, frequency-dependent population genetics. The novelty here is that selection on one locus depends on the history of gene frequencies at the other, “niche construction”, locus. In an extension, gene frequencies at one locus affect an environmental variable with its own dynamics that in turn influences the second locus. As one would guess, the models display a range of potentially interesting dynamics, though generalisations and broad conclusions are sparse. We guess the aim of the chapter is to illustrate that environmental feedbacks can be potent and general agents of evolutionary change, but the restriction of the theory to such a narrow model, with very technical explanation, risks losing the few readers who we suspect will stay the course (did we really need a rederivation for haplodiploids?). Perhaps aware of the dangers of getting bogged down in detail, the argument then moves to proving a case for the universality of niche construction. Invoking the second law of thermodynamics and Maxwell's Demon, the authors lead us through a challenging thesis that concludes that the persistence of life on earth requires both natural selection and niche construction, thereby justifying some of the bold claims for their new theory. We think they are technically correct, but we are concerned that the demonstration of the inescapability of niche construction, as defined here, does not guarantee that it will actually tell us new and important things about the world, as the theory of natural selection has. The remainder of the book explores the implications of niche-construction thinking for evolutionary biology, ecology, and the human sciences, and in our view is the most successful part. Though it is rare for the authors to offer new analysis and insight, their sideways look at many issues from the niche-construction viewpoint often offers interesting new angles on old problems, and suggests new avenues of enquiry that may be the book's greatest legacy. A good example of this is their convincing and timely argument that a more explicit recognition of evolution's role in environmental feedbacks will help to unify population/community ecology and ecosystem science. The chief argument for the prosecution is that niche construction is common but not pervasive, and that wherever ecologists and evolutionists have found interesting examples of it, they have developed appropriate theory and concepts to understand its ramifications. For example, some of the clearest examples of niche construction occur in plant succession where, as F.E. Clements realised nearly a hundred years ago, early-succession plants frequently modify their environment in ways that allow other species to replace the pioneers. Interestingly, the strict Clementsian theory of facilitation, niche construction avant la lettre , has given way to a more pluralistic theory of succession. It is a great pity that the authors give so little space to plants and plant ecology, as it here that some of the finest examples of niche construction are found, as well as the best-developed conceptual framework for studying the roles of environmental feedback. Other areas where biologists have well-developed theories of the influences and impacts of niche construction include co-evolutionary theory, where the environmental feedbacks are largely biotic, and Dawkins' theory of the extended phenotype. Very close to some of the arguments discussed here, and generously acknowledged, is the idea developed by Jones, Lawton, and colleagues of ecosystem engineers, species that have a major impact on the abiotic environment experienced by a large number of species. A major strength of the book is that it reveals common processes and patterns underlying disparate biology in consistently interesting ways. Its chief contribution is thus not to tell us new things about how nature works but to link together many different aspects of ecology under an umbrella of theory that may in the future lead to new insights. Do they deliver on their grand claims? Time will tell, but our view is that they don't. They engagingly admit that for their project to succeed the new theory must earn its keep by producing significant new biology—something which has yet to occur. However, the great need for the biological and human sciences to integrate across subdisciplines, as the authors bravely attempt here, makes this a hugely worthwhile book. Its breadth of scope and its boldness in creating syntheses have resulted in a stimulating and challenging read.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC406402.xml
529436	Information assessment on predicting protein-protein interactions	Background Identifying protein-protein interactions is fundamental for understanding the molecular machinery of the cell. Proteome-wide studies of protein-protein interactions are of significant value, but the high-throughput experimental technologies suffer from high rates of both false positive and false negative predictions. In addition to high-throughput experimental data, many diverse types of genomic data can help predict protein-protein interactions, such as mRNA expression, localization, essentiality, and functional annotation. Evaluations of the information contributions from different evidences help to establish more parsimonious models with comparable or better prediction accuracy, and to obtain biological insights of the relationships between protein-protein interactions and other genomic information. Results Our assessment is based on the genomic features used in a Bayesian network approach to predict protein-protein interactions genome-wide in yeast. In the special case, when one does not have any missing information about any of the features, our analysis shows that there is a larger information contribution from the functional-classification than from expression correlations or essentiality. We also show that in this case alternative models, such as logistic regression and random forest, may be more effective than Bayesian networks for predicting interactions. Conclusions In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology (GO) functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed by Jansen et al . Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. We also found that the data discretizations used in the Bayesian methods decreased classification performance.	Background Proteins transmit regulatory signals throughout the cell, catalyze large numbers of chemical reactions, and are crucial for the stability of numerous cellular structures. Interactions among proteins are key for cell functioning and identifying such interactions is crucial for deciphering the fundamental molecular mechanisms of the cell. As relevant genomic information is exponentially increasing both in quantity and complexity, in silico predictions of protein-protein interactions have been possible but also challenging. A number of techniques have been developed that exploit combinations of protein features in training data and can predict protein-protein interactions when applied to novel proteins. Our study is motivated by a study by Jansen et al . [ 1 ], who proposed a Bayesian method to use the MIPS [ 2 ] complexes catalog as gold standard positives and lists of proteins in separate subcellular compartments [ 3 ] as gold standard negatives. The various protein features considered in this method include time course mRNA expression fluctuations during the yeast cell cycle [ 4 ] and the Rosetta compendium [ 5 ], biological function data from the Gene Ontology [ 6 ] and the MIPS functional catalog, essentiality data [ 2 ], and high-throughput experimental interaction data [ 7 - 10 ]. The MIPS and Gene Ontology functional annotations are used for quantifying the functional similarity between two proteins. The MIPS functional catalog (or GO biological process annotation) can be thought of as a hierarchical tree of functional classes (or a directed acyclic graph (DAG) in the case of GO). Each protein is either a member or not a member of each functional class, such that each protein describes a "subtree" of the overall hierarchical tree of classes (or subgraph of the DAG in the case of GO). Given two proteins, one can compute the intersection tree of the two subtrees associated with these proteins. This intersection tree can be computed for the complete list of protein pairs (where both proteins of each pair are in the functional classification), and thus a distribution of intersection trees is obtained. Then the "functional similarity" between two proteins is defined as the frequency at which the intersection tree of the two proteins occurs in the distribution. Intuitively, the intersection tree gives the functional annotation that two proteins share. The more ubiquitous this shared functional annotation is, the larger is the functional similarity frequency; the more specific the shared functional annotation is, the smaller is the functional similarity frequency. The essentiality data represents a categorical variable that denotes whether zero, one or both proteins in a protein pair are essential. The supplementary online material of [ 1 ] provides more details about the quantification of these variables. Their Bayesian method predicts protein-protein interactions genome-wide by probabilistic integration of genomic features that are weakly associated with interactions (mRNA expression, essentiality and localization). The model was used for two separate predictions of probabilistic interactomes (PI), one of which (PIE) is built on four high-throughput experimental interaction data sets, and the other (PIP) on the mRNA expression, Gene Ontology, MIPS functional and coessentiality data. Within the PIP sub-network, different genomic features are assumed to be independent in prior. In addition, this method involved discretizing the raw data into groups and representing the two mRNA expression profiles (cell cycle and Rosetta compendium data) by their first principal component for computational convenience. Our current study focuses on assessing the contributions of different types of genomic data towards predicting protein-protein interactions. This may help us to understand which genomic features have the closest biological relationship with protein-protein interactions and hence to construct a better prediction model. As prediction rules involving less relevant information may have lower prediction accuracy, our analysis can give us insights into how to construct more parsimonious models with comparable or better prediction accuracy. A potential disadvantage of the Bayesian network approach may be that the data discretization can obscure information contained in the raw genomic data. Thus, in addition to assessing the information content of the data sources, we also propose alternative non-Bayesian models that fully utilize the data without discretization. These methods, such as logistic regression and random forests, do not require prior knowledge, and we can evaluate the importance of the different genomic features in the context of these methods. Results and discussions To accurately and quantitatively assess the information contributions of different genomic features, we construct in essence a simplified problem that has some but not all of the elements of the original study. Here, we only look at a subset of the data from [ 1 ] comprising the 18 million protein pairs in total and approximately 8,000 gold standard positives and 2.7 million gold standard negatives. This subset (see Additional File 1 ) contains 2,104 positives and 172,409 negatives. In this subset, we have complete information for each feature and we can thus quantitatively assess the relative contributions of the different features on this set. This data set can be downloaded from . In doing so, we find that some of the features have stronger influence on the overall prediction. While this might be true for the larger problem as well, there are a number of caveats that one has to keep in mind, such as that the features that are present in this subset might not be the strongest in the whole set of 18 million protein pairs. Alternative models Here, we construct models for predicting protein-protein interactions that, given the gold standards, are basically dichotomous classifiers. Multiple logistic regression [ 11 ] is one commonly used model for such an application [ 12 , 13 ]. An alternative, more sophisticated supervised learning approach that we apply is the random forest algorithm [ 14 ]. Note that, although not our focus here, all these methods can be used to compute the estimated probabilities for predicted protein-protein interactions. Logistic regression The logistic model has the advantage that it provides an estimated probability that a pair of proteins interact, and is readily available in standard statistical packages. In this paper, the logistic regression analysis was generated using PROC LOGISTIC in SAS/STAT software, Version 9 of the SAS System. Moreover, we can evaluate the importance of different genomic features by variable selections. Among many available schemes, we chose stepwise variable selection that is widely used in standard packages. Stepwise selection is a greedy search algorithm that selects variables with the best marginal prediction power given the current model. To quantify the importance of the predictor variables to the model fitting, we can use the deviance measure -2(log L 1 - log L 0 ), where L 0 is the likelihood of the final model given by the stepwise selection, and L 1 is the likelihood of the reduced model by removing all terms that involve the corresponding predictor variable from the final model. However, this measure only considers the prediction power of variables for the training sample but not for any random test samples. Therefore, this measure can be biased due to its dependence on the training sample. We consider, similarly as in [ 1 ], all the main effects and interaction terms among the genomic features in the PIP (indirect evidence for protein-protein interactions) and the PIE (direct experimental protein-protein interaction measurements) respectively. Table 1 presents all the terms remained in the final model and their orders to enter the final model. Table 2 shows the deviance measure of predictor variables. The Gavin data, Gene Ontology and MIPS functional similarity features, and the cell cycle gene expression data are the most important genomic evidences for predicting protein-protein interactions according to the deviance measure, whereas the three other high-throughput experimental data sets are less relevant or even do not have significant effects to be included in the final model. However, the logistic model is restricted by its linear form and may not provide an optimal solution to the prediction problem. And it will be more objective to evaluate the variable importance according to its prediction accuracy for any random test samples. In the following, we present the results from using the random forest, a more sophisticated supervised learning algorithm. Random forest The "random forest" method [ 14 ] is a supervised learning algorithm that has previously been successfully applied to many genomic studies. It has been implemented in the randomForest package of R [ 15 ]. A random forest is an ensemble of many classification trees generated from bootstrap samples of the original data. It is well known that random forests avoid overfitting and usually have better classification accuracy than classification trees. A natural way to evaluate the importance of the feature variables with the random forest algorithm is to measure the increase of the classification error when those variables are permuted. Intuitively, the more important variables will, when permuted, produce larger classification errors. The importance score provided by the random forest is a more accurate estimate of the classification error that considers the situation of random test samples. Therefore, this importance score provides a more objective evaluation of the relative merit of different genomic features on protein-protein interaction prediction. Moreover, the intrinsic tree structure of the random forest easily takes into account the interactions among the different variables and avoids complications caused by missing data that occurred in many other modeling procedures. We performed our random forest analysis by growing 5,000 trees. Figure 1 shows the importance measures of the genomic evidences used in the random forest algorithm. The result agrees mostly with that of the logistic regression in that the MIPS and Gene Ontology functional similarity features are found to be very important, whereas most of the high-throughput experimental data sets have negligible effects. However, different from the result from logistic regression, the Gavin data set is shown to be less important than MIPS and Gene Ontology functional similarity features after considering the situation of random test samples. These observations motivated us to perform a more thorough information assessment of the genomic evidences considered. We first compared the performance of different classification methods (random forest, logistic regression and Bayesian network), and then evaluated the importance of the different genomic datasets within the framework the best method (the method with the lowest classification error). Comparison of three methods We conducted 7-fold cross validations on the subset with complete information (described above) on all the features for random forest, logistic regression and the Bayesian network method. Figure 2 displays their receiver operating characteristic (ROC) curves, where we observe a better performance of the random forest over the other two and similar performances between logistic regression and the Bayesian network. Information assessment Information assessment of different genomic data may help us understand their relationship with protein-protein interactions, and form a guideline for future model development. MIPS and gene ontology functional similarity data We saw that the MIPS and Gene Ontology functional similarities were the two most important information sources under both the logistic regression and random forests methods. Histograms of the MIPS and GO functional similarity data (Figures 3 and 4 ) show that they are very different for the gold standard positives and negatives; protein pairs in the gold standard positives are associated with smaller functional similarity values than the gold standard negatives. This pattern explains why the functional similarity features have such a strong impact on classification accuracy in the model fitting, as observed in Figure 2 . However, the vast number of protein pairs in the gold standard negatives are likely to be those that have not been thoroughly studied by researchers, and henceforth are observed to belong to large functional categories that actually should be further divided into more specific categories. This conjecture suggests that the information from MIPS and Gene Ontology function data is possibly caused by selection biases other than intrinsic biological relevance. It deserves further investigations of the relationship between the gold standards and the MIPS and Gene Ontology functional similarity data. In the following paragraphs, we show quantitatively that the MIPS and Gene Ontology functional similarities are the dominating information contributors for predicting protein-protein interactions, while other genomic features have negligible benefit and can not provide credible predictions by themselves. We examine the performance of random forests using three different genomic feature sets: (i) all genomic features included, (ii) MIPS and Gene Ontology functional similarities only, and (iii) genomic features other than the MIPS and Gene Ontology functional similarities. The random forest performance is evaluated with the classification error ( Err ) defined as follows. Denote Err 1 as the proportion of protein pairs misclassified in the gold standard positives, and Err 2 the counterpart for the gold standard negatives. Then we define the classification error as the average of Err 1 and Err 2 . Err is a balanced error rate across gold standard positives and negatives. Suppose the joint probability density functions of the predictor features X are f 1 ( X ) and f 2 ( X ) for the gold standard positives and negatives, respectively. Denote a classifier by C ( X ). Then the classification error can be written as where I ( A ) is an indicator function equal to 1 when A is true and 0 otherwise. A minimal classification error Err min can be computed by minimizing (1) across the space of X . It is easy to see that is achieved at C ( X ) = I ( f 1 ( X ) > f 2 ( X )). With this formula, we can estimate the optimal (minimum) classification error based on any estimates of f 1 ( X ) and f 2 ( X ). In our study, f 1 ( X ) and f 2 ( X ) are estimated by their empirical density functions. Table 3 presents the optimal classification error using the MIPS and Gene Ontology functional similarity data. Using the MIPS and Gene Ontology functional similarity data sets alone results in a highly accurate classification with an optimal error of only 0.28%. Table 3 also shows the effects of the data discretizations that were originally used in the Bayesian network method ("grouped"). The significant discrepancy between optimal classification errors using the raw data and the discretized data ("grouped") suggests that the discretization causes serious loss of information. Other genomic features We also estimated the classification errors using the other genomic features within the random forest framework. Table 4 shows that adding the other genomic evidences in the complete-information subset provides only negligible benefit or even reduces the classification accuracy. Moreover, we compared the ROC curves (Figure 5 ) of the random forest method using all genomic information, only the MIPS and GO functional similarities, and the genomic information other than MIPS and GO. Figure 5 shows that we barely gain any by considering other genomic information if the MIPS and GO are available; classifications without the MIPS and GO functional similarity data are poor on the complete-information subset. Note, however, that the subset of full interaction data which have the strongest expression correlations is not necessarily the complete-information set considered. Hence, we would expect that expression correlations might be a stronger source of information in other context. Conclusions In the restricted problem posed by the complete-information subset, we identified that the MIPS and Gene Ontology functional similarity datasets as the dominating information contributors for predicting the protein-protein interactions under the framework proposed in [ 1 ]. Random forests based on the MIPS and GO information alone can give highly accurate classifications. In this particular subset of complete information, adding other genomic data does little for improving predictions. The MIPS and GO information, however, is only available for a small proportion of the ~18M protein pairs. We considered alternative non-Bayesian methods such as logistic regression and random forest for predicting protein-protein interactions. These existing methods do not require prior information needed for the Bayesian approach, and can fully utilize the raw data without discretization. The logistic model performs similarly as the Bayesian method in terms of classifications and, like the Bayesian method, produces estimated probabilities that two proteins interact. As a dichotomous classifier, the random forest method outperforms the other methods considered and efficiently uses the information, although it is computationally more expensive. In particular, its importance measure provides a more objective assessment of different genomic features on predicting protein-protein interactions than simply considering contributions to model fitting. These findings are motivation to look for other, more sensible data resources and superior models. We found that the data discretizations used in the Bayesian methods decreased classification performance. We note here that the genomic features datasets investigated here themselves are highly processed versions of the datasets they were derived from and that there may be better ways to take the original data into account. Another caveat is that the predictions might be just defining groups of proteins that have the same genomic properties as the protein complexes in the MIPS data. This does not necessarily mean that they really represent protein complexes. Rather, they may represent groups of proteins that have the same properties as protein complexes. In this analysis we have looked at the relative weights of various features in predicting protein-protein interactions based on the previous study in [ 1 ]. We looked at a particular subset of the data where we had complete information and we were able to show that, for this particular subset of the full information, we are able to show that the functional classification features in the MIPS functional catalog and Gene Ontology were the most informative and that particular machine learning algorithms, such as random forests were more effective than Bayesian networks. However, one has to keep in mind that in the full problem there is the issue of incomplete information. On data sets with incomplete information Bayesian approaches maybe more effective because they can easily handle the missing information. Further careful studies such as these will be needed to determine what the optimum machine learning method is and the optimum features are in presence of incomplete information. It will be also of great interest to consider other genomic features such as phylogenetic profiles [ 16 ] and local clustering information [ 17 ]. This is just the first step in that direction. Methods Logistic regression Denote the gold standards by random variable Y and the other genomic features by X 1 , X 2 , ..., X n . Let Y = 1 when two proteins interact, i.e., they are in the same complex, and Y = 0 when not. The logistic model is of the form where the random vector X consists of X 1 , X 2 , ..., X n and their interaction terms. Stepwise variable selection The stepwise selection procedure starts from a null model. At each step, it adds a variable with the most significant score statistics among those not in the model, then sequentially removes the variable with the least score statistic among those in the model whose score statistics are not significant. The process terminates if no further variable can be added to the model or if the variable just entered into the model is the only variable removed in the subsequent elimination. Here, the score statistic measures the significance of the effect of a variable. ROC curve analysis Receiving operator characteristic (ROC) curve [ 18 ] is a graphical representation used to assess the discriminatory ability of a dichotomous classifier by showing the tradeoffs between sensitivity and specificity . Sensitivity is calculated by dividing the number of true positives (TP) through the number of all positives, which equals the sum of the true positives and the false negatives (FN); specificity is calculated by dividing the number of true negatives (TN) through the number of all negatives, which equals the sum of the true negatives and the false positives (FP). Sensitivity = TP /( TP + FN ), Specificity = TN /( TN + FP ). The ROC curve plot shows 1 - specificity on the X axis and sensitivity on the Y axis. A good classifier has its ROC curve climbing rapidly towards upper left hand corner of the graph. This can also be quantified by measuring the area under the curve. The closer the area is to 1.0, the better the classifier is; and the closer the area is to 0.5, the worse the classifier is. Authors' contributions NL and BW conducted the major part of the data analysis, and created all the tables and figures, under the supervision of HZ. RJ and MG provided the data sets for the analysis and contributed to the discussion on the comparisons of different methods. All authors read and approved the final manuscript. Supplementary Material Additional File 1 The complete-information subset in ZIP file. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC529436.xml
524489	Vaccine candidates derived from a novel infectious cDNA clone of an American genotype dengue virus type 2	Background A dengue virus type 2 (DEN-2 Tonga/74) isolated from a 1974 epidemic was characterized by mild illness and belongs to the American genotype of DEN-2 viruses. To prepare a vaccine candidate, a previously described 30 nucleotide deletion (Δ30) in the 3' untranslated region of DEN-4 has been engineered into the DEN-2 isolate. Methods A full-length cDNA clone was generated from the DEN-2 virus and used to produce recombinant DEN-2 (rDEN-2) and rDEN2Δ30. Viruses were evaluated for replication in SCID mice transplanted with human hepatoma cells (SCID-HuH-7 mice), in mosquitoes, and in rhesus monkeys. Neutralizing antibody induction and protective efficacy were also assessed in rhesus monkeys. Results The rDEN2Δ30 virus was ten-fold reduced in replication in SCID-HuH-7 mice when compared to the parent virus. The rDEN-2 viruses were not infectious for Aedes mosquitoes, but both readily infected Toxorynchites mosquitoes. In rhesus monkeys, rDEN2Δ30 appeared to be slightly attenuated when compared to the parent virus as measured by duration and peak of viremia and neutralizing antibody induction. A derivative of rDEN2Δ30, designated rDEN2Δ30-4995, was generated by incorporation of a point mutation previously identified in the NS3 gene of DEN-4 and was found to be more attenuated than rDEN2Δ30 in SCID-HuH-7 mice. Conclusions The rDEN2Δ30 and rDEN2Δ30-4995 viruses can be considered for evaluation in humans and for inclusion in a tetravalent dengue vaccine.	Background The increased prevalence of disease caused by the mosquito-borne dengue (DEN) viruses (four serotypes; DEN-1 – DEN-4) has intensified the effort to generate a vaccine that would both confer protection and be economically feasible for use in countries with limited resources for healthcare [ 1 ]. Dengue fever and dengue hemorrhagic fever and shock (DHF/DSS) are a severe disease burden for tropical and semitropical countries inhabited by more than 2.5 billion people [ 2 ]. Risk factors for the more severe disease, DHF/DSS, include the strain of virus, age and genetic background of the host, and secondary infection by a DEN serotype different from that which caused the primary infection [ 2 ]. Increased risk associated with secondary infection by a different DEN serotype is believed to be caused both by increased virus replication resulting from antibody-dependent enhancement and by augmented immune activation induced by the secondary infection [ 3 , 4 ]. Typically, regions with DHF/DSS have all four DEN serotypes circulating simultaneously, and an effective DEN vaccine must contain a tetravalent formulation that confers protection against each of the four DEN serotypes. Immunity to the DEN viruses is primarily mediated by neutralizing antibodies directed against the envelope (E) glycoprotein, and most vaccine strategies aim to induce antibody against this major protective antigen. Live attenuated tetravalent vaccines appear to be the best vaccine candidates since they are economical to manufacture and they induce long-term immunity with the live attenuated yellow fever virus vaccine serving as a successful model flavivirus vaccine [ 5 ]. Several strategies to produce live attenuated tetravalent vaccines are being pursued including attenuation of viruses by conventional passage in tissue culture or introduction of defined attenuating mutations into recombinant DEN viruses [ 6 - 9 ]. In addition, chimeric dengue viruses are being evaluated that contain the E protein of a DEN virus on a background of either an attenuated DEN virus from a different serotype or a more distantly related, but attenuated, flavivirus [ 10 - 12 ]. We have previously described attenuated and immunogenic monovalent vaccine candidates for DEN-1, DEN-2, DEN-3, and DEN-4 that were generated by two distinct recombinant methodologies. Using the first methodology, nucleotides 10478–10507 were deleted from the 3' UTR (Δ30) of a wild type cDNA clone for DEN-4 to generate a vaccine candidate, rDEN4Δ30, which is safe, attenuated, and immunogenic in rhesus monkeys and humans [ 13 ]. Incorporation of the Δ30 mutation into an infectious cDNA clone of DEN-1 wild type virus at a site homologous to that in DEN-4 attenuated DEN-1 for rhesus monkeys and is currently being evaluated in humans [ 14 ]. The Δ30 mutation did not confer attenuation upon DEN-3 for reasons that have not been defined [ 15 ]. Thus, this approach has yielded live attenuated virus vaccine candidates for both DEN-1 and DEN-4. Using a second methodology, antigenic chimeric viruses have been generated by replacing the membrane protein (M) and E structural genes of rDEN4Δ30 with those from DEN-2 or DEN-3 [ 12 , 15 ]. These antigenic chimeric viruses were attenuated and immunogenic in rhesus monkeys and represent vaccine candidates for DEN-2 and DEN-3. We have also described a set of point mutations that can attenuate wild type rDEN-4 for SCID mice transplanted with human liver cells (SCID-HuH-7) or for rhesus monkeys [ 16 , 17 ]. Such mutations identified in rDEN-4 could be introduced into conserved sites of cDNA clones for other DEN serotypes to fine-tune the level of attenuation of vaccine candidates. We have found it prudent to pursue several strategies to develop a live attenuated virus vaccine for each dengue serotype recognizing that it has been a challenge to achieve a satisfactory balance between attenuation and immunogenicity [ 15 , 18 - 20 ]. Thus, in addition to the antigenic chimeric DEN-2 vaccine candidate described above, a second approach was pursued in the present study that involved the construction of an infectious cDNA clone of a wild type DEN-2 virus isolated in Tonga [ 21 ], and the generation of DEN-2 vaccine candidates by the sequential introduction of defined attenuating mutations into the recombinant version of the DEN-2 Tonga/74 wild type virus. The rDEN2Δ30 vaccine candidate was evaluated for replication in SCID-HuH-7 mice, mosquitoes, and rhesus monkeys. In addition, an attenuating point mutation, previously described in DEN-4, was introduced into the rDEN2Δ30 virus, and this rDEN2Δ30 derivative was characterized in SCID-HuH-7 mice. Methods Cells and viruses Vero cells (African green monkey kidney) were propagated in OptiPro SFM (Invitrogen, Grand Island, NY) supplemented with 4 mM L-glutamine (Invitrogen). HuH-7 cells (human hepatoma) were maintained in D-MEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS), 1 mM L-glutamine and 0.05 mg/ml gentamicin (Invitrogen). C6/36 cells (Aedes albopictus mosquito cells) were maintained at 32°C in Minimal Essential Medium (MEM) containing Earle's salts and 25 mM HEPES buffer (Invitrogen) and supplemented with 10% FBS, 2 mM L-glutamine, and 0.1 mM non-essential amino acids (Invitrogen). A dengue virus type 2 isolate, Tonga/74, was provided by Dr. Duane Gubler (CDC, Fort Collins, CO). The virus was isolated during a 1974 dengue outbreak in the South Pacific island of Tonga [ 21 ]. The virus was isolated by inoculation of patient sera into Aedes albopictus mosquitoes, and subsequent passage in C6/36 cells before determination of genomic sequence. Sequence analysis Viral RNA was isolated from DEN-2 Tonga/74 wild type virus using the QIAamp Viral RNA mini kit (Qiagen, Valencia, CA). Reverse transcription was performed using random hexamer primers and the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Overlapping PCR fragments of approximately 2000 base pairs were generated using DEN-2 specific primers and Advantage cDNA polymerase (ClonTech, Palo Alto, CA). Both strands of the resulting PCR fragments were sequenced directly on a 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) using DEN-2 specific primers in BigDye terminator cycle sequencing reactions (Applied Biosystems) and the results were assembled into a consensus sequence. To determine the nucleotide sequence of the genomic 5' and 3' regions, the 5' cap nucleoside of the viral genome was removed with tobacco acid pyrophosphatase (Epicentre Technologies, Madison, WI), followed by circularization of the genome using RNA ligase (Epicentre Technologies). An RT-PCR fragment spanning the ligation junction was generated and sequenced using DEN-2 primers. For the DEN-2 Tonga/74 consensus sequence, GenBank accession number AY744147 was assigned. Genetic construction of rDEN-2 Tonga/74 cDNA clone cDNA fragments of DEN-2 Tonga/74 were generated by reverse-transcription of the genome as indicated in Figure 1 . Each fragment was subcloned into a plasmid vector and sequenced to verify that it matched the consensus sequence as determined for the virus. This yielded seven cloned cDNA fragments spanning the genome. Cloned fragments were modified as follows: Fragment X, representing the 5' end of the genome was abutted to the SP6 promoter; Fragment L was modified to contain a Spe I restriction site at genomic nucleotide 2353; Fragment R was modified to contain a Spe I restriction site also at genomic nucleotide 2353, and, to stabilize the eventual full-length clone, two additional mutations at nucleotides 2362 – 2364 and 2397 were created to ensure that translation stop codons were present in all reading frames other than that used to synthesize the virus polyprotein; Fragment A was modified at nucleotide 3582 to ablate a naturally occurring Spe I restriction site and at nucleotide 4497 to ablate a naturally occurring Kpn I restriction site; Fragment C was modified at nucleotide 9374 to ablate a naturally occurring Kpn I restriction site; and Fragment Y, representing the 3' end of the genome was abutted to a Kpn I restriction site. All mutations introduced into the cloned cDNA fragments were translationally-silent, thereby preserving the wild-type polyprotein sequence. Each fragment was added incrementally between the Asc I and Kpn I restriction sites of DEN-4 cDNA clone p4 (GenBank accession number: AY648301) to generate a full-length DEN-2 cDNA clone (p2) with the same vector background successfully used to generate rDEN-4 and rDEN4Δ30 virus [ 13 ]. cDNA clone p2 was sequenced to confirm that the virus genome region matched the DEN-2 Tonga/74 consensus amino acid and nucleotide sequence, with the exception of the translationally-silent modifications noted above. The Δ30 mutation which removes nucleotides 10541–10570 was introduced into Fragment Y to generate Fragment YΔ30. To create p2Δ30, the Fragment Y region of p2 was replaced with Fragment YΔ30 (Figure 1 ). The genomic region of each full-length cDNA was sequenced as described above and GenBank accessions were assigned as follows (cDNA clone: accession numbers): p2: AY744148, p2Δ30: AY744149. Using site-directed mutagenesis, an attenuating amino acid change characterized in the NS3 gene of DEN-4 (nt 4995–7; a.a. 158, Ser→Leu) was introduced into the p2Δ30 cDNA clone [ 17 ]. A mutagenic oligonucleotide was designed to change DEN-2 NS3 amino acid 158 from Ser (AGT) to Leu (CTA) and used to construct the cDNA clone, p2Δ30-4995 (accession number: AY744150), which was sequenced for confirmation of nucleotide changes. Recovery of rDEN-2 viruses cDNA clones were linearized with Acc 65I (isoschizomer of Kpn I which cleaves leaving only a single 3' nucleotide) and were transcribed in vitro using the AmpliCap SP6 Message Maker kit (Epicentre Technologies, Madison, WI). Purified transcripts were then transfected into Vero or C6/36 cells. Viruses recovered in C6/36 cells were passaged 3 times in Vero cells, and all viruses were biologically cloned by terminal dilution in Vero cells. The genomes of recombinant viruses used to infect rhesus monkeys were completely sequenced as described above to identify adventitious mutations that had accumulated during transfection and biological cloning. Replication in SCID-HuH-7 mice Four to six week-old SCID mice (Tac:Icr:Ha(ICR)- Prkdc scid ) (Taconic, Germantown, NY) were injected intraperitoneally with 10 7 HuH-7 cells suspended in 0.2 ml phosphate-buffered saline. Tumors were detected in the peritoneum, and mice were infected by direct inoculation of the tumor with 10 4 PFU of virus in 0.05 ml Opti-MEM (Invitrogen). On day 7 post-infection, serum was obtained from cardiac blood and stored at -70°C. Virus titer in serum samples was determined by plaque assay in Vero cells. Replication, immunogenicity, and protection in rhesus monkeys The DEN-2 viruses were evaluated in rhesus macaques using established methods [ 13 ]. DEN virus sero-negative monkeys were injected subcutaneously with 10 5 PFU virus diluted in L-15 medium (Invitrogen) or with a mock inoculum. Serum was collected on days 0–6, 8, 10, 12 and 28 after inoculation and stored at -70°C. Virus titer was determined for each serum sample by plaque assay in Vero cells and serum neutralizing antibody titer was determined for serum from days 0 and 28 by plaque reduction neutralization test. On day 28, monkeys were challenged with 10 5 PFU of DEN-2 Tonga/74, and serum was collected on days 29–34, 36, and 56. Virus titer was determined for serum from days 28–34 and 36 and serum neutralizing antibody titer was determined for serum from day 56. Virus replication in mosquitoes Replication in Aedes aegypti and Toxorynchites amboinensis mosquitoes was evaluated as previously described [ 22 ]. Briefly, A. aegypti were fed blood meals containing serial 10-fold dilutions of virus. After 21 days, viral antigen was detected in head and midgut preparations by immunoflourescence assay using DEN-2-specific hyperimmune mouse ascitic fluid and fluorescein isothyocyanate conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD), and the mosquito infectious dose-50% (MID 50 ) was determined. T. amboinensis were inoculated intrathoracically with a 0.2 ul dose containing serial ten-fold dilutions of virus and incubated for 14 days. Head preparations were made and antigen visualized as described above. Results Generation and sequence analysis of recombinant DEN-2 Tonga/74 viruses A full-length cDNA clone, p2, was constructed that matched the genomic consensus sequence of the American genotype DEN-2 isolate, Tonga/74, with the exception of translationally-silent modifications made to facilitate cloning (Figure 1 ). The previously described Δ30 deletion mutation was incorporated into the p2 cDNA clone to form p2Δ30 [ 13 ]. The rDEN-2 virus was recovered in C6/36 and Vero cells, but the presence of the Δ30 mutation limited recovery to only C6/36 cells. After passage in Vero cells, adaptation mutations were identified by sequence analysis as had been described for other DEN viruses [ 23 ]. Both rDEN-2 and rDEN2Δ30 viruses accumulated a single nucleotide change in NS4B at nt 7169 encoding a Val→Ala change at amino acid position 115 as has been observed for rDEN-3 (Table 1 ) [ 15 ]. The same nucleotide change was previously reported to occur at the homologous site following passage of DEN-4 in Vero cells resulting in a Leu→Ser change (Table 1 ) [ 23 ]. Inclusion of the 7169 mutation into the p2Δ30 cDNA permitted recovery in both C6/36 and Vero cells (data not shown). The rDEN2Δ30 virus reached a virus titer of 6.6 log 10 PFU/ml in Vero cells. Replication of rDEN-2 viruses in SCID-HuH-7 mice As an initial evaluation of replication of the DEN-2 Tonga/74 virus and the rDEN-2 viruses, replication in SCID mice transplanted with HuH-7 human hepatoma cells (SCID-HuH-7 mice) was tested. Wild-type viruses from each DEN serotype have been shown to replicate to approximately 6.0 log 10 PFU/ml serum in SCID-HuH-7 mice, and an att phenotype in SCID-HuH-7 mice has been shown to be a predictor of reduced replication in rhesus monkeys [ 12 , 14 , 15 , 17 ]. The parent DEN-2 Tonga/74 virus replicated efficiently in SCID-HuH-7 mice and reached a mean titer in serum of 5.9 log 10 PFU/ml (Table 2 ) similar to that previously observed with the DEN-2 New Guinea C (NGC) prototype strain [ 12 ]. The rDEN-2 virus replicated to the same level as the wild-type isolate, while rDEN2Δ30 was 10-fold restricted in replication. This reduction was statistically significant (Tukey-Kramer post-hoc test; P < 0.05), and was similar to that observed for the well-characterized rDEN4Δ30 virus [ 17 ]. Replication of rDEN-2 viruses in mosquitoes The DEN-2 viruses were evaluated for infectivity of Aedes aegypti fed on an infectious bloodmeal (oral infectivity only) and for Toxorynchites amboinensis inoculated intrathoracically (Table 3 ). At the doses tested neither DEN-2, rDEN-2, or rDEN2Δ30 were detected in the midgut or head of A. aegypti mosquitoes which had fed on an infectious bloodmeal 21 days earlier. The inability to infect the midgut led to a lack of infection in the head tissue. This indicates that the DEN-2 Tonga/74 viruses are poorly infectious for A. aegypti mosquitoes by oral infectivity, as has been demonstrated for multiple DEN-2 viruses of the American genotype [ 24 , 25 ]. In contrast the DEN-2 NGC prototype strain, an Asian genotype member, was highly infectious in A. aegypti mosquitoes when tested previously but it was not included here as a concurrent control [ 12 ]. The defect in rDEN-2 infectivity for A. aegypti was further investigated by directly inoculating the same virus stocks intrathoracically into T. amboinensis and measuring the ability of the viruses to infect the head tissues. Both rDEN-2 and rDEN2Δ30 were highly infectious by intrathoracic inoculation (Table 3 ). The Δ30 mutation did not alter the infectivity of rDEN-2 following intrathoracic inoculation, a property also previously observed for DEN-1, -3 and -4 [ 14 , 15 , 22 ]. These results indicate that the lack of infectivity for A. aegypti was likely caused by the inability of the DEN-2 Tonga/74 viruses to establish a midgut infection and that the viruses retained the ability to infect head tissues. Replication, immunogenicity, and protective efficacy in rhesus monkeys The replication (viremia), immunogenicity, and protective efficacy of the DEN-2 viruses in monkeys were studied. Monkeys inoculated with the DEN-2 Tonga/74 wild-type isolate were viremic for an average of 4.5 days with a mean peak titer of 2.1 log 10 PFU/ml (Table 4 ). Inoculation with rDEN-2 resulted in detectable viremia for 4.0 days with a mean peak titer of 1.9 log 10 PFU/ml. While the levels of rDEN2Δ30 replication (2.8 days viremia; mean peak titer of 1.7 log 10 PFU/ml) were lower than DEN-2 and rDEN-2, the differences were not as dramatic as had been observed for rDEN1Δ30 and rDEN4Δ30 when compared to their parent viruses [ 13 , 14 ]. The level of neutralizing antibodies induced by the rDEN2Δ30 virus was also less than that induced by the wild-type DEN-2 viruses, a finding consistent with the decreased replication exhibited by this vaccine candidate. Therefore, by three quantitative measures, duration and peak titer of viremia and the level of neutralizing antibodies induced, rDEN2Δ30 appeared to be attenuated when compared to DEN-2 Tonga/74. When vaccinated monkeys were challenged with DEN-2 Tonga/74, all monkeys were protected, as indicated by the lack of viremia (Table 4 ). The 4995 mutation further attenuates rDEN2Δ30 in SCID-HuH-7 mice Based on the limited attenuation conferred upon rDEN-2 by the Δ30 mutation in rhesus monkeys, we sought to construct a further attenuated derivative of rDEN2Δ30. To further attenuate rDEN2Δ30, an att mutation that has been characterized in another DEN serotype was imported into a homologous region in DEN-2. One such mutation, the 4995 mutation in DEN-4 NS3 at amino acid 158 (Ser→Leu), was previously incorporated into the DEN-4 vaccine candidate, rDEN4Δ30, and found to further attenuate the virus for SCID-HuH-7 mice and rhesus monkeys [ 17 ]. Site directed mutagenesis was used to introduce a Ser→Leu mutation at amino acid 158 of NS3 in rDEN2Δ30-7169, and the rDEN2Δ30-4995 virus was recovered in C6/36 cells and propagated in Vero cells reaching a virus titer of 6.2 log 10 PFU/ml (Table 1 ). Importantly, the resulting Leu codon would require two nucleotide changes to revert to one of the six odons encoding a Ser residue. Replication in the SCID-HuH-7 mouse model was used as an initial assessment of the rDEN2Δ30-4995 virus phenotype. Table 5 includes results from three separate experiments (including those from Table 1 ) and confirms the approximate 10-fold reduction in replication conferred by the Δ30 mutation upon rDEN-2 replication in SCID-HuH-7 mice. The rDEN2Δ30-4995 virus had a mean peak virus titer of 4.6 log 10 PFU/ml which was only a modest reduction from that of rDEN2Δ30, 5.2 log 10 PFU/ml. However, comparison of a large number of samples indicated that the reduction in virus titer conferred by the NS3 4995 mutation upon rDEN2Δ30 was statistically significant (rDEN2Δ30-4995 versus rDEN2Δ30; Tukey-Kramer post-hoc test; P < 0.05). The virus titer of rDEN2Δ30-4995 virus in SCID-HuH-7 mice was over 60-fold reduced from that of the rDEN-2 parent virus. Discussion Development of a live-attenuated tetravalent dengue vaccine has been complicated by two major factors. First, monovalent vaccine candidates that exhibit a satisfactory balance between attenuation and immunogenicity have been difficult to identify [ 15 , 18 - 20 ]. Second, satisfactorily attenuated tetravalent vaccine formulations that induce a broad neutralizing antibody response against each of the four DEN serotypes have been difficult to develop [ 6 , 10 , 20 , 26 ]. For these reasons, we have sought to develop multiple vaccine candidates for each DEN serotype to increase the likelihood that a vaccine with a satisfactory balance between attenuation and immunogenicity will be identified. To produce a live-attenuated DEN-2 vaccine candidate, we previously generated an antigenic chimeric virus, rDEN2/4Δ30, expressing the M and E structural genes of the DEN-2 NGC strain on the attenuated rDEN4Δ30 background [ 12 ]. The vaccine candidates described in the present study, rDEN2Δ30 and rDEN2Δ30-4995, could serve as alternates to this antigenic chimeric virus if evaluation of the rDEN2/4Δ30 virus in humans, either as a monovalent vaccine or as a component of a tetravalent vaccine, indicates that it lacks a balance between attenuation and immunogenicity. It was hoped that each of the four components of a tetravalent vaccine, consisting of DEN-1, -2, -3, and -4 wild type viruses, each with the common 30 nucleotide deletion mutation in the 3' UTR, would exhibit a similar level of attenuation in animal models [ 13 - 15 ]. Unfortunately, the level of attenuation conferred by the Δ30 mutation upon each of the four serotypes has proven to be variable. In rhesus monkeys, the rDEN2Δ30 virus appears to have an intermediate attenuation phenotype in between that of the attenuated rDEN1Δ30 and rDEN4Δ30 and the non-attenuated rDEN3Δ30 [ 13 - 15 ]. Although rDEN2Δ30 was slightly attenuated compared to its DEN-2 parent virus in rhesus monkeys, the reduction in replication was less than that of rDEN1Δ30 and rDEN4Δ30. While the latter two viruses had detectable viremia in only 50% of monkeys, a mean number of viremic days of less than one day, and a mean peak viremia of less than 1.0 log 10 PFU/ml [ 13 , 14 ], the rDEN2Δ30 virus infected 100% of the rhesus monkeys and reached a peak virus titer of 1.7 log 10 PFU/ml. However, the 10-fold reduction of replication of rDEN4Δ30 and rDEN2Δ30 in SCID-HuH-7 mice, compared to that of their respective wild type parents, was similar. To date, rDEN4Δ30 is the only Δ30 vaccine candidate that has been tested in humans, and it was found to be both safe and immunogenic [ 13 ]. If the level of attenuation in SCID-HuH-7 mice serves as a better guide to attenuation in humans, rDEN2Δ30 might be satisfactorily attenuated in humans since its level of attenuation for SCID-HuH-7 mice and that of the rDEN4Δ30 vaccine candidate are comparable. To construct a further attenuated derivative of rDEN2Δ30, the 4995 mutation present in the NS3 gene of DEN-4 at amino acid 158 (Ser→Leu) was introduced into the homologous region of the NS3 protein of rDEN2Δ30 [ 17 ]. Although the 4995 mutation results in a single amino acid change and thus may be susceptible to reversion, the mutant leucine codon selected for insertion into rDEN2Δ30-4995 would require two nucleotide changes to revert to a serine codon. Introduction of the 4995 mutation into rDEN4Δ30 resulted in a 100-fold greater reduction of replication in SCID-HuH-7 mice [ 17 ]. In rDEN2Δ30, its introduction resulted in nearly a 10-fold reduction in virus titer, a smaller but still statistically significant reduction. These results provide a second example of the difficulty in predicting the precise level of attenuation following import of an attenuating mutation into a different DEN serotype. Nevertheless, the rDEN2Δ30-4995 vaccine candidate is more attenuated than its rDEN2Δ30 parent and warrants evaluation in rhesus monkeys and humans. Epidemiologic and molecular pathogenesis studies of DEN-2 strains support the concept that the DEN-2 Tonga/74 virus, from which the vaccine candidates were derived, may naturally have a lower level of virulence than other DEN-2 viruses. If the DEN-2 Tonga/74 parent virus is naturally attenuated to some degree, only a small incremental increase in attenuation might be required to satisfactorily attenuate it for humans. Gubler et al. investigated the 1974 outbreak of DEN-2 infection in the Pacific island of Tonga [ 21 ]. In comparison to a subsequent DEN-1 outbreak, the 1974 DEN-2 outbreak was distinguished by mild disease with few hemorrhagic sequelae, low viremia, and an overall slow spread of virus infection [ 21 ]. The weak DEN-2 outbreak was proposed to be a result of the circulation of a strain with an inherently low level of virulence [ 21 ]. Since the Tonga/74 outbreak, additional evidence has emerged that supports the suggestion that there are at least two circulating lineages of DEN-2 viruses that differ in virulence [ 27 - 29 ]. The DEN-2 Tonga/74 virus is a member of the DEN-2 American genotype, which as a group appear to possess lower virulence than that of the Asian genotype of DEN-2 viruses [ 28 , 29 ]. Despite the presence of the American DEN-2 genotype viruses and limited co-circulation of DEN-1 and DEN-3 viruses in the Americas in the 1960s and 1970s, the first major epidemic of DHF/DSS in the Americas occurred only after the introduction of a DEN-2 Asian genotype virus in 1981 [ 27 - 30 ]. It was thought that genetic differences might have contributed to this difference in virulence and evidence to this effect has been forthcoming. Rico-Hesse and colleagues have defined genetic elements within the genome of DEN-2 American genotype viruses which distinguish them from members of the Asian genotype [ 31 ]. In addition, using chimeric rDEN-2 American/Asian viruses, introduction of three genetic elements (a point mutation in the E gene, the 5' UTR, and the 3' UTR) of the American genotype was found to confer reduced virus replication in dendritic cells and monocytes upon an Asian genotype rDEN-2 [ 32 ]. The Tonga/74 virus shares each of these three attenuating genetic determinants specific to the American genotype [ 31 , 32 ], which provides a possible explanation for its lower virulence in humans. The rDEN2Δ30 vaccine candidate, whose parent is the DEN-2 Tonga/74 American genotype virus, thus contains naturally occurring and experimentally introduced attenuating mutations. Thus, the small incremental increase in attenuation provided by the Δ30, with or without the 4995 mutation, might prove to satisfactorily attenuate the DEN-2 Tonga/74 for humans. The American genotype DEN-2 viruses exhibit decreased infectivity for Aedes mosquitoes in comparison to Asian DEN-2 viruses [ 24 , 25 ]. Consistent with these observations, the wild type New Guinea C Asian DEN2 virus was highly infectious for Aedes mosquitoes in our laboratory [ 12 ] whereas the Tonga/74 American genotype virus was poorly infectious by the oral route (present study). In fact, the increased prevalence of DEN-2 viruses of the Asian genotype in the Americas has been suggested to be a result of their enhanced transmission [ 28 ]. However, the active circulation of American genotype viruses over many decades indicates that mosquito transmission does occur and large epidemics have been associated with viruses of this genotype [ 21 , 27 ]. At the doses tested, neither the DEN-2 Tonga/74 isolate nor the recombinant viruses were found to infect the midgut or head of Aedes aegypti mosquitoes fed an infectious blood meal. Since rDEN-2 and rDEN2Δ30 viruses were infectious by intrathoracic inoculation of Toxorynchites mosquitoes, the lack of infectivity for A. aegypti was likely caused solely by the inability of the DEN-2 Tonga/74 viruses to establish a midgut infection. Decreased infectivity for Aedes mosquitoes could serve to help limit transmission of the vaccine virus. Conclusions The live-attenuated DEN-2 virus candidates described here, rDEN2Δ30 and rDEN2Δ30-4995, have several properties desired in a live attenuated virus vaccine for humans. First, both viruses reached a titer over 6.0 log 10 PFU/ml in Vero cells that would permit economical manufacture. Second, the viruses are derived from the DEN-2 Tonga/74 strain, a member of the American genotype, which has been associated with decreased virulence. Third, rDEN2Δ30 was attenuated for replication in SCID-HuH-7 mice and slightly attenuated for rhesus monkeys while inducing a protective neutralizing antibody response. Fourth, rDEN2Δ30-4995 was more attenuated in SCID-HuH-7 mice than rDEN2Δ30. Fifth, the DEN-2 Tonga/74 strain, like other members of the American genotype, is poorly infectious for Aedes aegypti mosquitoes which would help to limit uncontrolled transmission of the vaccine virus. Competing interests The authors declare that they have no competing interests. Authors' contributions J.B. recovered viruses, conducted animal studies, and drafted the manuscript. C.H. and S.W. constructed the DEN-2 cDNA clone and C.H. performed sequencing. K.H. performed mosquito studies. B.M. and S.W. supervised the study and participated in planning and design. All authors read and approved the manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC524489.xml
340963	PLoS Medicine	Announcing the next top-tier open-access journal from the Public Library of Science	Open access is gaining momentum. Authors are submitting papers in ever-increasing numbers to open-access journals. Several prominent research sponsors, including the Wellcome Trust, the Max Planck Society, the Centre National de la Recherche Scientifique (CNRS), and the Institut National de la Santé et de la Recherche (INSERM), have recently pronounced that open access is the best way for the researchers they support to publish their work. Several established commercial and not-for-profit publishers have announced plans to experiment with open-access models for some or all of their journals. Delighted and encouraged, we gear up for the launch of PLoS Medicine this autumn—the next step in our efforts to bring the benefits of open access to the entire scientific and medical community. We aim to make PLoS Medicine a premier journal, providing open access to the best medical research to researchers, to physicians and other caregivers, and to the public. The case for open access to medical research is even stronger than it is for basic research in biology. There are more interested parties: patients and their advocates; biotechnology and pharmaceutical companies that develop drugs and medical devices; doctors, nurses, and other healthcare providers; and health policy-makers at the national and international levels. The goal of the medical research enterprise is—or should be—scientifically, ethically, and socially responsible medicine, which means research that will benefit patients worldwide. The reality looks somewhat different. Large investments into basic research have not yet lived up to their full potential to save lives and improve their quality. Doctors, patients, and their advocates do not have ready access to the combined peer-reviewed evidence from medical research. The prices for the latest drugs often put them out of reach of patients in poor countries or poor patients in countries without universal healthcare systems. Moreover, research focuses disproportionately on the potentially lucrative treatments for diseases of wealthy societies, shortchanging the poorer countries, which bear the greatest burden of disease. A medical journal by itself cannot change this reality. But with the help of researchers and practicing physicians around the world who recognize the need and opportunity for change, we seek to create a journal that promotes medical research and practice that is both scientifically rigorous and compassionate. Open access to this literature will strengthen the medical research community by giving all stakeholders free and immediate access to the latest medical research, along with new and more powerful search tools and links between the literature and other relevant information. PLoS Medicine will be an international, modern, general medical journal, covering all areas in the medical sciences, from basic studies to large clinical trials and cost-effectiveness analyses. We will concentrate on human studies that enhance our understanding of disease epidemiology, etiology, and physiology; the development of prognostic and diagnostic technologies; and trials that test the efficacy of specific interventions and those that compare different treatments. We will publish original research and commentary that promotes translation both of basic research into clinical investigation and of clinical evidence into practice. A truly broad medical journal is an ambitious project, but we want PLoS Medicine to promote an integrated understanding of the patient—to make it easy for people to read outside their specialty area. “Doctors are systems biologists,” as one medical researcher put it, and inspiration can often be found in unfamiliar territory. Articles published in PLoS Medicine will be rigorously peer-reviewed. Academic and professional editors, supported by expert peer-reviewers, will select those studies that drive research forward—in this case, toward medical applications and benefits for patients. This issue of PLoS Biology contains two “human” studies that met our criteria for excellence and originality, a paper by Howard Chang and colleagues (found at DOI: 10.1371/journal.pbio.0020007 ) on the microarray analysis of tumors and one by Sarah Rowland-Jones and coworkers (found at DOI: 10.1371/journal.pbio.0020020 ) that examines how HIV exhausts the capacity of the immune system. Similar papers submitted in the future will be published in PLoS Medicine , alongside studies that have more direct implications for clinical practice. This issue also contains several articles describing more basic advances with medical implications: a study by Terry van Dyke and colleagues (found at DOI: 10.1371/journal.pbio.0020022 ) describing a new mouse model for breast cancer, a report on a novel approach to drug synthesis by Chaitan Khosla and coworkers (found at DOI: 10.1371/journal.pbio.0020031 ), and an article by Stephen Dowdy et al. (found at DOI: 10.1371/journal.pbio.0020036 ) on targeted modulation of p53 activity. PLoS Biology will continue to solicit and publish such articles, but we will bring them—and similar ones published elsewhere—to the attention of the readers of PLoS Medicine . Like PLoS Biology , PLoS Medicine must be a community journal to achieve its goals. If you are a researcher or an individual anywhere in the world who has a stake in medical research and if the goals of PLoS Medicine outlined here resonate with you, please contact us. PLoS Medicine will accept submissions beginning in April 2004, and we are looking for advocates who will help to spread the word about open access and PLoS Medicine in the global medical community and for investigators who will submit excellent research, review submitted articles, and contribute editorials and commentaries. PLoS Medicine is and will stay a work in progress, and we want to consult with as many people as possible, both before the launch and as PLoS Medicine evolves. Sign up to join the PLoS Medicine community at http://www.plos.org/medicine and help us to make the best of medical research and practice accessible to a global audience.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC340963.xml
538261	Noise filtering and nonparametric analysis of microarray data underscores discriminating markers of oral, prostate, lung, ovarian and breast cancer	Background A major goal of cancer research is to identify discrete biomarkers that specifically characterize a given malignancy. These markers are useful in diagnosis, may identify potential targets for drug development, and can aid in evaluating treatment efficacy and predicting patient outcome. Microarray technology has enabled marker discovery from human cells by permitting measurement of steady-state mRNA levels derived from thousands of genes. However many challenging and unresolved issues regarding the acquisition and analysis of microarray data remain, such as accounting for both experimental and biological noise, transcripts whose expression profiles are not normally distributed, guidelines for statistical assessment of false positive/negative rates and comparing data derived from different research groups. This study addresses these issues using Affymetrix HG-U95A and HG-U133 GeneChip data derived from different research groups. Results We present here a simple non parametric approach coupled with noise filtering to identify sets of genes differentially expressed between the normal and cancer states in oral, breast, lung, prostate and ovarian tumors. An important feature of this study is the ability to integrate data from different laboratories, improving the analytical power of the individual results. One of the most interesting findings is the down regulation of genes involved in tissue differentiation. Conclusions This study presents the development and application of a noise model that suppresses noise, limits false positives in the results, and allows integration of results from individual studies derived from different research groups.	Background DNA microarrays have become a useful tool in biomedical research as they can be used to determine the relative expression of thousands of genes in a given sample. Such expression profiles could predict genetic predisposition to disease, serve as a set of diagnostic markers, define better drug treatments options for existing diseases (pharmacogenomics), or mark the precise nature of disease progression. A major limitation of this technology is the lack of uniform data mining strategies. This study integrates complementary approaches to more effectively analyze Affymetrix GeneChip microarray data derived from several different types of solid tumors. If the noise is consistent and reproducible it can be filtered from the data and some false positives can be eliminated. There are two principal sources of noise in microarray experiments: biological noise and technical noise. Biological noise consists of variation among patients and tumor locations, variation in the cellular composition of tumors, heterogeneity of the genetic material within tumor due to genomic instability. Technical noise consists of differences in sample preparation and experiment variables which include nonspecific cross hybridization, differences in the efficiency of labeling reactions and production differences between microarrays. Biological noise cannot be corrected but it can be accounted for with statistics using replicates of the treatments or conditions. However, the noise derived from experimental techniques is reproducible and its boundaries can be modeled. It has been observed that in differential gene expression comparisons of any given gene, there is a greater variance in the fold-change calculation at lower signal intensities [ 1 , 2 ], and when comparing replicate samples, lower expression values tend to have greater variance in signal intensity. This suggests that larger errors can occur when lower signals are used to compute fold-changes in differential comparisons. Fold change, computed in this way, can lead to extraneous inclusions in lists of significantly up-regulated or down regulated genes. For example, a fold change of two calculated from intensities of 25 and 50 may not be as trustworthy as a fold-change of two determined between intensity values of 2,000 and 4,000. Thus, the purpose of error boundary modeling is to reduce the influence of less trustworthy fold-change calculations in the analysis of differential microarray data. The efficacy of coupling a noise boundary model to an analysis method has been previously shown for two color cDNA arrays [ 3 - 6 ]. The principal concerns when using microarray data derived from different labs to identify cancer markers is that chip-to-chip normalization cannot eliminate differences in cRNA synthesis and labeling, hybridization protocols, scanner settings and image processing software. Variable RNA quality can influence the amount of individual cRNAs generated. The laser power on scanners can differ causing saturation of high intensity probe sets and may have a more variable estimation of the very low expressed transcripts. Two studies [ 7 , 8 ] have successfully classified different types of cancer by their molecular profile on microarrays using hierarchical clustering and support vector machine (SVM) techniques. Both studies found that their markers comported a high number of genes whose expression differed among the normal tissues of origin. The approach taken in this paper is that the cancer samples are compared first to their normal tissue and then the most discriminating genes for each cancer vs. normal comparison are compared between cancers. This circumvents normalization problems due to lab-specific parameters (scanner settings, labeling, hybridization variables) and tissue specific artifacts, as each cancer biopsy is compared to its corresponding normal tissue processed by the same research group, in the same environment. These environmental parameters and artifacts are assumed to be similar for the normal and cancer biopsies and should cancel out. This allowed the selection of the genes that best discriminated between the normal and tumor samples. These classifiers were then evaluated to see if they were specific to the different types of cancers. Since gene expression measurements of individual Affymetrix GeneChips probe sets frequently do not follow a normal distribution, a non-parametric analysis was used. The commonly used t-test tests to see if two populations have a different mean but does not test the overlap of the populations. Selecting markers with minimal overlap in their expression between the normal and tumor states would improve their predictive value. We developed a method to find such markers using an un-weighted voting scheme. This non-parametric method for marker selection was chosen so that no assumptions on the shape of the data distribution were required. The computed noise boundary makes the selection criteria more stringent, eliminating many false positives signals and highlighting genes that are differentially expressed most consistently in comparisons between a cancer and its corresponding normal tissue. This integrative approach can yield a signature of distinct transcripts distinguishing a variety of solid tumors. The objectives of this work were three fold: 1). develop a noise boundary for GeneChip data, 2). develop an algorithm for selecting markers with minimal overlap in their expression between the normal and tumor states, 3). integrate the analysis of previously published data from different sources. Results and discussion Noise boundary model The noise boundary model was created to evaluate the reliability of calculated fold changes. Data from normal tissue biopsies obtained from public data sets were used to infer the noise boundary to be used when comparing the normal tissue to their corresponding cancer biopsies. The cancer biopsies were not used in designing the noise model as they are likely to be more variable than normal tissue. Three different signal extraction methods, MAS5 [ 9 , 10 ], dChip [ 11 , 12 ] and RMA [ 13 ] were compared and MAS5 was chosen as it showed stable results and is widely used. A complete analysis of the three methods is included in the supplemental data [see Additional file 2 ]. A fold-change threshold boundary was drawn for each comparison between normal tissues for each of the cancers studied to model the noise inherent to the method. The data was first sorted according to the average intensity of the values of the probe-sets for two replicate chips. If there is no noise in the technique or the biology, one should expect to have all the fold changes be 1. However when plotting the fold changes against the average intensity for the probe-sets we observed that the data formed a volcano plot with considerable scattering for low intensity and a progressive tapering of the fold changes at high intensities. As there is a lot of noise in estimating the low end expression a cut-off is needed to eliminate part of that noise. Then, as the samples were biological replicates, we assumed that most of the genes were not differentially expressed; a certain percent of the genes should not change and a percentile cut off was set up to eliminate spurious variations. The data was then binned into fixed width bins including 200 expression values. For modeling purposes, the percentile was plotted against the inverse of the average bin intensity to reveal a linear relationship that can be characterized with a slope and intercept. A sensitivity analysis to optimize the noise boundary percentile and low intensity cut off parameters was performed and is presented in the supplemental data [see Additional file 2 ]. The low intensity cutoff was set to 100 and bins with a mean expression value lower than 100 were excluded. The 80 th percentile of the fold-change, chosen as the noise boundary, was calculated for each bin. Figure 1 shows the 80 th percentile error boundaries for the five different tissues as a function of the inverse of the average bin intensity. To decrease the effect of saturation on the regression, the gene expression values in the top 8% were eliminated (this correspond to the 5 highest bins intensities in figure 1 ). The noise boundary was found to be tissue dependant and the slope and intercept were calculated for each tissue (Table 1 ). For a fold change to be considered reliable, it has to be greater than the noise boundary threshold for the same average intensity: Nonparametric microarray data analysis: Er Algorithm In microarray experiments, the number of replicates is often small and the distributions of gene expression are not normal for all genes. For the same difference in mean, depending on the distribution of the data, the overlap of two distributions can be dramatically different. Ideal markers would be genes with no overlap in their distribution; the consistency of change is therefore more significant than the amplitude. To address the problem of low numbers of replicates and multiple testing on 12,000 genes, the noise boundary model was incorporated with non-parametric data mining. The noise boundary eliminates noise that is proportional to the probe intensity measured. The combination of the non-parametric voting scheme with the noise model will be referred to as the directional change assessment algorithm. For each transcript, the ratio of expression intensities (fold change) of each cancer biopsy to each normal biopsy was determined. Those ratios were recorded and evaluated against the noise boundary model. If a ratio was above the ratio given by the noise boundary, the direction of the fold-change as increased (+) or decreased (-) expression was recorded. If the ratio was below the value given by the noise boundary for the average of the intensities, then the fold-change was considered insignificant and assigned a no change (0) direction. For each probe-set For each sample c i in the cancer class For each sample n j in the normal class If c i> n j then r = c i /n j else r = n j /c i If r>noise_boundary ((c i +n j )/2) And If c i> n j Pos_score = Pos_score + 1 If r>noise_boundary ((c i +n j )/2) And If c i ≤ n j Neg_score = Neg_score + 1 Else NoChange_score = NoChange_score + 1 We designed an index, called event ratio to summarize the overlap in distribution between the cancer and normal samples. This Er index is described by: Where the #comparisons is equal to the number of cancer samples multiplied by the number of normal samples. This method counts direction and evaluate the overlap of the distributions normalized to the number of comparisons. The Er index ranges from 0 to 1. As the direction of change for a gene becomes more consistent, Er approaches 1. Conversely, if the Er score is close to 0.5, then the gene is inconsistent with regard to its directionality and thus cannot be considered a reliable marker for disease classification. As the score approaches 0, the transcript direction and fold change cannot be reliably estimated as it is within the noise level of the technique. The software is available at . To test the validity of this approach, the samples were shuffled 100 times between the categories (cancer and normal) and the Er computation was repeated. Each time the data was shuffled, the probe sets were sorted by descending Er score and the probe set information was discarded and replaced by its rank. The average and standard deviation of the ranks was then computed and compared to the results obtained for the cancer versus normal biopsies. For all the comparisons performed, higher Er scores were obtained in the case of cancer versus normal classifications than with randomly shuffled sets. An illustration of the results obtained with the breast cancer versus normal biopsies can be seen in figure 2 . The average Er score per rank converged rapidly, and was consistent after shuffling the dataset 50 times (figure 3 ). Comparison of the Noise Boundary-Er Algorithm to standard analysis techniques To compare the Er algorithm including the noise model to other commonly used analysis methods, the replicate set from the Latin square dataset was used [ 14 ]. In this dataset fourteen specific RNAs were exogenously added to the hybridization mixture in two fold increasing concentrations. The T-test performed on this data identified all fourteen genes as well as 161 presumed false positives with a significant p-value (below 0.01). Therefore, the percentage of true positives is only 8% of the genes found significant in the result. This reflects the multiple-testing problem when using the t-test in this way. If twelve thousand tests are performed simultaneously on 12,000 genes with a type I error of 0.01 (the test is falsely considered significant one time every 100 tests), we can expect 120 (= 12,000*0.01) probe sets to be below the p-value 0.01 simply by chance. The Hochberg/Simes [ 15 ] method addresses this issue. They both found 16 genes to have a significant fold change with 11 of the 14 true positives (approx 69% true positives in the result). Another correction technique for multi-testing is the Bonferroni [ 15 , 16 ] method which found seven genes to be significant including six out of the fourteen true positives (approx. 86% true positives in the result). The SAM [ 17 ] method found 21 significant genes including 12 out of the 14 true positive (57% true positives in the result) for a delta of 1.54, and a Pi0Hat of 0.96. Although they were able to identify 85% of the exogenously added transcripts, their false positive rate was underestimated. SAM estimated a median false positive rate of 4.58%, but found 9 out of 21 probe-sets to be significant while they were not exogenously added (false positive rate of 43%). The Er model described above with a cut-off of 0.9 identified 12 genes with 8 of them being true positives (66% true positives in the result). These data suggest that the Er model is well within the separation levels of those standards techniques. However the results and performance of the different techniques might be dataset dependant. The replicate set from the Latin square dataset has little inherent noise. Even the chips at different control concentrations can be considered as technical replicates as only 14 out of 12,000 genes were supplemented. Using the noise model to remove noise from a noisier dataset might prove even more useful. It would be interesting to compare those methods on multiple datasets, but at the time of this study, this is the only dataset with an absolute knowledge of true and false positives. It is worth noting that the supplemented RNAs were added at a fold variance in concentration but that the actual intensity found averaged only 1.53 fold. All those methods greatly decrease the number of false positives compared to the t-test alone but some true positives were also missed. This is partly due to the fact that the control RNAs were added at concentrations testing the limits of detection. Cancer-specific biomarkers The Er model was used to compare each cancer biopsy to its corresponding normal tissue. In the absence of error modeling, the directional change algorithm identified 1,910 probe-sets that had an Er score above 0.9 in ovarian cancer, 1,355 in breast cancer, 1,730 in oral cancer and 322 in prostate cancer. Incorporation of error modeling dramatically reduced the number of probe-sets with Er scores above 0.9 to 272 for ovarian, 177 for breast, 129 for oral cancer and 2 for prostate cancer [see Additional file 3 ]. For lung cancer biopsies, the distinct sub-classes were compared against normal tissues and 15 probe-sets with an Er value above 0.9 in all comparisons were uncovered. The advantage of determining Er scores for differentially expressed cancer transcripts is that it provides a statistical metric that can be used to underscore markers that are unique to a particular cancer. Although the Er is not a statistical test and an Er score can vary in its significance depending on the number of samples studied, we selected genes with a high Er index in one cancer type (Er> = 0.9) and low in the others (Er<0.6). As Affymetrix HG-U95A and Hu133A contain different probe-set numbers for the same gene, the SOURCE software [ 18 ] from Stanford University was used to match the probe set to their cluster ID using the UniGene Build 167. Cluster IDs were then matched between chip types using Microsoft Access. No universal marker encompassing all the cancer vs. their normal tissue was found. This result is consistent with the result from Ramaswamy et al. [ 7 ] using 14 common tumor types including breast, prostate, ovarian and lung cancer. Nonetheless, caveolin-1 (CAV1) was found down regulated in 90% of breast, ovarian, and lung tumors, and in 80% of the prostate cancers. This gene is also down-regulated in large diffuse B-cell lymphoma [ 19 ], is associated with a region of the chromosome 7 q31 frequently deleted in tumors [ 20 ], and has been shown to have a tumor suppressing activity when restored [ 21 , 22 ]. The number of genes found to be reliable markers varied greatly between cancer types [see Additional File 1 ]. Prostate and lung cancer had the smallest number of such markers and were the 2 datasets with the most samples. The only prostate marker identified, SIM2, is a transcription factor involved in regulation of transcription during development [ 23 ]. This gene has also been found differentially expressed in colon and pancreatic cancer [ 24 ], and an antisense inhibition of SIM2-s expression in a colon cancer cell line restored growth inhibition and apoptotic cell death [ 24 ]. Two genes, AGER and MARCO, were found to be under expressed in all the lung cancer types compared to other cancer types. The advanced glycosylation end product-specific receptor (AGER or RAGE) has been previously reported down-regulated in non small cell lung carcinoma [ 25 ]. AGER is a receptor for amphoterin which mediates cell differentiation [ 26 ], and is highly expressed in lungs. Down-regulation of AGER may be a critical step in lung tumor formation as it is down regulated in all the different subtypes of lung cancer studied here. On the other hand, AGER seems to be up-regulated in pancreatic cancer and its level correlates to the metastatic potential of the cancer cell line [ 27 ]. The second gene specific to lung cancer is MARCO which is expressed by alveolar macrophages in the lung and is involved in inflammation and pathogen clearance [ 28 , 29 ]. A decrease of MARCO RNA in the sample may be due to a decrease in the number of macrophages inside the tumor compared to the normal tissue. Thirty nine probe sets were found to have an Er score above 0.9 in ovarian cancer and lower than 0.6 for the other cancers. Two of these genes, Janus kinase 1 (JAK1) and a zinc finger homeobox (ZFHX1B), which are involved in the TGF β signaling pathway regulating cell growth, were down regulated. PAX8, a gene important in development which had been identified in an earlier study [ 30 ], was found to have consistently increased expression. Three genes involved in cell growth or maintenance, MLLT2, PRSS11, FOXO3A, were down regulated. Breast cancer profiles have several interesting features. First, 16 ribosomal protein genes have decreased expression: L34 is involved in translational control [ 31 ], S27 in signal transduction, and RPS4X in development and cell cycle control. As the genes coding for those ribosomal proteins are located on different chromosomes the down regulation of these ribosomal proteins could be due to methylation of the ribosomal DNA [ 32 , 33 ]. All of the markers for breast cancer are down regulated except for inosine monophosphate dehydrogenase 1 (IMPDH1), increased by two fold, which is involved in the biosynthesis of purine nucleotides. Breast cancer has distinct sub-groups which some are hormone dependant for growth, others being very aggressive with an Her-2 amplification. The cancer samples in this study [ 8 ] are likely to be a mix of these subtypes. This might explain why the well known markers for a particular sub group do not appear in those results. However, the particular sub-classification of those 16 breast cancer samples is not known [ 8 ]. In oral cancer [see Additional File 1 ], many genes involved in differentiation of epithelial cells are found to be specific markers for this cancer. Keratin 4 and 13 (KRT4 and 13), and the small proline-rich protein 1B (SPPR1B) involved in epidermal differentiation, are all down regulated, as well as cellular adhesion genes desmoglein 1 and 3 (DSG1 and 3). The matrix metalloproteinase 13 (MMP13) gene encoding collagenase was specifically up regulated in this cancer. In the original study the up regulation of MMP1 and down regulation of KRT4 was confirmed by RT-PCR [ 34 ]. Conclusions The method described here provides improved non-parametric approaches to microarray data analysis. After applying the noise boundary model, markers were selected according to their consistency for up-regulation or down-regulation using a voting scheme comparing normal versus cancer biopsies. Tissue-specific expression differences were eliminated by comparing the cancer samples to the normal biopsies from the same tissue. The genes with the greatest differential expression between cancer and normal biopsies were then compared between cancer types. This differs from previous studies [ 7 , 8 , 35 ] which directly compare results among different cancers. Groups of markers with consistent differential expression among ovarian, breast, prostate and lung cancer were found. Many of these markers are related to de-differentiation of the tissue and were highly specific to their tissue of origin. Also, tumors arising from cells with the same embryogenic origin tend to have the same genes required for cancer progression. This confirms a previously described oncodevelopmental connection [ 36 ]. Methods All of the microarray data used in this analysis was derived from RNA isolated from biopsies and hybridized on Affymetrix GeneChips HG-U95A, HG-U95Av2 or HG-U133A. All the research groups used the same standard procedure for labeling the cRNA, hybridization and scanning the GeneChips [ 37 ]. The datasets were obtained from several different sources: Data from 24 breast cancer biopsies were from Su et al.[ 8 ], and the three corresponding normal breast tissue biopsies were provided by Garret Hampton from the Genomics Institute of the Novartis Research Foundation. For prostate cancer, the dataset was derived from 21 tumors and 8 normal biopsies [ 38 ] whereas the ovarian cancer dataset originated from 14 tumor and four normal biopsies [ 39 ]. Finally, the lung cancer dataset consisted of biopsies from 61 samples of lung adenocarcinoma, 20 lung carcinoids, six small cell lung cancer, 21 squamous lung cancers, and 17 normal lung tissues [ 40 ]. Out of the 61 adenocarcinoma samples, 19 were replicates and 52 were sub-divided into five categories according to Bhattacharjee et al.(2001) [ 40 ]: seven in cluster 1, nine in cluster 2, 15 in cluster 3, 13 in cluster 4, and eight samples of colon metastasis. The Oral cancer dataset consisted in 4 normal and 16 oral cancer biopsies [ 34 ]. The directional change assessment and the noise model algorithms were programmed using Python, and the comparison for markers was performed with Excel. Authors' contributions VA and PT conceived the study. VA carried out the analysis and drafted the manuscript. MC performed the programming. JC helped with the evaluation of raw image analysis methods. MR supervised the programming and analysis. PS and JD participated in the study design and management. Supplementary Material Additional File 2 Assessing the noise level and trust threshold for differential expression on Affymetrix GeneChips. This document compares the noise from MAS5, RMA and dChip and presents the sensitivity analysis for the noise model parameters using the Latin square replicate data set. Click here for file Additional File 3 Gene markers for prostate, breast, ovarian, oral, and lung cancer. This file presents the Top Er scores for each cancer studied when compared to its normal tissue. There is a result table with gene information for each cancer on separate tabs. Click here for file Additional File 1 Gene markers that distinguish between prostate, breast, ovarian, oral, and lung cancer. This file presents the Er scores of genes expression levels that are consistently up or down in a given cancer compared to its normal tissue (Er>0.9) but not in any of the other four cancers (Er<0.6). Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC538261.xml
521081	Elevated midbrain serotonin transporter availability in mixed mania: a case report	Background Results obtained from brain imaging studies indicate that serotonin transporter (SERT) and dopamine transporter (DAT) densities are altered in major depression. However, no such studies have been published on current mania or hypomania. Case presentation In this single photon emission computed tomography (SPECT) study with [ 123 I]nor-β-CIT we present a case with simultaneous symptoms of major depression and hypomania. She had an elevated serotonin transporter availability (SERT) in the midbrain and elevated dopamine transporter availability (DAT) in the striatum, which normalised in a one-year follow-up period during which she received eight months of psychodynamic psychotherapy. Conclusions To our knowledge, this is the first report on SERT and DAT associated with mania. In our case the availability of both SERT in the midbrain and DAT in the striatum were elevated at baseline and declined during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Symptoms of hypomania in the case were alleviated during psychotherapy. Clinical recovery was also reflected in the Hamilton Depression Rating Scale (HDRS) scores.	Background Decreased serotonin transporter densities (SERT) have been recorded from the brains of depressed patients [ 1 , 2 ], which have also been reported to normalise during psychotherapy [ 3 , 4 ]. A few studies have also revealed altered levels of dopamine transmission in major depression [ 5 , 6 ]. However, in a search of the PubMed database we found no serotonin-specific brain imaging studies on mania or hypomania. In this paper we present a case with simultaneous symptoms of major depression and hypomania. The structural diagnostic classifications DSM-IV and ICD-10 are unable to diagnose this kind of mixed state, even though similar cases have been presented earlier in the literature [ 7 ]. Materials and methods Case The index subject was a 25-year-old female with a history of two periods of major depression according to the DSM-IV-R criteria. She had also had hypomanic periods that did not completely fulfil the criteria of mania, and at baseline she fulfilled the criteria of hypomania, moderate depression and dysthymia (296.32, 300.40). She was overactive, restless and irritated and coped with a reduced amount of sleep. She had difficulty in concentrating on one thing at a time, and she appeared very lively and talkative. She was therefore diagnosed with bipolar mood disorder type II. In other words, our index patient had a mixed mania without fulfilling the criteria of full-blown mania during the baseline scan. Controls The 6 female depressed controls were diagnosed to have severe or moderate depression (296.22, 296.23, 296.32 or 296.33). Three of them also fulfilled the criteria for dysthymia (300.40) and three had previously suffered periods of major depression. They were participants of our larger study on the effects of psychotherapy on SERT and DAT densities to be reported in full elsewhere. The group of 10 healthy controls consisted mainly of employees of Kuopio University Hospital and medical students. Depression was an exclusion criterion for the control group. Controls had previously received no psychotropic medication or other psychiatric treatment and were physically healthy. Psychiatric evaluation Psychiatric diagnosis was based on clinical assessment and verified for all study subjects by a trained independent psychiatrist using the Structured Clinical Interview for DSM-IV-R (SCID-I) [ 8 ]. The severity of depression was assessed with the 17-item Hamilton Depression Rating Scale (HDRS) [ 9 ]. Setting, therapist and therapy The depressed controls (see Table for sociodemographic characteristics) received psychotherapy for 12 months in an outpatient clinic of the Department of Psychiatry of Kuopio University Hospital. The case discontinued the therapy after eight months because she felt it was no longer useful to her. The case and the depressed controls had received no psychiatric treatment or medication prior the SPECT imaging and they were treated without medication. Psychotherapy sessions occurred twice a week, 80 sessions per year. The psychotherapists had formal postgraduate professional training in psychodynamic psychotherapy. The study design was approved by the ethics committee of Kuopio University Hospital. Table 1 Characteristics of the participants. Values of the controls are means ± SD. Case Depressed controls n = 6 Healthy controls n = 10 Age, y 25 27.2 (5.9) 26.3 (5.9) Duration of depressive episode, months 9 10.6 (6.9) - SERT availability in midbrain at baseline 1.51 1.08 (0.12) 1.28 (0.12) SERT availability in midbrain at 12 months 1.36 1.23 (0.20) - DAT availability in striatum at baseline 2.75 2.48 (0.35) 2.45 (0.25) DAT availability in striatum at 12 months 2.61 2.70 (0.45) - HDRS score at baseline 15 17.67 (3.44) - HDRS score at 12 months 5 12.66 (5.09) - Abbreviations: DAT: Dopamine transporter, HDRS: Hamilton Depression Rating Scale SD: Standard deviation, SERT: Serotonin transporter. Participants of the study were right-handed and all were female. The depressed and the healthy controls were age-matched with the case (Table). The healthy controls were not followed up. Imaging procedure SPECT imaging was performed on the Wednesday after the menstrual bleeding that preceded the psychotherapy and on 12-month follow-up. A dose of 185 MBq of [ 123 I] nor-β-CIT (supplied by MAP Medical Technologies OY, Tikkakoski Finland) was diluted in a volume of 10 ml physiological saline. The specific activity was higher than 1.8 × 10 11 Bq/μmol [ 10 ]. The dose was slowly injected into the right antecubital vein in a dimly lit and quiet room. Serial SPECT scans (5 min, 6 h and 24 h) were performed on a dedicated Siemens MultiSPECT 3 gamma camera with fan-beam collimators (Siemens Medical Systems; Hoffman Estates I11., USA) [ 11 ]. Head positioning was monitored during acquisition by using two position lasers. Data analysis The SPECT scans were decay-corrected and reconstructed with Butterworth-filtered back-projection in a 128 × 128 matrix with a pixel size of 3 × 3 mm, and were attenuation-corrected with Chang's algorithm [ 10 , 11 ]. The imaging resolution was 8–9 mm. The SPECT slices were consecutively summarised to the slice thickness of 6 mm and re-aligned using a Siemens semi-automatic brain quantification program and the Talairach coordinates [ 12 ]. The slices were rotated and re-aligned so that transaxial (x-direction), sagittal (y-direction) and coronal (z-direction) slices were at right angles to each other. Region of interest placement was based on a Siemens semi-automatic brain quantification program. The lower threshold of 60% of the maximum count was used to reduce the volume averaging and partial volume errors. Regions of interest were the cerebellum, striatum and the midbrain. It was assumed that the cerebellum (reference region) corresponds to a two-compartment model (unbound tracer in arterial blood and free plus non-specifically bound tracer in the tissue) [ 13 ]. The specific binding in ml/ml for SERT and DAT was calculated using a graphical plot [ 13 ]. The slope of this plot is equal to the distribution volume ratio: (Region - Cerebellum)/Cerebellum = V D - 1. The striatal uptake was pooled. Functional neuroanatomy by means of SPECT was confirmed using magnetic resonance imaging (MRI) within two weeks of the SPECT imaging. If any cerebral focal abnormalities or organic brain diseases were detected by MRI scan, the patient was excluded from our study. The participants of our study thus had normal findings. Reproducibility of SPECT The reproducibility of the SPECT scan had been previously studied with eleven healthy subjects (5 males and 6 females; age range 20–39 y). SPECT studies were performed twice with a 12-month interval. The correspondence between the studies was good, with the mean difference being 0.00 ± 0.08 (SD = standard deviation) for SERT (mean and SD: 1.27 ± 0.11 and 1.27 ± 0.14, respectively) and 0.04 ± 0.18 for DAT (mean and SD: 2.49 ± 0.28 and 2.45 ± 0.27, respectively). The intraclass correlation coefficient was 0.82 (p < 0.01) and 0.79 (p < 0.01), respectively (unpublished data). Statistics The Student's t-test was used to compare the depressed controls and the healthy controls and paired samples t-test to compare SERT densities of the depressed controls at baseline and on follow-up. A p-value of less than 0.05 was considered as the criterion for statistical significance. Results The background characteristics of the case and the controls are presented in the table. The case and the depressed controls had HDRS scores of 14 or more. The midbrain SERT availability did not correlate with HDRS scores in depressive controls either at baseline or on follow-up. Neither did the change in the HDRS score correlate with changes in SERT or DAT capacities under therapy. The case had an elevated SERT availability in the midbrain at baseline, while the depressed controls had decreased levels compared to the healthy controls (t = 3.17, p < 0.01; Table and Figure 1 ). The SERT availability of the index case was two standard deviations (SD) higher than the mean SERT availability of the depressed controls and almost two SD higher than the mean SERT availability of the healthy controls. The MRI scans of the case and the controls were normal. At the twelve-month follow-up, the HDRS scores of both the case and the depressed controls had decreased. The mean decrease in HDRS scores in depressed patients during the follow-up period was 5 (SD 3.3; t = 3.7, p = 0.02). The SERT availability in the midbrain had decreased in the case by 9.9% (Fig. 2 ) and increased in the depressed controls by 12.5% (t = 3.00, p = 0.03) during the one year of psychotherapy. Figure 1 SERT densities of the healthy controls, depressed controls and of the index subject at baseline. Figure 2 Transaxial slices of [ 123 I]nor-β-CIT scans in the index subject at baseline (A and B) and after 12 months (C and D). A and C indicate midbrain SERT binding and B and D striatal DAT binding. A 10% step colour scale is shown on the right and the cerebellum was used in its normalization. DAT densities in the striatum were elevated in the case (Table, Fig. 2 .) and slightly elevated in the depressed controls at baseline compared to the healthy controls. On 12-month follow-up the DAT availability in the striatum of the case had decreased by 5.1%, while DAT densities in the depressed controls had increased by 8.8%. The increase in DAT binding was not significant (data not shown). Discussion To our knowledge, this is the first report on SERT and DAT associated with a current mania. In our case the densities of both SERT in the midbrain and DAT in the striatum were elevated at baseline and they decreased during psychotherapy, while the SERT and DAT of the depressed controls increased during psychotherapy. Clinical recovery was also reflected in the change in the HDRS score. Furthermore, the index case had no symptoms of hypomania after the psychotherapy. Ichimiya et al. [ 14 ] found increased SERT binding of the radioligand in the thalamus in a sample of patients with either major depression (n = 7) or bipolar disorder (n = 6) compared to healthy controls (n = 21). In their study the bipolar patients were either depressed or euthymic prior to brain imaging. There were no significant differences in binding potentials in the midbrain compared to the mood-disorder patients and the healthy controls. Bipolar patients had slightly higher binding potentials in the midbrain than the healthy controls, while patients with major depression had slightly lower binding potentials than the healthy controls. However, these differences were not significant. The patients of Ichimiya and co-workers were euthymic or depressed, so we cannot compare our results with theirs. It is clinically well known that anti-depressive medication, including serotonin-selective drugs, may aggravate mania [ 15 ]. If the serotonergic functions had already been elevated in mania and had then been further enhanced by anti-depressive medication, it is reasonable that this might further increase the manic symptoms. In animal models, the administration of an amino-acid mixture lacking the cathecolamine precursors tyrosine and phenylalanine decreases the availability of plasma tyrosine to the brain, which further diminishes cathecolamine synthesis [ 16 ]. In a few clinical human studies, decreasing tyrosine availability has had a positive effect on the symptoms of acute mania [ 17 , 18 ]. Tyrosine depletion has also led to decreased dopamine functions in healthy volunteers [ 19 ]. In positron emission tomography, a tyrosine-free amino-acid mixture increased striatal binding of the dopamine receptor ligand in healthy volunteers, which may reflect lowered presynaptic dopamine release [ 20 ]. SPECT studies revealing increased amphetamine-induced dopamine release in bipolar disorder patients have also been published [ 21 ]. The above-mentioned results indicate that acute mania may be associated with elevated dopaminergic functions in the central nervous system. A decrease in the DAT availability of the striatum was associated with clinical recovery in our case, which may support this hypothesis. The DAT densities of the depressed controls were only slightly above the level of the healthy controls. Laasonen-Balk et al. [ 5 ] have previously observed a greater increase in DAT densities in depressed patients. Because of the small sample size of this study there was insufficient statistical power to draw any conclusions in this issue. Our results support previous findings that the SERT densities of depressed patients are lower than in healthy controls [ 1 , 2 , 4 ]. Previous studies on neurotransmitters in association with mania have mostly dealt with dopamine. We cannot completely rule out the bias of nor-β-cit binding to the noradrenergic transporters (NORT). The noradrenergic cell-body rich nucleus ceruleus is close to our target region. This is assumed to contain the nucleus raphe, which is mostly comprised of serotonergic cell-bodies. The dopamine cell-body rich substantia nigra is also located close to our region of interest. However, nor-β-cit is considered to be more serotonin-specific than previous radioligands [ 10 ]. Three studies have been published on the association between SERT availability and the serotonin transporter genotype. Two of these were performed on healthy subjects [ 22 , 23 ], while the third concerned abstinent alcoholics and healthy controls [ 24 ]. In the study of Van Dyck and co-workers the short homozygotes had a significantly greater SERT availability than the long-short heterozygotes, which indicates a complex relationship between the genotype and SERT availability. Furlong et al. [ 25 ] found an association between promoter allele 2 of the SERT gene and bipolar disorder. Studies combining brain imaging and data of genotype are also recommended in the evaluation of bipolar disorder. PET would be a more valid method if adequate radioligands were available [ 14 ], as PET provides absolute rather than relative values for transporter availability. Conclusion We have found no previous imaging studies showing that serotonin plays a role in mania. Further research is needed to determine whether our findings could be generalised to all manic states. Competing interests None declared. Authors' contributions TT planned the study and analysed and interpreted the data. MJ also interpreted the data. PIS initiated and planned the study and organised the psychotherapy setting. HM took care of the imaging procedures. PA organised the psychotherapy setting. RV was responsible for the MRI scans and the interpretation of the scans. JK performed SPECT analyses. JT participated in the design of the study. JL initiated and planned the study project. All authors read, critically revised and approved the final manuscript. Abbreviations DAT: Dopamine transporter HDRS: Hamilton Depression Rating Scale SD: Standard deviation SERT: Serotonin transporter SPECT: Single photon emission computed tomography Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC521081.xml
368157	A Spontaneous, Recurrent Mutation in Divalent Metal Transporter-1 Exposes a Calcium Entry Pathway	Divalent metal transporter-1 (DMT1/DCT1/Nramp2) is the major Fe 2+ transporter mediating cellular iron uptake in mammals. Phenotypic analyses of animals with spontaneous mutations in DMT1 indicate that it functions at two distinct sites, transporting dietary iron across the apical membrane of intestinal absorptive cells, and transporting endosomal iron released from transferrin into the cytoplasm of erythroid precursors. DMT1 also acts as a proton-dependent transporter for other heavy metal ions including Mn 2+ , Co 2+ , and Cu 2 , but not for Mg 2+ or Ca 2+ . A unique mutation in DMT1, G185R, has occurred spontaneously on two occasions in microcytic (mk) mice and once in Belgrade (b) rats. This mutation severely impairs the iron transport capability of DMT1, leading to systemic iron deficiency and anemia. The repeated occurrence of the G185R mutation cannot readily be explained by hypermutability of the gene. Here we show that G185R mutant DMT1 exhibits a new, constitutive Ca 2+ permeability, suggesting a gain of function that contributes to remutation and the mk and b phenotypes.	Introduction Spontaneous mutations in mice and rats have provided important information about mammalian iron homeostasis (reviewed in Andrews 2000 ). Interestingly, three independent, autosomal recessive mutants have been shown to have the same amino acid substitution in a key iron transport molecule. Two strains of mutant microcytic (mk) mice (MK/ReJ- mk , SEC/1ReJ- mk ) and Belgrade ( b ) rats have severe iron deficiency attributable to a G185R mutation in divalent metal transporter-1 (DMT1) ( Fleming et al. 1997 ; Andrews 2000 ). Based on the phenotypes of these animals and the properties of DMT1 detailed below, we and others concluded that DMT1 is essential for intestinal absorption of Fe 2+ and for unloading of transferrin-derived iron from transferrin cycle endosomes ( Fleming et al. 1997 , 1998 ; Gunshin et al. 1997 ; Picard et al. 2000 ). It is intriguing that no other DMT1 mutations have been described in mammals, and no features of the DNA sequence suggest that the G185 codon would be hypermutable in two species. We speculated that a novel characteristic of the G185R DMT1 protein might account for this remarkable pattern of remutation. Trace metal ions including Fe 2+ , Mn 2+ , Cu 2+ , Zn 2+ , and Co 2+ are required cofactors for many essential cellular enzymes. They cannot cross the plasma membrane through simple diffusion, and active uptake requires specific transporters. DMT1 is the only molecule known to mediate cellular iron uptake in higher eukaryotes. It is structurally unrelated to known Zn 2+ and Cu 2+ transporters, but DMT1 can transport those and other divalent metal ions ( Gunshin et al. 1997 ), and it appears to be the major mammalian Mn 2+ transporter ( Chua and Morgan 1997 ). DMT1 is predicted to have 12 transmembrane (TM) segments ( Figure 1 A). It is expressed on the apical brush border of the proximal duodenum ( Canonne-Hergaux et al. 1999 ) and in transferrin cycle endosomes ( Su et al. 1998 ; Gruenheid et al. 1999 ). It appears to function by coupling a metal entry pathway to a downhill proton gradient, taking advantage of the acidic pH in both of those sites. An earlier study proposed a 1:1 stoichiometry of metal ion and proton cotransport ( Gunshin et al. 1997 ). Figure 1 Wild-Type DMT1-Expressing Cells Exhibit a Proton Current and a Proton-Dependent Mn 2+ -Induced Current (A) The G185R mutation is in the fourth of 12 putative TM domains in both mouse (shown) and rat DMT1 proteins. (B) 55 Fe 2+ uptake was greatly reduced for G185R in comparison to wild-type DMT1, although the protein expression levels were comparable (inset). (C–E) Representative currents induced by protons (pH 4.2) and Mn 2+ (100 μM) at +50 mV (open triangles; some of the datapoints have been removed for clarity) and −130 mV (open circles) in a wild-type DMT1-transfected CHO-K1 cell. Whole-cell currents were elicited by repeated voltage ramps (−140 to +60 mV, 1,000 ms), shown in (E), with a 4 s interval between ramps. Holding potential (HP) was +20 mV. Neither control solution (10mM Ca 2+ /140 mM Na + /[pH7.4]) nor isotonic Ca 2+ (105 mM) solution induced significant current. Representative I-V relations are shown in (E). Current responses from a vector (pTracer)-transfected cell are shown in (D). (F) pH-dependence of the E rev of the wild-type DMT1 current in the presence or absence of 300 μM [Mn 2+ ] o . In the absence of Mn 2+ , the pH dependence of the E rev can be fitted by a line with a slope 58 mV/pH unit. In the presence of 300 μM Mn 2+ , the relationship was nonlinear, especially at higher pH. E H , H + equilibrium potential. Note that the currents were not leak-subtracted. Ca 2+ is not a measurable substrate for wild-type DMT1 ( Gunshin et al. 1997 ; Tandy et al. 2000 ), even though it is at least 1,000 times more abundant in plasma than trace metals. Surprisingly, we found that the G185R mutation ( Figure 1 A) dramatically increases the Ca 2+ -permeability of DMT1, functionally converting DMT1 into a Ca 2+ channel. In light of the important and ubiquitous role of Ca 2+ in cell signaling ( Berridge et al. 2003 ), this gain of function offers a likely explanation for the remutation. Interpretations of recent structural data have already suggested that permeation pathways exist within some transporters ( Hirai et al. 2002 ), blurring the distinction between transporters and ion channels ( DeFelice and Blakely 1996 ). Our finding, that a single amino acid substitution in a presumed transporter can expose a channel pathway, strongly supports this notion and provides new insight into what must be viewed as a continuum between transporter and channel activities. Results We studied wild-type DMT1 and the G185R mutant proteins by whole-cell patch–clamp in transiently expressing CHO-K1 and HEK-293T cells and in doxycycline-inducible DMT1-HEK-On and G185R-HEK-On cells. Consistent with previous studies, DMT1 expression significantly increased cellular 55 Fe 2+ uptake at low pH ( Figure 1 B). As reported in Xenopu s oocytes ( Gunshin et al. 1997 ), reduction of extra-cellular pH in the absence of metal (nominal free [Fe 2+ ] o of approximately 0.05 μM) induced large inward currents in DMT1-expressing cells ( Figure 1 C and 1 D). This current is referred to as a substrate-free “leak” pathway and is representative of “drive-slip” phenomena seen in DMT1 and a related yeast metal transporter, SMF1p ( Sacher et al. 2001 ), as well as many other transporters ( Nelson et al. 2002 ). Because we found that protons also activated an endogenous diisothiocyanostilbene 2,2-disolphonic acid (DIDS)-sensitive anion conductance (unpublished data) that was strongly outwardly rectifying ( Figure S1 ), we used SO 4 2– to replace most of the Cl – ([Cl – ] o = 5 mM) in low-pH bath solutions. With elimination of the background Cl – current, the proton-evoked current was inwardly rectifying (hyperbolic) ( Figure 1 E). The large proton-induced current caused significant DMT1-specific intracellular acidification ( Gunshin et al. 1997 ). In whole-cell recordings of DMT1 currents, we routinely observed slow inactivation (or decay) after a proton-induced current reached its peak (see Figure 1 C). While the extent of the slow inactivation varied from cell to cell, it usually reached a relative steady state within 100 s. Addition of 100 μM Fe 2+ (data not shown) or Mn 2+ induced an additional current with less pronounced slow inactivation ( Figure 1 C). Because Fe 2+ is readily oxidized to Fe 3+ in the absence of substantial concentrations of reducing agents (e.g., ascorbate), and Fe 3+ is not transported by DMT1 ( Gunshin et al. 1997 ; Picard et al. 2000 ), we have used Mn 2+ as an Fe 2+ surrogate since both metals induced similar currents ( Gunshin et al. 1997 ; unpublished data). The observed Mn 2+ deficiency of b rats in vivo ( Chua and Morgan 1997 ) also supports its use in this role. H + alone or H + /Mn 2+ induce distinct currents in DMT1. No significant voltage- or time-dependent fast inactivation was seen when the DMT1-mediated H + /Mn 2+ current (I DMT1 ) was recorded ( Figure S2 ). The amplitude of additional Mn 2+ -induced current was dependent on [Mn 2+ ] o , with a measurable response at [Mn 2+ ] o < 1 μM (pH 4.2). In the presence of 100 μM Mn 2+ (pH 4.2), the additional Mn 2+ -induced current was typically half the amplitude of the proton-induced current. Addition of Mn 2+ alone (100 μM) at pH 7.4 did not induce any additional current. Since H + or H + /Mn 2+ induced two currents with distinct kinetics in DMT1-expressing cells, the underlying charge-carrying ion species and their relative contributions to the macroscopic currents were investigated. We monitored the reversal potential (E rev ) and the current amplitude in ion-substitution experiments. Replacement of Na + with N -methyl-D-glucamine (NMDG + ) did not significantly change the E rev of H + or H + /Mn 2+ -induced currents, although the net current amplitude was slightly increased ( Figure S3 ). On the other hand, the current amplitude (data not shown) and E rev of the proton current were strongly affected by [H + ] o (see Figure 1 F). The slope of E rev versus pH was 58 mV/decade, is consistent with an H + -permeable pore. The large positive displacement in E rev from E H (see Figure 1 F) may result in part from leak and capacitance-charging, but the carrier mechanism is not well understood. In contrast, when Mn 2+ was introduced, the slope of the curve fitted to E rev versus pH deviated considerably from the theoretical slope for a H + -permeable electrode (see Figure 1 F). Replacement of Na + by NMDG + did not significantly affect the Mn 2+ -induced response (see Figure S3 ). Our interpretation of this deviation is that DMT1 transport stoichiometry is variable ( Chen et al. 1999 ; Sacher et al. 2001 ; Adams and DeFelice 2002 ) or has a fixed but very low permeation ratio (P Mn /P H ) ( Hodgkin and Horowicz 1959 ). P Mn /P H can be estimated from the slope of E rev versus pH based on an extended Goldman–Hodgkin–Katz equation ( Lewis 1979 ) with two permeable ions (H + and Mn 2+ ). At pH 4.2, the slope of E rev versus pH did not differ significantly with or without Mn 2+ (see Figure 1 F). Therefore, we estimate that at pH 4.2 the contribution of H + to I DMT1 is much larger than that of Fe 2+ /Mn 2+ (P Mn /P H < 0.01), in contrast to the 1:1 stoichiometry proposed previously ( Gunshin et al. 1997 ). Importantly, no Ca 2+ permeability was observed, even in isotonic (105 mM) Ca 2+ solution (see Figure 1 C). In G185R-expressing cells, we observed a large inward current in control bath solution (10 mM Ca 2+ and 140 mM Na + ) at pH 7.4 ( Figure 2 A), though no significant current was detected with wild-type DMT1 under similar conditions (see Figure 1 E). This inward current mediated by G185R mutant DMT1 (I G185R ) was stable over minutes with no slow inactivation (see Figure 2 A), in contrast to the DMT1-mediated proton current (see Figure 1 C). We observed I G185R in more than 85% of enhanced green fluorescent protein (EGFP)-positive cells transfected with the pTracer-G185R construct and in stable, doxycycline-induced G185R-HEK-On cells, but never in cells transfected with wild-type DMT1 ( Figure 1 C) or with 30 DMT1 mutations at other positions ( n > 300 cells; unpublished data). The inwardly rectifying current was cationic, since Ca 2+ and Na + substitution by NMDG + completely abrogated the current (see Figure 2 A and 2 B). The current and rectification profiles were not significantly changed when ATP and Mg 2+ were omitted from the intracellular solution, or when Na + or K + replaced Cs + as the primary intracellular cation. Figure 2 G185R-Expressing Cells Display a Constitutive [Ca 2+ ] o -Dependent Cationic Current (A–B) Large inward currents were evoked by control solution (10mM Ca 2+ /140 mM Na + [pH 7.4]) in G185R-transfected cells. The current was inhibited by lowering the solution pH to 5.8 without altering other ions. Further reducing the pH to 4.2 induced I DMT1 -like current (enhanced by adding 100 μM Mn 2+ ). No significant inward current was seen in NMDG + (Na + -free, Ca 2+ -free) solution. (C) Time- and voltage-dependent kinetics of I G185R recorded in control solution in response to voltage steps. (D) Current densities (mean ± SEM, n = 15) of I G185R in control solution mea-sured at various voltages and normalized by cell capacitance. (E) Time- and voltage-dependent kinetics of I G185R in the presence of 105 mM Ca 2+ . (F) Ca 2+ is more permeant than Na + in G185R-expressing cells. We found that low pH strongly inhibited I G185R (by approximately 90% at pH 5.8; Figure 2 A), in contrast to both wild-type DMT1 currents, which were activated at low pH. However, further reduction to pH 4.2 revealed a current ( Figure 2 A and 2 B) that was similar to the proton current of wild-type DMT1. Addition of Mn 2+ at pH 4.2 enhanced the inward current, as with wild-type DMT1 ( Figure 2 A and 2 B). The proton current and Mn 2+ -induced response displayed similar patterns of inactivation and further activation as in wild-type DMT1-transfected cells, but both currents were much smaller than their wild-type counterparts. Consistent with this result and our previous uptake studies ( Su et al. 1998 ), we found that G185R cells had much lower Fe 2+ uptake (approximately 10% measured at 16 min) compared to wild-type DMT1 at similar protein expression levels (see Figure 1 B). I G185R rectified more steeply with voltage than I DMT1 , probably due to pronounced time- and voltage-dependent fast inactivation ( Figure 2 C; see Figure S2 for comparison). Fast inactivation was enhanced when [Ca 2+ ] o was increased to 105 mM ( Figure 2 E), strengthening the notion that I G185R was fundamentally distinct from the currents mediated by wild-type DMT1. In control bath solution (10 mM Ca 2+ , 140 mM Na + [pH 7.4]), I G185R was 64 ± 7 pA/pF at −140 mV (mean ± SEM, n = 15; Figure 2 D) compared to less than 2 pA/pF in mock and DMT1-transfected cells. I G185R reversed at approximately +20 mV with very little current above 0 mV ( Figure 2 D), whereas the E rev of I DMT1 was approximately +50 mV at pH 4.2. The dependence of I G185R on holding potential was also distinct from I DMT1 (see below). We next investigated the cation selectivity of I G185R . The amplitude of I G185R was strongly dependent on [Ca 2+ ] o ( Figure 2 F). With 10 mM Ca 2+ in the bath, replacement of 140 mM NMDG + by 140 mM Na + only slightly (by approximately 15%) increased the current, indicating that Ca 2+ permeated the plasma membrane of G185R-transfected cells much more readily than Na + . As shown in Figure 3 A and 3 B, increasing [Ca 2+ ] o not only augmented the current amplitude but also shifted E rev toward depolarized potentials. The slope of this shift (25 mV per decade) was close to the slope of 29 mV per decade predicted by the Nernst equation for a Ca 2+ -selective electrode ( Figure 3 C). The relative permeability of various divalent cations was studied under bi-ionic conditions (pipette solution containing Na + and Glutamate; see Materials and Methods ). After adding 10 mM test divalent cations to the NMDG + solution, we recorded currents using step voltages from two holding potentials (-60 mV and +40 mV). We determined G185R-specific currents by measuring the reversal potentials of the currents subtracted from two holding potentials (see Figure 4 A and 4 B) and corrected for the junction potential. The permeability sequence was Ca 2+ > Sr 2+ > Ba 2+ as calculated ( Equation 2 ; see Materials and Methods ) and illustrated in Figure 3 E. For divalent cations, we found that the highest conductance was to Ca 2+ , followed by Sr 2+ and Ba 2+ ( Figure 3 D). While Ca 2+ , Sr 2+ , and Ba 2+ currents were relatively stable over time, currents mediated by Mn 2+ and Mg 2+ were transient ( Figure 3 D), the simplest explanation for this behavior being a block by these two weakly permeant ions. The monovalent permeability was calculated using Equation 1 ( see Materials and Methods ), yielding a selectivity sequence Li + > Na + > K + > Cs + ( Figure 3 E). Under these conditions, P Mn was insignificant. The cationic permeability sequence ( Figure 3 E) of I G185R was similar to L-type voltage-gated Ca 2+ channels (VGCCs) ( Sather and McCleskey 2003 ), but I G185R was less Ca 2+ -selective (P Ca /P Na of approximately 10) than VGCCs (P Ca /P Na of approximately 1,000). Single i G185R channels were not observed in cell-attached patches. Analysis of membrane current noise at −100 mV predicted a single-channel chord conductance of 0.4 ± 0.1 pS ( n = 5; unpublished data), too small to be observed under most patch–clamp conditions. Figure 3 Ca 2+ Permeability of I G185R (A) Whole-cell I-V relations in the presence of [Ca 2+ ] o are indicated. (B) Enlarged view of (A) to show the E rev measurement. (C) [Ca 2+ ] o dependence of E rev . The slope was fit by linear regression to 25 mV per decade, close to the 29 mV per decade predicted for a Ca 2+ -selective electrode (dotted line). (D) Currents through G185R in various isotonic divalent solutions. I-Vs are shown in the inset. Note that currents induced by isotonic Mg 2+ and Mn 2+ were transient. (E) Relative permeability of various divalent and monovalent cations . The reversal potentials of I G185R in 10 mM test divalent cations were measured under bi-ionic conditions as described in Materials and Methods . The permeability was calculated using Equations 1 and 2. (F) [Ca 2+ ] i changes estimated by Fura-2 fluorescence in response to an elevation of [Ca 2+ ] o from 1 to 30 mM. The results were averaged from five (HEK-On) and seven (G185R) independent experiments ( n = 3–13 cells each). To minimize potential endogenous depletion-activated and/or TRP-mediated Ca 2+ influx, cells were bathed in the presence of 50 μM SKF96365 and 50 μM 2-APB. The F340/F380 ratio was recorded and converted into estimated [Ca 2+ ] i based on an ionomycin-induced Ca 2+ calibration. Figure 4 Voltage Dependence and Pharmacological Properties of I G185R (A) Whole-cell currents recorded in 105 mM [Ca 2+ ] o were dependent on holding potential before the voltage ramps (−140 to −120 mV shown). For clarity, only the first 20 ms of the 4 s-long holding potential is shown. (B) Voltage dependence of I G185R in control solution and 105 mM [Ca 2+ ] o . I DMT1 (dotted line) exhibited no depen-dence on the holding potential. Abbreviations: V 1/2 , half activation voltage. κ, slope factor. (C and D) Sensitivity of I G185R to various pharmacological agents and cation channel blockers. I G185R was relatively insensitive to RR, 2-APB, or SKF96365, but was blocked by 1mM La 3+ or Cd 2+ (D). Using the Ca 2+ indicator dye Fura-2, we demonstrated G185R-mediated Ca 2+ influx by monitoring intracellular Ca 2+ levels in response to an elevation of [Ca 2+ ] o ( Figure 3 F). To minimize the contributions of endogenous Ca 2+ -influx and/or store release, we bathed cells in the presence of 50 μM SKF96365 and 50 μM 2-APB. Upon raising [Ca 2+ ] o , [Ca 2+ ] i rose from 105 nM to 240 nM in doxycycline-induced G185R-HEK-On cells, significantly higher than in control HEK-On cells treated with doxycycline. Thus, the permeability of G185R to Ca 2+ is capable of increasing [Ca 2+ ] i . I G185R displayed hyperpolarization-induced inhibition ( Figure 4 A and 4 B) ( Bakowski and Parekh 2000 ). The half-maximal activation voltages (V 1/2 ) were −33 mV and −10 mV for control and isotonic Ca 2+ solutions, respectively ( Figure 4 B). The voltage-dependence of I G185R was Ca 2+ -independent, since the Na + and Li + currents in nominal [Ca 2+ ] o also exhibited a similar voltage dependence. By contrast, I DMT1 lacked this voltage dependence ( Figure 4 B). I G185R was not enhanced under low-divalent conditions (less than 10 nM), nor was it blocked by antagonists of known Ca 2+ -permeant channels. In particular, the current was not blocked by ruthenium red (RR), Ca 2+ -release activated Ca 2+ channel (CRAC) blockers SKF96365 and 2-APB ( Kozak et al. 2002 ; Prakriya and Lewis 2002 ) ( Figure 4 C), or the L-type VGCC blocker nifedepine (10 μM). Divalent cations, including DMT1 substrates (Cd 2+ , Ni 2+ , Co 2+ ), inhibited I G185R . La 3+ (1 mM; Figure 4 C) and Cd 2+ (1 mM) blocked I G185R in a similar voltage-dependent manner ( Figure 4 D). Thus, I G185R is distinct from known Ca 2+ -permeant channels such as VGCCs, transient receptor potentials (TRPs), and CRAC currents, based on its current–voltage (I-V) relation, kinetics, permeation properties, and pharmacological sensitivity. To investigate whether G185R-induced Ca 2+ permeability might play a physiological role in the mutant animals, we recorded from intestinal enterocytes isolated from both wild-type and homozygous mk mice. We studied cells from the proximal 1 cm of the mouse duodenum, where DMT1 expression is highest and iron absorption is maximal ( Gunshin et al. 1997 ; Canonne-Hergaux et al. 1999 ). Because DMT1 expression is very low in iron-replete, wild-type mice, but induced in iron-deficient mice ( Canonne-Hergaux et al. 1999 ), we isolated enterocytes from mice that had been made iron-deficient by prolonged feeding of an iron-deficient diet, and confirmed DMT1 induction by Western blotting using a DMT1-specific antibody (unpublished data). We were able to record I DMT1 -like currents in mature enterocytes that stained positive for alkaline phosphatase (I > 80 pA at −130mV, n = 7 out of 20 cells; representative data shown in Figure 5 A and 5 B). Figure 5 DMT1-Like and G185R-Like Currents in Enterocytes Isolated from Wild-Type and mk / mk Mice, Respectively (A) Enterocyte currents isolated from an iron-deficient wild-type mouse (−Fe). Reducing bath pH (140 mM NaCl) induced a slowly desensitizing inward current that was further enhanced by addition of Mn 2+ . (B) Both proton and H + /Mn 2+ currents were inwardly rectifying. (C and D) An mk enterocyte expressed a large constitutive inward current in control bath solution. Reducing the bath pH (140 mM NaCl) first inhibited and then activated another inward current insensitive to the holding potential. This slowly-desensitizing current displayed a less steeply rectifying I-V as shown in (D). Mice homozygous for the mk mutation express large amounts of G185R DMT1 protein in the duodenum. Although much of it is mislocalized to the cytoplasm ( Canonne-Hergaux et al. 2000 ), we expected that some would be present in the plasma membrane. Accordingly, and in contrast with wild-type enterocytes, we recorded a large, constitutive inward current in most mature mk enterocytes ( n = 6 out of 8 cells; Figure 5 C and 5 D), which displayed the same conductance as seen in G185R-transfected cells. The I-V relationship, step current response, dependence on holding potential, ion selectivity and insensitivity to RR, and SKF96365 or 2-APB were indistinguishable from those of transfected I G185R . Furthermore, H + inhibited the I G185R -like current in mk enterocytes, and the H + /Mn 2+ -induced DMT1-like current at pH 4.2 ( Figure 5 C and 5 D) was insensitive to holding potential, as observed in transfected cells. Based on these observations, we conclude that the major current observed in mk enterocytes was I G185R . Although our preparation did not allow us to distinguish apical versus basolateral localization, the large size of the current in mk cells was consistent with plasma membrane localization of G185R protein. Discussion We conclude that expression of G185R in transfected cells and in vivo in mk mice is associated with the appearance of a novel Ca 2+ permeation pathway that has the properties of a Ca 2+ channel. One interpretation is that a Ca 2+ channel pathway through the DMT1 protein is exposed or augmented by the G185R mutation. Another possibility is that Ca 2+ conduction occurs through an associated Ca 2+ -permeable protein. We favor the first possibility because the Ca 2+ conductance has been observed in diverse cell lines expressing G185R DMT1 (CHO-K1, HEK293T, and HEK-On cell lines) and in mk enterocytes. A putative associated protein, if present in these different cell types, would have to be activated in a G185R-dependent manner. We did not find evidence of an associated protein when we immunoprecipitated wild-type or G185R DMT1 from transfected CHO-K1 cells (unpublished data). Furthermore, a distinct DMT1 mutant, G185K, also displayed Ca 2+ permeability, but this mutant was less selective for Ca 2+ over Na + (unpublished data). G185R mutations have occurred at least three times in rodents, which suggests that G185R not only inactivates DMT1, but may confer an unknown selective advantage. Because it has arisen in inbred colonies, the postulated selective advantage must either make the animals more viable than other DMT1 mutants with impaired iron transport or more likely to be noticed by those managing the animal colonies. In parallel with these studies, we have generated knockout mice homozygous for a null DMT1 allele ( Dmt1 –/– ; H. Gunshin and N. C. Andrews, personal communication). Although detailed phenotypic characterization has not yet been completed, we have noted that Dmt1 –/– mice invariably die by the end of the first week of life, in contrast to mk/mk mice, which are poorly viable but can survive for more than a year (H. Gunshin and N. C. Andrews, personal communication). This suggests that the small amount of residual function of G185R DMT1, perhaps in combination with its gain-of-function Ca 2+ conductance, contributes to viability. Two previous studies support the notion that the gain-of-function reported here is an advantage. Elevated intracellular [Ca 2+ ] has been reported to increase nontransferrin-bound iron uptake through an undefined transport system that has characteristics distinct from DMT1 ( Kaplan et al. 1991 ). This might ameliorate the iron-transport defect caused by inactivation of DMT1, either in the intestine or in erythroid precursors. The transferrin cycle is essential for iron uptake by erythroid precursor cells ( Levy et al. 1999 ), and DMT1 mediates at least some of the transfer of iron from transferrin cycle endosomes to the cytoplasm ( Fleming et al. 1998 ; Gruenheid et al. 1999 ; Touret et al. 2003 ). Elevated [Ca 2+ ] i has been reported to accelerate iron uptake through the transferrin cycle, apparently through activation of protein kinase C ( Ci et al. 2003 ). Thus, the influx of Ca 2+ might potentiate the residual DMT1 iron-transport activity. Accordingly, 55 Fe uptake by mk/mk reticulocytes has been reported to be approximately 45% of the level observed in wild-type reticulocytes ( Canonne-Hergaux et al. 2001 ), higher than expected for a severe loss-of-function mutation. In summary, we have found that a single point mutation (G185R) in a 12-TM transporter protein conferred new Ca 2+ -selective permeability. Previous studies have suggested that channels, pumps, and transporters may share some common mechanisms for ion translocation ( Gadsby et al. 1993 ; Fairman et al. 1995 ; Cammack and Schwartz 1996 ; for review see references in Lester and Dougherty 1998 ; Nelson et al. 2002 ). The “channel mode” has been proposed to explain the “drive-slip” mechanism as part of the transport cycle. In this sense, wild-type DMT1 may simply be a proton channel with limited permeability for certain divalent metal ions. By mutating a single residue, G185R, it becomes an unambiguously Ca 2+ -permeant ion channel. Our findings may add new insight into mechanisms of Ca 2+ entry and transporter function. The notion that the 12-TM proteins can be ion channels may inform the search for candidate Ca 2+ and/or cationic channels and facilitate the molecular characterization of many unidentified native conductances. We initiated these studies to investigate why a unique DMT1 mutation, G185R, has occurred independently at least twice in mice and once in rats ( Fleming et al. 1997 , 1998 ). The multiple occurrences of this spontaneous mutation suggested that it might confer some type of selective advantage. We speculate that the proposed Ca 2+ entry gain of function helps to account for this remarkable pattern of remutation. Further investigation of this hypothesis will require direct and detailed comparison of DMT1 -null and mk mice. Materials and Methods Molecular biology The DMT1 cDNA used in this study was derived from one of four alternatively-spliced DMT1 gene transcripts. The G185R mutation was generated by using M13 phage and the oligonucleotide GTCCCCCTGTGGGGC C GAGTCCTCATCACCA. Wild-type DMT1 and the G185R mutant were tagged with a C-terminal FLAG epitope and subcloned into pTracer-CMV2 (Invitrogen, Carlsbad, California, United States). CHO-K1 or HEK293T cells transiently transfected with DMT1 and G185R were used for the 55 Fe uptake assay and Western blot analysis. To obtain a stable G185R-expressing cell line, the G185R-encoding DMT1 gene was subcloned into pRevTRE (Clontech, Palo Alto, California, United States), a retroviral vector that drives expression from a Tet-responsive element. All constructs were confirmed by sequencing. DMT1 Western blot analyses were performed with an anti-FLAG M2 monoclonal antibody (Sigma, St. Louis, Missouri, United States) and, in some cases, with a goat polyclonal antibody raised against human DMT1 (Santa Cruz Biotechnology, Santa Cruz, California, United States). Mammalian cell electrophysiology Wild-type and G185R mutant DMT1 were subcloned into an EGFP-containing vector (pTracer-CMV2, Invitrogen) for transient expression in CHO-K1 and HEK293T cells. Cells were transfected using Lipofectamine 2000 (Invitrogen). Transfected cells, cultured at 37°C, were plated onto glass coverslips and recorded 24 (DMT1) or 30 (G185R) hrs after transfection. A stable cell line (HEK293 Tet-On TM , or HEK-On) was generated, and expression was induced by adding 1–10 μg/ml doxycycline into the culture medium. Unless otherwise stated, the pipette solution contained 147 mM cesium, 120 mM methane-sulfonate, 8 mM NaCl, 10 mM EGTA, 2 mM Mg-ATP, 20 mM HEPES (pH 7.4). Bath solution contained 140 mM NaCl, 10 mM CaCl 2 , 10 mM HEPES, 10 mM MES, 10 mM glucose (pH 7.4). Unless otherwise stated, the low pH solutions contained only nominal free Ca 2+ (1–10 μM). Data were collected using an Axopatch 2A patch–clamp amplifier, Digidata 1320, and pClamp 8.0 software (Axon Instruments, Union City, California, United States). Whole-cell currents were digitized at 10 kHz and filtered at 2 kHz. The permeability to monovalent cations (relative to P Na ) was estimated according to Equation 1 from the shift in E rev upon replacing [Na + ] o in nominally Ca 2+ -free bath solution (150 mM XCl, 20 mM HEPES, 10 mM glucose [pH 7.4]]), where X + was Na + , K + , Cs + , or Li + . For the permeability to divalent cations (relative to P Na ), bi-ionic conditions were used; Y 2+ was Ca 2+ , Ba 2+ , or Sr 2+ ( Equation 2 ). The internal pipette solution contained 100 mM Na-gluconate, 10 mM NaCl, 10 mM EGTA, 20 mM HEPES-Na (pH 7.4 adjusted with NaOH, [Na + ] total = 140). The external solution was 140 mM NMDG-Cl, 10 mM Y 2+ Cl 2 , 20 mM HEPES (pH 7.4 adjusted with HCl). The permeability ratios of cations were estimated from the following equations ( Lewis 1979 ): where R, T, F, V, and γ are, respectively, the gas constant, absolute temperature, Faraday constant, E rev , and activity coefficient. The liquid junction potentials were measured and corrected as described by Neher (1992 ). Uptake assay The assay buffer contained 25 mM Tris, 25 mM MES, 140 mM NaCl, 5.4 mM KCl, 5 mM glucose, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 . Ascorbic acid was adjusted to 1 mM and the pH was adjusted to 5.8. Most assays were performed with 20 μM Fe 2+ at pH 5.8 unless otherwise indicated. A 50-fold 55 Fe stock was made immediately before the assay with 1 mM 55 Fe (with a 1:20 molar ratio for 55 FeCl 3 and FeSO 4 ) and 50 mM nitrilotriacetic acid. About 30 h after transient transfection, CHO-K1 or HEK293T cells were washed and harvested with PBS (for CHO-K1 cells, trypsin treatment was required). Cells were resuspended in glass test tubes at 0.5–1 million/ml in 490 μl assay buffer at 30°C. The reaction was started by adding 10 μl of 55 Fe stock and stopped at 4, 8, and 16 min by quickly filtering the reaction mix on a nitrocellulose filter (HAWP02500; Millipore, Billerica, Massachusetts, United States). Filters were washed twice with 2 ml of assay buffer, dried, and radioactivity counted by liquid scintillation spectrometry. Calcium imaging Cells were loaded with 2 μM Fura-2 AM in culture medium at 37°C for 30 min. Low levels of G185R protein were expressed in the absence of doxycycline in G185R HEK-On cells (Western blotting; unpublished data). Therefore, doxycycline-treated HEK-On cells not expressing DMT1 were used as controls in imaging experiments. We recorded Fura-2 ratios (F340/F380) on an UltraVIEW imaging system (Olympus, Tokyo, Japan). A standard curve for Fura-2 ratio versus [Ca 2+ ] was constructed according to Grynkiewicz et al. (1985 ). Isolation of enterocytes Homozygous mk mice ( Fleming et al. 1997 ) were housed in the barrier facility at Children's Hospital (Boston, Massachusetts, United States). Husbandry and use were according to protocols approved by the Animal Care and Use Committee. Wild-type iron-deficient mice were provided by J.-J. Chen (Massachusetts Institute of Technology, Cambridge, Massachusetts, United States). Mouse enterocytes were isolated using a modified protocol provided by Dr. F. Sepulveda ( Monaghan et al. 1997 ). In brief, 1 cm of the proximal duodenum was excised, rinsed with cold PBS, and soaked for 5 min at 37°C in a solution containing 7 mM K 2 SO 4 , 44 mM K 2 HPO 4 , 9 mM NaHCO 3 , 15 mM Na 3 Citrate, 10 mM HEPES, and 180 mM glucose (pH 7.4). The tissue was then incubated with gentle shaking for 3 min in a similar solution containing 7 mM K 2 SO 4 , 44 mM K 2 HPO 4 , 9 mM NaHCO 3 , 10 mM HEPES, 180 mM glucose, 1 mM DTT, and 0.2 mM EDTA (pH 7.4). The mucosal cells were gently squeezed from the duodenum with forceps into 5 ml of ice-cold DMEM/F12 medium, pelleted at 800 × g for 4 min, resuspended in 5 ml of prewarmed DMEM/F12 with 0.5 mg/ml collagenase type 1A, and incubated at 37°C for 10 min. Cells isolated by this procedure have been shown previously to be primarily of villus origin and hence are mature enterocytes. We confirmed this by alkaline phosphatase staining. Diluted cells were filtered through a 40-μm nylon cell mesh (BD Biosciences, Palo Alto, California, United States). The cells were then washed with DMEM/F12, resuspended in 20 ml of ice-cold DMEM and kept at 4°C. They were plated on coverslips coated with Cell-Tak TM (BD Biosciences) and maintained on ice before patch–clamp recording at room temperature. Data analysis Group data are presented as mean ± SEM. Statistical comparisons were made using analysis of variance and the t -test with Bonferroni correction. A two-tailed value of p < 0.05 was taken to be statistically significant. Supporting Information Figure S1 CHO-K1 Cells Express an Endogenous Proton-Activated Chloride Channel (A) Anion dependence of pH-induced response in a DMT1-expressing cell. Outward current usually appears later than the inward current. (B) Currents generated in response to a voltage ramp. (C) pH-induced outwardly rectifying current in a nontransfected CHO-K1 cell. A similar current was seen also in HEK293T and HEK-On cells, with properties similar to the cloned ClC-7 channel ( Diewald et al. 2002 ). This current exhibits the same anion depen-dence as in (A) (data not shown) . We attributed the outward currents shown in (A) and (B) to this endogenous Cl – current. Therefore, for our recordings on DMT1, SO 4 2\– was usually used to replace most of the Cl – ([Cl – ] o = 5 mM) for all low-pH bath solutions. (718 KB PDF). Click here for additional data file. Figure S2 Time- and Voltage-Dependent Kinetics of H + /Mn 2+ Current of DMT1 Whole-cell currents were generated by voltage steps from −140 to +80 mV in 20 mV steps, 400 ms. The interval between steps was 1,000 ms. (1 MB PDF). Click here for additional data file. Figure S3 Na + -Dependence of DMT1 H + and H + /Mn 2+ Currents Replacement of extracellular Na + by NMDG + slightly increased the proton current (approximately 20%) and this was further augmented by adding 300 μM Mn 2+ . The concentrations used were Na + and NMDG + , 140 mM, (pH 4.2); Mn 2+ , 300 μM. (141 KB PDF). Click here for additional data file. Accession Numbers The GenBank ( www.ncbi.nlm.nih.gov/GenBank/ ) accession number for DMT1 is AF029758.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC368157.xml
528728	Long-term age-dependent behavioral changes following a single episode of fetal N-methyl-D-Aspartate (NMDA) receptor blockade	Background Administration of the N-methyl-D-aspartate (NMDA) antagonist ketamine during the perinatal period can produce a variety of behavioral and neuroanatomical changes. Our laboratory has reported reliable changes in learning and memory following a single dose of ketamine administered late in gestation. However, the nature of the drug-induced changes depends on the point during embryonic development when ketamine is administered. Embryonic day 18 (E18) rat fetuses pre-treated with ketamine (100 mg/kg, i.p. through the maternal circulation) and taught a conditioned taste aversion (CTA) learn and remember the CTA, whereas E19 fetuses do not. The current study sought to determine if long-term behavioral effects could be detected in animals that received ketamine or a saline control injection on either E18 or E19. Rat behavior was evaluated on two different measures: spontaneous locomotion and water maze learning. Measurements were collected during 2 periods: Juvenile test period [pre-pubertal locomotor test: Postnatal Day 11 (P11); pre-pubertal water maze test: P18] or Young-adult test period [post-pubertal locomotor test: P60; post-pubertal water maze test: P81]. Results Water maze performance of ketamine-treated rats was similar to that of controls when tested on P18. Likewise, the age of the animal at the time of ketamine/saline treatment did not influence learning of the maze. However, the young-adult water maze test (P81) revealed reliable benefits of prenatal ketamine exposure – especially during the initial re-training trial. On the first trial of the young adult test, rats treated with ketamine on E18 reached the hidden platform faster than any other group – including rats treated with ketamine on E19. Swim speeds of experimental and control rats were not significantly different. Spontaneous horizontal locomotion measured during juvenile testing indicated that ketamine-treated rats were less active than controls. However, later in development, rats treated with ketamine on E18 were more active than rats that received the drug on E19. Conclusion These data suggest that both the day in fetal development when ketamine is administered and the timing of post-natal behavioral testing interact to influence behavioral outcomes. The data also indicate that the paradoxical age-dependent effects of early ketamine treatment on learning, previously described in fetuses and neonates, may also be detected later in young adult rats.	Background Administration of ketamine or other NMDA receptor blocking drugs [ 1 ] may bring with it both beneficial and problematic outcomes. Ketamine's use as a dissociative anesthetic is well established in clinical practice [ 1 ] and more recently, it has also been proposed as a neuroprotectant against hypoxic-ischemic brain damage in neonatal rats [ 2 ]. However, in adult animals, NMDA receptor blockade is known to produce psychotomimetic side effects [ 3 ], impair memory formation [ 4 - 7 ], and may produce neurotoxicity [ 3 , 8 - 11 ]. This neurotoxicity is evidenced by vacuolization of cortical neurons [ 3 , 10 ] and has also been linked to programmed cell death (apoptosis) during development [ 12 - 14 ]. The toxic effects of NMDA receptor blockade are apparently dependent on the dose of the drug, administration regimen, and the age of the animal treated. For example, vulnerability to MK-801-induced cortical vacuolization is not evident in fetal animals but rather begins at approximately the time of puberty [ 8 ]. On the other hand, apoptogenic effects of ketamine have been seen following drug administrations during the last trimester of pregnancy [ 12 ]. Further, the selection of an acute or chronic dosing regimen may also modulate the neurobehavioral outcomes and the permanence of the neurological changes that can be expected [ 9 , 13 , 15 - 18 ]. Recent experiments from our laboratory have focused on age-dependent effects of a single dose of ketamine on fetal learning and memory. Rat fetuses can learn conditioned taste aversions (CTAs) and exhibit taste-mediated conditioned motor responses (CMRs) [ 19 , 20 ], which can be modulated in complex ways by exposure to ketamine at different times during the perinatal period [ 20 , 21 ]. For example, ketamine will either cause a potentiation or a blockade of memory formation in rats, depending on the specific day during fetal development when the drug is administered. Rat fetuses that receive ketamine on E18 (30-minutes before CS-US pairing) are able to learn and remember CTAs and CMRs quite well. However, rat fetuses that receive ketamine before CTA training just one day later, on E19, exhibit an amnesia for these conditioned responses [ 21 , 22 ]. We have referred to this phenomenon as the "ketamine paradox" [ 21 ]. These previous studies have only tested the durability of the ketamine paradox over a period of up to 2 weeks [ 22 ] and have looked at a very narrow range of behavioral measures – all with gustatory components. However, there are some indications that early treatment with NMDA receptor blocking drugs can have long-term behavioral implications. For example, neonatal treatment with MK-801 can produce long-lasting behavioral radioprotection in rats with x-ray-induced hippocampal damage [ 23 ]. Likewise, Maier et al. [ 24 ] have reported that MK801-induced NMDA receptor antagonism in young rats (P7-17) extends the sparing of hindlimb function after spinal transection in older animals. However, treatment with an NMDA receptor antagonist [(+)HA-966] for a longer time, later in neonatal development (P10-20), impaired motor and cognitive behaviors in adult rats [ 25 ]. Several questions remain. What is the range of behaviors that can be influenced by ketamine treatment during the perinatal period? How long lasting are the different behavioral effects of ketamine administered late in gestation? The current study extends our original observations and reports how a single injection of ketamine on either E18 or E19 modulates spontaneous locomotor activity and performance in a water maze. We tested the rats as juveniles and then as young adults. Our data suggest several age-dependent effects of early ketamine treatment – effects that depend on not only the length of time between drug administration and behavioral testing but also on the day in embryonic development when the NMDA receptor antagonist was administered. Results Water maze P18 water maze test Over the10 trials of the water maze test, subjects significantly reduced their latencies to mount the hidden platform [F(9, 102) = 4.233; p < 0.0001] indicating the gradual learning of the maze. However, as Figure 1A illustrates, the animals never gained real proficiency on this task. The latencies decreased most dramatically within the first 3 trials and therefore we undertook a more in-depth analysis of this portion of the study. We also noticed that, as the animals swam, they would sometimes stop and tread water against the side of the tank. Therefore, we undertook an analysis of the stop time/trial during the first 3 water maze trials (see Figure 2 ). The time spent treading water decreased significantly over the first 3 trials [F(2, 111) = 18.359; p < 0.001]. There was also a significant drug effect indicating that ketamine-treated rats spent less time treading water than did saline-treated controls [F(1, 153) = 15.972; p < 0.001]. This effect was independent of the age at which the rats received ketamine. Figure 1 Time to mount a hidden platform during the first (juvenile; panel A) and second (young-adult; panel B) water maze test. Rats were treated (through the maternal circulation) with either 100 mg/kg ketamine HCl (i.p.) or saline on either E18 or E19 and then tested later on P18 and P81. The data presented here illustrate raw latencies without taking into account the time the animals spent treading water (i.e., not making forward progress). Panel A: P18 rats generally decreased their latencies to mount the hidden platform over the 10 trials. This effect was most prominent over the first 3 trials. The behavioral changes induced by drug or age manipulations were not statistically significant. Panel B: P81 rats all readily re-learned the location of the hidden platform as there was a significant reduction in time to mount the platform over the 10 trials. The analysis (see text) also revealed a significant Age X Drug interaction which was most prominent on trial 1 (see also Figure 3). Data were analyzed using a three-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19) X Time] with time blocks as the repeated time factor and compensation for unequal Ns. Variance indicators are the Standard Error of the Mean (SEM). Figure 2 Time spent treading water (i.e., not making forward progress) during the P18 water maze test. There was a general decline in the time spent treading water as the animals learned the maze. Ketamine-treated rats spent the least time exhibiting this behavior. Rats treated with the NMDA-receptor blocking drug on E18 stopped swimming for the shortest period and made more-constant progress towards the hidden platform. Data were analyzed using a three-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19) X Time] with time blocks as the repeated time factor and compensation for unequal Ns. Variance indicators are the Standard Error of the Mean (SEM). Time spent treading water at the side of the tank may be interpreted as an alternative, futile, escape strategy and not necessarily as an indicator of learning the position of the hidden platform. Subsequent analyses subtracted out stop-times in order to provide the most accurate portrayal of maze learning during the first 3 trials. With stop-time removed, the declining time-to-platform [F(2, 102) = 3.501; p = 0.034] and swimming distance [F(2, 102) = 4.632; p = 0.012] over the first 3 trials indicated a learning of the maze during this initial exposure to the apparatus. However the behavioral changes induced by drug or age manipulations were not statistically significant. The number of trials in which the rat failed to mount the platform within 90 seconds was not significantly different among the 4 treatment groups. Likewise, swim speed did not decrease significantly over the first 3 maze trials indicating that the animals were not fatiguing as they undertook multiple swims. Once the rat mounted the hidden platform we timed how long the subject remained there before it was removed (maximum of 30 seconds). An analysis of these data during the first 3 maze trials revealed neither a significant influence of subject age nor drug treatment. P81 water maze test An analysis of the second water maze test indicated that there was a significant reduction in time [F(9, 157) = 26.868; p < 0.001] to mount the hidden platform over the 10 trials (see Figure 1B ). The analysis also revealed a significant Age X Drug interaction [F(1, 401) = 5.15; p = 0.024] but no significant main effects of drug or age at treatment. Rats had previous experience with the maze (see P18 maze data) and inspection of the data indicated that, after the first trial, latencies in all groups converged and dropped dramatically. As in the P18 water maze analysis, stop times (i.e., time spent treading water) during this first trial were significantly lower in ketamine-treated rats [Drug effect: F(1, 62) = 6.645; p = 0.012]. A subsequent examination of the data excluded the time spent treading water in swim-time, swim-distance and swim-speed analyses. On the first trial (see Figure 3 ), ketamine-treated rats found the platform significantly faster than saline controls [Drug effect: F(1, 67) = 7.28; p = 0.009] and swam shorter distances to do so [Drug effect: F(1, 67) = 8.89; p = 0.004]. Animals injected on E18 were generally quicker to find the platform [Treatment Age effect: F(1, 67) = 10.55; p = 0.002] and swam more direct routes to the platform [Treatment Age effect: F(1, 67) = 5.89; p = 0.018] than were animals injected on E19. Figure 3 Time to mount the hidden platform (top panel) and swimming distance to the platform (bottom panel) during the first trial of the second (young-adult) water maze test. Rats were treated (through the maternal circulation) with either 100 mg/kg ketamine HCl (i.p.) or saline on either E18 or E19 and then tested later on P81. The temporal data presented here do not include time spent treading water but rather represent only the time that the rats were making forward progress. Ketamine-treated rats found the platform significantly faster than saline controls and swam shorter distances to do so. Group comparisons indicated that rats treated with ketamine on E18 or E19 reached the hidden platform significantly faster and swam shorter distances than saline-treated controls (* = p ≤ 0.05; NS = non-significant group differences). Further, E18 rats treated with ketamine later exhibited a shorter latency to reach the hidden platform than did E19 rats treated with ketamine. Data were analyzed using a two-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19)]. Individual group comparisons were accomplished by using t-tests employing the Bonferroni compensation for multiple comparisons. Variance indicators are the Standard Error of the Mean (SEM). Post-hoc analyses indicated that rats treated with ketamine on E18 reached the hidden platform significantly faster and swam shorter distances than saline-treated controls as well as rats treated with ketamine on E19 (see Figure 3 ). Rats treated with ketamine (on either E18 or E19) also exhibited significantly fewer failures to reach the hidden platform (within the 90-second limit/trial) than did saline control animals [t(66) = 1.86, p = 0.034 (one-tail test)]: ketamine-treated Mean ± SEM = 0.71 ± 0.05 failures/10 trials; saline-treated Mean ± SEM = 0.24 ± 0.07 failures/10 trials. The short latencies to mount the platform cannot be attributed to faster swimming speeds. On the first, second and third water maze trials (i.e., the only ones analyzed for swim speeds), P81 rats that were treated with ketamine on E18 did not swim significantly faster than any of the animals in the other treatment groups. For example, the swim speeds for trial 1 were: [E18/ketamine: 34.30 ± 3.59 cm/sec; E18/saline: 37.97 ± 1.85 cm/sec; E19/ketamine: 30.47 ± 2.40 cm/sec; E19/saline: 32.92 ± 2.23 cm/sec (Mean ± SEM)]. Fatigue did not seem to play a role in the group differences since swim speed remained stable in all groups over the first 3 water maze trials on P81. Locomotion A single ketamine treatment during the perinatal period had long-lasting effects on spontaneous locomotor movements. Ketamine's effects depended on the age of behavioral testing as well as the age of the drug treatment. Horizontal movements (i.e., line crossings) were more prominently influenced by perinatal ketamine than were vertical movements (rearing). P11 locomotor tests P11 rats treated prenatally with ketamine showed habituation to the open-field test chamber and exhibited reduced horizontal movements overall (see Figure 4 ). After being placed in the activity chamber, P11 rats decreased their horizontal movements (i.e., line crossings analyzed in 6, 5-minute blocks) significantly over the 30-minute test session [F(5, 405) = 80.66, p < 0.0001]. In fact, locomotor activity of all treatment groups was reduced to very low levels (typically <5 line breaks/min) after the first 5 minutes (Figure 4B ). Figure 4 Spontaneous horizontal locomotor activity of P11 rats treated (through the maternal circulation) with either 100 mg/kg ketamine HCl (i.p.) or saline on either E18 or E19. Panel A illustrates the entire 30-minute test. In only the initial 5-minute observation period, rats treated with ketamine in utero crossed significantly fewer lines than did the saline control animals. After this initial period of habituation, indicators of horizontal movement declined and group scores converged. Panel B is a minute-by-minute illustration of the first 5 minutes of locomotor activity. In a time-dependent manner, rats treated with ketamine in utero crossed significantly fewer lines than did the control animals. Habituation to the open field is represented by a rapid decline in movement. * = Significantly different from E18/saline group; + = significantly different from E19/saline group. Data were analyzed using a three-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19) X Time] with time blocks as the repeated time factor and compensation for unequal Ns. If the repeated-measure ANOVA revealed a significant trial effect (indicating a change over time) a two-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19)] was run to analyze the group differences during a particular trial. Individual group comparisons were accomplished by using the Tukey-Kramer test for Multiple Comparisons. An α = 0.05 was used throughout these analyses. Variance indicators are the Standard Error of the Mean (SEM). Ketamine-treated rats were also generally less active than saline-injected controls [F(1, 81) = 7.79, p = 0.007]. However, there was a significant interaction between drug treatment and the block of time in which the locomotor measurement was made [F(5, 405) = 6.26, p = 0.0002]. At the end of 5 minutes, P11 rats reduced their spontaneous locomotion to approximately 20% of its original level. For this reason, we performed a minute-by-minute analysis of the first 5 minutes in the open field apparatus (see Figure 4B ). Once again, there was a significant decrease in horizontal movement over the first 5 minutes in the chamber [F(4, 336) = 143.13, p < 0.001]. Generally, ketamine treatment caused a significant decrease in line crossings as compared to saline-injected controls [Drug effect = F(1, 84) = 12.77, p = 0.0006]. A Drug X Time Block interaction [F(4, 336 = 3.21, p = 0.01] revealed that the largest group differences were exhibited within the first 3 minutes (see Figure 4B for individual group comparisons). There was not a significant difference in the spontaneous horizontal locomotor responses of E18- and E19-ketamine treated rats. P60 locomotor tests Horizontal movements of ketamine-treated rats tested on P60 varied depending on when, during the fetal period, they had received the drug (see Figure 5 ). As was the case during the P11 tests, horizontal movements decreased significantly over the 30-minute test [F(5, 340) = 163.12, p < 0.0001]. There was both an overall effect of subject age at time of drug injection [F(1, 68) = 4.37, p = 0.04] and a Treatment Age X Drug interaction [F(1, 68) = 10.37, p = 0.002]. Over the course of this 30-minute test (Figure 5A ), E18 fetuses treated with ketamine were generally more active in their horizontal movements than were animals treated with saline on this day of embryonic development. Further, rats exposed to ketamine on E18 exhibited significantly more horizontal movement than did E19 rats treated with either saline or ketamine. Rats injected with saline on E18 or E19 did not exhibit significant differences in line crossings when tested on P60. Figure 5 Horizontal locomotion of P60 rats treated (through the maternal circulation) with either 100 mg/kg ketamine HCl (i.p.) or saline on either E18 or E19. Panel A illustrates the entire 30-minute test. E18 rats treated with ketamine were significantly more active than were animals in the other treatment groups. These effects were most prominent at particular time periods. * = Significantly different from E18/saline group; # = significantly different from E19/ketamine group. + = significantly different from E19/saline group. Panel B is a minute-by-minute illustration of the first 5 minutes of locomotor activity. In all treatment groups, there is a significant decline in locomotion over the first 5 minutes of testing. There is also a significant interaction between drug treatment and subject age at time of treatment – indicating that rats treated with ketamine on E18 are more active than both E18 saline-control rats and E19 rats that received ketamine. Data were analyzed using a three-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19) X Time] with time blocks as the repeated time factor and compensation for unequal Ns. If the repeated-measure ANOVA revealed a significant trial effect (indicating a change over time) a two-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19)] was run to analyze the group differences during a particular trial. Individual group comparisons were accomplished by using the Tukey-Kramer test for Multiple Comparisons. An α = 0.05 was used throughout these analyses. Variance indicators are the Standard Error of the Mean (SEM). An analysis that focused on the first 5-minutes of this P60 locomotor test (Figure 5B ) revealed a significant interaction between drug treatment and subject age at time of treatment [F(1, 70) = 14.13, p < 0.001]. Multiple comparisons revealed that rats treated with ketamine on E18 were more active than both E18 saline-control rats and E19 rats that received ketamine. These data reveal a very different pattern of horizontal locomotor responses exhibited by ketamine-treated rats depending on the day during fetal development that they received the drug. There were no significant group differences in rearing movements on P60. Discussion The data presented here suggest several age-dependent effects of early ketamine treatment – effects that depend on not only the day of behavioral testing but also the day in embryonic development when the NMDA receptor antagonist was administered. During the initial stage of the second water maze test (on P81), rats treated with ketamine on E18 found the hidden platform more quickly than did animals receiving the same treatment on E19. Moreover, they exhibited enhanced maze performance compared to both groups of saline-treated rats. It is important to note that ketamine treatment on E18 did not induce faster swim speeds. Rather, the animals swam more direct routes to the hidden platform. Effects of ketamine on spontaneous open-field locomotion were also age-dependent. In neonatal animals (P11), ketamine administration in utero reduced subsequent spontaneous movement. This effect was subtle (i.e., only in evidence within the first 3-minutes of testing) and did not depend on the subject's age at the time of the drug's administration. However, when these animals were re-tested on P60, the rats that received ketamine on E18 both moved more than the rats that received ketamine on E19 and also moved more than saline-injected controls. First, these data reveal long-term behavioral effects of a single dose of ketamine administered in utero . Drug-induced effects on water maze learning were observed over 11 weeks after birth and locomotor effects were documented 9 weeks post partum . These findings are consistent with others indicating that early NMDA receptor blockade may produce behavioral alterations that are detectable in adulthood [ 23 , 25 , 26 ]. Second, these data are consistent with the hypothesis that the behavioral effects of NMDA receptor blockade depend on the day in embryonic development when the antagonist is administered. In particular, some of the findings reported here extend our previous work indicating that ketamine treatment on E18 may have different effects than administration of the drug on E19 [ 21 ]. The same dose of ketamine administered in the current study potentiated conditioned motor responses of neonates if the drug had been given (through the maternal circulation) on E18. However, ketamine impaired acquisition of this learned response if it was administered one day later on E19 [ 21 ]. Similar age-dependent effects have been reported using different behavioral indicators of learning [ 22 , 27 ]. The effects of early ketamine treatment on locomotion are apparently not consistent throughout postnatal development. Ketamine reduced locomotor movements in P11 rats but later (P60) selectively enhanced locomotion of animals that received the drug on E18. The reasons for this change in responding are unclear. In order to accommodate the different size of the animals at P11 and P60, there were differences in the dimensions of the open field chambers used at each test. Also, the chamber walls were clear during the P11 test and opaque at P60. But beyond these differences in apparatus, maturation clearly brings with it a variety of capabilities and propensities many of which can modulate motor responding. For example, at the end of 5 minutes, P11 rats reduce their spontaneous locomotion to approximately 20% of its original level. However, P60 rats are 80% as active during this same time period. These data indicate a general tendency for young rats to habituate (or fatigue) more rapidly than older rats. Other behavioral studies have revealed toxin-induced performance impairments that reveal themselves only at certain stages of early postnatal development but not at older ages [ 28 ]. More recently, Beninger et al. [ 29 ] reported that rats administered MK-801 on P3 and tested pre- (P35) and post-pubertally (P56) exhibited different locomotor responses to amphetamine depending on the time of the behavioral test. Our measures of swim speed may offer some insights regarding the relative motor capacities and motivation of our animals. Swim speeds did not significantly differ between animals previously treated with ketamine or saline. Likewise, fetal age at the time of the drug treatment did not influence speed of swimming. Instead, rats reduced their latencies to mount the platform by swimming more-direct routes. Thus, the water maze data reported here are less likely a reflection of the animal's capacity or motivation to get to the platform and more likely a reflection of learning ability. It should be noted that water maze performance may be influenced by a number of factors beyond cognitive ability. For example, drug-induced alteration of visual acuity, motivation or motor capacities can alter performance of this task [ 30 ]. The literature suggests that early NMDA receptor blockade may alter development of the visual system [ 31 , 32 ]. But measures of actual visual acuity following a single occurrence of NMDA receptor blockade in the developing brain are lacking. The available data suggest that visual plasticity is more significantly altered by NMDA receptor antagonism than are visual maps [ 33 ] or neural activity per se [ 34 ]. Our water maze procedures did not necessarily place demands on the rat's visual system. The location of the hidden platform was not changed from trial to trial or between the P18 and P81 tests. Therefore, once the platform was located, our subjects could have adopted motor strategies to reach the goal on subsequent trials. Although water maze performance is typically cited as an indicator of spatial learning, our paradigm does not eliminate the possibility that other types of learning may also be involved. We used accepted statistical methods to avoid spurious inflation of sample size and to control for litter effects [ 35 , 36 ] (see Methods section below). However, due to constraints of resources, the small number of litters employed may have reduced our power to detect subtle differences in performance. Thus, this report should be viewed as a conservative account of drug- and age-influenced changes in behavior. It is worth noting that it was initial responding on the water maze and in the locomotor test chamber that was most sensitive to our fetal ketamine treatment. ketamine-treated rats exhibited significantly faster times to reach the hidden platform (at age P81) – but only on the first trial. Likewise, P11 rats treated with ketamine as fetuses, exhibited fewer locomotor movements than did saline controls – but only during the first 5 minutes of our test. Our laboratory [ 37 ], as well as other investigators [ 38 ] have reported a role for NMDA receptors in the determination of novelty. The current data seem to suggest that these effects may extend to various behavioral testing paradigms. Moreover, since our tests were conducted weeks after fetal ketamine treatment, our data indicate the persistence of ketamine's effects on initial responding. What neural, or other, mechanisms might subserve the behavioral phenomenon outlined here? NMDA receptor populations and physiology are neither static nor mature during the perinatal period and blockade of these receptors during particular days of development may produce quite different effects [ 39 , 40 ]. For example, Sircar [ 41 ] has shown that the binding of [ 3 H] MK-801 (a potent/selective NMDA receptor antagonist) in synaptosomal membranes is differentially altered by glutamate (and other) agonists during various periods of development. These data build on previous findings [ 42 ] indicating a dramatic change in the number of PCP-binding sites in fetal rat brain between the ages of E18 and E19. This is the same time frame in which ketamine's effects on memory change so significantly. Could these developmentally linked changes in NMDA receptor populations and functional roles mediate the ketamine paradox as well as the behavioral phenomena presented here? The identification of several NMDA receptor subtypes with different functional roles and different patterns of expression during the perinatal period may also eventually reveal aspects of the ketamine paradox's physiological substrate [ 43 - 54 ]. Could a drug-induced change in the population and/or distribution of NMDA receptor subtypes mediate the ketamine paradox as well as long-term behavioral effects? The current data do not address this point directly. However, NR2B NMDA receptors (which are known to be involved in learning, in general, and taste memory formation, in particular) [ 55 - 57 ] have been identified as being especially sensitive to upregulation following pharmacological antagonism [ 58 , 59 ]. Further, other laboratories have reported that NMDA exposure can produce a reduction in NMDA receptors within 4 hours of exposure [ 60 ]. Our data suggest the potential usefulness of studies aimed at correlating ketamine-induced changes in NMDA receptor subtype populations with behavioral outcomes recorded at several times in development. Such experiments are currently underway in our laboratory. It should also be noted that ketamine can influence maternal and fetal physiology in ways that go beyond the drug's well-known effects on NMDA glutamate receptors [ 1 ]. Pulmonary vasodilator responses have been recorded following ketamine administration [ 61 ]. The drug can also alter uterine tone in pregnant ewes by increasing cardiac output and mean arterial pressure [ 62 ]. Although these cardiovascular effects were slight and transient, they may have contributed in yet-unknown ways to some of the long-term behavioral changes we report here. Likewise, ketamine not only affects NMDA receptors but may also inhibit non-NMDA glutamate receptors [ 63 ], the high-affinity states of the dopamine D2 receptor, and other G-protein-linked receptors [ 64 ]. While ketamine's actions on NMDA receptors are certainly prominent, acute changes in vascular tone and the drug's actions on other brain receptors are capable of influencing fetal development in ways not addressed by the current experiments. If early ketamine exposure influences NMDA receptor populations or functioning, post-synaptic second messenger pathways would also be engaged as mediators of behavioral change [ 65 ]. Downstream calcium and calmodulin signaling, calcium-dependent kinases, and ultimately changes in gene expression are known to produce synaptic restructuring [ 66 ]. This cascade of NMDA-receptor-initiated biochemical events provides a likely avenue for further investigation as we examine the physiological substrate of the behavioral phenomena described here. The variables of subject age and ketamine dose interact in complex ways to produce predictions of neurotoxicity. If NMDA antagonists are used to suppress neuronal activity during a critical developmental period of synaptogenesis, the timing and sequence of synaptic connection is disrupted [ 67 ]. This causes neurons to receive an internal signal to commit suicide – a form of programmed cell death called apoptosis [ 68 ]. Ketamine, and other NMDA receptor blocking drugs, are reported to produce these neurotoxic effects [ 69 ] under certain circumstances. In the rat, the period of brain sensitivity is largely confined to the postnatal period (i.e., from P1 to P14) [ 70 ]. Our single dose of ketamine was administered on either E18 or E19, i.e., subject ages that, to the best of our knowledge, have not been systematically manipulated in studies aimed at investigating ketamine's ability to produce apoptosis. These studies should be done to confirm the role that apoptosis may/may not play in producing the long-term behavioral changes we report here. In addition to subject age, the dose of ketamine is another important factor in determining the likelihood of apoptosis induction as well as the generalizability of our data to clinical settings. In order to produce an increase in apoptotic neurons in neonatal rats, ketamine must be administered in multiple injections over a period of 9 hours [ 13 ]. Our study used a single dose of ketamine (delivered to a pregnant rat) that was significantly higher (100 mg/kg) that those used previously in neonates [ 69 ]. However, from previous biochemical studies we know that our dosing regimen in pregnant rats [ 27 ] produced a concentration of fetal brain ketamine roughly comparable to that seen in blood following repeated doses of 20 mg/kg administered to neonatal rats (14 μg/g) [ 69 ]. These blood levels were approximately seven-fold greater than anesthetic blood levels in humans [ 71 , 72 ]. Therefore, by extrapolation, we may predict that our dose of ketamine produced tissue levels of the drug that significantly exceeded those typically produced in human patients who encounter the drug in a clinical setting. Of course, this does not eliminate the possibility that the human recreational use of ketamine (street name: "Special K") [ 68 ] may produce blood and brain levels that are significantly higher than those encountered in the clinic. Nor does it exclude the possibility of differing drug sensitivities of rats and humans. Both these factors will influence the clinical relevance of the studies reported here. Conclusions These studies were aimed at determining the long-term behavioral effects of ketamine administration on E18 and E19 as a means of assessing the durability, intensity and generalizability of the ketamine paradox [ 21 ]. Our previous work indicated that ketamine administration enhanced the formation of a conditioned taste aversion in E18 fetuses but not those treated on E19 or later [ 20 - 22 , 29 ]. The current data reveal several subtle, but consistent, residual behavioral changes produced by of a single large dose of ketamine administered during the rat's late pre-natal period. In terms of ketamine effects on spontaneous locomotion, we found that, irrespective of the day of fetal dosing employed, ketamine reduced horizontal movements when animals were tested on P11. However, when the animals were tested later, on P60, rats that had received ketamine on E18 differentiated themselves from the E19 ketamine-treated animals (and saline-treated controls) by exhibiting an increase in locomotion – especially in the early minutes of behavioral testing in the open field. Ketamine's long-term influence on water maze learning/retention was limited in scope, but palpable, on the first trial of the P81 test. Despite the reliable enhancement of CTA learning that has been reported in fetuses and neonates treated with ketamine on E18 [ 29 ], this same dosing regimen produced limited improvements in learning/retention of a water maze when the animals were tested as young adults. The usefulness of ketamine as a cognitive enhancer, administered in the perinatal period, appears to be limited not only by its known toxic effects at critical stages of development [ 68 ] but also by its influence on spontaneous movement and the drug's weak memory-enhancing properties over the long term. Methods Subjects The subjects were Sprague-Dawley rats (male and female) obtained from timed pregnant female rats supplied by Zivic Laboratories (Zelienople, PA). Litters (N = 2/treatment group) were not culled and ranged in size from 9 to 13 pups. [See behavioral testing sections below for details about the number of animals in each treatment group.] The variable number of subjects/group was due to different litter sizes and several logistical constraints that did not always allow the testing of all rats in each litter. Statistical adjustments were made in order to compensate for the unequal Ns in the treatment groups (see below). The date of conception (i.e., the date a vaginal plug was first detected) was designated as "embryonic day 0" (E0). Postnatal day 0 (P0) was the day of birth (typically E21.5). The pregnant animals from which our subjects were derived were individually housed in plastic 'shoe-box' cages (44.45 cm long × 21.59 cm wide × 20.32 cm high). After birth, perinatal rats were housed with the dam until they were sexed and weaned between P21-25 (this is within recommended weaning dates, see [ 73 ], at which point the pups were group-housed (separated by sex) in the standard-sized cages described above. Throughout the experiment home cage temperature was maintained at 23–26°C under a 12:12-h light:dark cycle (lights on at 0600 h). The Baldwin-Wallace College Institutional Animal Care and Use Committee approved these experiments. The animals involved in this study were procured, maintained and used in accordance with the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals , prepared by the Institute of Laboratory Animal Resources – National Research Council. Drug treatments Pregnant dams received ketamine HCl (100 mg/kg, i.p.; Sigma Chemical Company), or an equal volume of physiological saline, (0.9% NaCl, i.p.) on either E18 or E19. Saline injections controlled for the stress of pre-natal manipulation. Thus, there were 4 treatment groups designated hereafter as follows: E18/ketamine, E18/saline, E19/ketamine, or E19/saline. Rats from two litters were used in each of the treatment groups. In order to separate out treatment effects from litter effects special statistical measures were employed (see Statistical Analyses , below) [ 35 , 36 ]. The dose of ketamine employed (100 mg/kg, i.p.) was selected based on previous experiments [ 21 ] in which the drug produced very different behavioral effects when administered to E18 or E19 fetuses through the maternal circulation. Using high-pressure liquid chromatography (HPLC) measures we have previously determined the level of ketamine (approximately 14 μg/g tissue) found in the brains of fetuses following a maternal injection of 100 mg/kg ketamine during the late pre-natal period [ 27 ]. Behavioral testing We recorded performance in a water maze and also spontaneous locomotor movements in an open field. Each of these behavioral tests was conducted twice, i.e., once in each of two different time periods: Juvenile Period (pre-pubertal), within the first three weeks after birth and Young-adult Period (post-pubertal), between 2–3 months of age. We selected these two behavioral test periods based on knowledge of the patterns of development of NMDA receptor subtypes. The NR2B receptor subtype has been implicated in learning and memory [ 55 ]. These receptors are present during the late pre-natal period and they rise steadily up to P20 when they achieve adult levels [ 43 , 74 ]. Therefore, our behavioral measures sampled times both before and after maturation of this receptor system. The behavioral tests conducted within the juvenile or post-pubertal periods were separated in time (by a minimum of 1 week) to reduce the influence of one on the other. Most, but not all, animals were tested and then retested. However, statistical analyses indicated that there were no differences between the animals that were tested once or twice. Therefore, these groups were combined for subsequent statistical analyses. Additional behavioral testing (i.e., conditioned taste aversion) was performed on some of these animals but there were some logistical problems with the experiment. These data did not reveal reliable group differences and are not reported here. Water maze At age P18, and then again at P81, we evaluated water maze performance by testing the following number of rats per group: P18 : E18/ketamine: (N = 12); E18/saline: (N = 16); E19/ketamine: (N = 14); E19/saline: (N = 15); P81 : E18/ketamine: (N = 12); E18/saline: (N = 22); E19/ketamine: (N = 17); E19/saline: (N = 20). Our data corroborate other studies indicating that rats younger than P20 are capable of learning a water maze [ 75 , 76 ]. The water maze was an oval stock-watering tank (manufactured by Rubbermaid, Inc.) measuring 94 cm × 74 cm × 60 cm deep. It was sized to shorten swim distances to the hidden platform and reduce the likelihood of fatigue in our young animals. The tank was filled to 39.5 cm (1 cm above a hidden platform) with water that was made opaque and white by adding 710 ml of evaporated milk (Nestle's Carnation ® brand). The water temperature was maintained at 26 ± 1°C. The escape platform was a clear plastic disc (12.5 cm diameter × 1.2 cm) mounted on a stand. The platform remained in the same location (approximately 10 cm from the side of the tank) throughout the test session. The edges of the platform had white rubberized tape attached in order to aid the rats as they mounted it. The tank was in a room lighted with fluorescent lights and surrounded by a rich array of laboratory furnishings. All test sessions were video recorded and the tapes were later used for analysis of latency to escape, stop time, time on platform, path length and swim speed (see Statistical Analyses below). "Escape latency" was defined as the time (in seconds) it took the rat, once in the water, to mount and gain balance on the platform. "Stop time" was the total time/trial that subjects spent treading water (i.e., not making forward progress). "Time on the platform" was defined as the time (up to 30 sec) the animal remained on the platform after initial mounting. "Path length" was the total distance (cm) swum before the subject mounted the platform. "Swim speed" was expressed in cm/sec and reflected the rate of forward progress towards the platform. At the beginning of each water maze test session, a rat was placed in the water facing the wall of the tank opposite the one near the hidden platform. A swim trial lasted until the rat reached the hidden platform or until 90 seconds had passed. If the animal reached the platform in the allotted 90 seconds, it was allowed to remain on the platform for up to 30 seconds and was then returned to a holding cage (a dry, plastic "shoe-box" cage). The holding cage sat upon a heating pad set at 33.5°C., producing a floor temperature of approximately 28.5 ± 1°C. If the animal did not reach the platform in 90 seconds it was removed from the water and returned to the cage. If the animal jumped off the platform before 30 seconds, it was removed from the water and returned to its holding cage. All rats were given a 60-second rest period before the next trial was initiated. Each rat experienced ten swim trials during each of the two test sessions. At the end of the 10 trials, each rat was thoroughly dried with a towel and a blow dryer and then returned to its home cage. Escape latencies and time on the hidden platform were recorded for each trial. Spontaneous locomotion At age P11, we measured the spontaneous locomotor activity of the following number of rat pups in each group: E18/ketamine: (N = 18); E18/saline: (N = 25); E19/ketamine: (N = 21); E19/saline: (N = 24). The test chamber consisted of a plastic 'shoe-box' cage (44.45 cm long × 21.59 cm wide × 20.32 cm high) with transparent walls and open top. This chamber had a grid on its floor composed of 3 × 6 squares (each measuring 7.1 cm × 7.1 cm). Young rats have limited abilities to thermoregulate [ 77 ]. Therefore, the activity chamber was placed on a heating pad set at 33.5°C., producing a floor temperature of approximately 28.5 ± 1°C. An individual pup was initially positioned in the center square of the chamber. Locomotor activity was recorded for 30 minutes. Test sessions were video recorded and tapes were later scored (see below). After each session, the animal was weighed and then returned to its home cage. In preparation for the next animal, the activity chamber was cleaned by spraying the cage with 50% ETOH, wiping it clean with paper towels, and allowing it to air-dry for approximately 10 minutes. At age P60, we again measured the spontaneous locomotor activity of the following number of rats in each group: E18/ketamine: (N = 12); E18/saline: (N = 21); E19/ketamine: (N = 17); E19/saline: (N = 22). For this second test, a larger test chamber (64 cm long × 46 cm wide × 42 cm high) was used. The walls were opaque plastic and the top open. This chamber had a grid on its floor that consisted of 3 × 3 rectangles (each measuring 21 cm × 15 cm). As before, rats were individually placed in the center square of the chamber at the beginning of the 30-minute test session. After each session, the animal was weighed and then returned to its home cage. In preparation for the next animal, the activity chamber was cleaned as described above. Videotapes of locomotor movements were later viewed and independently scored by observers blind to the experimental condition of the animal. We counted line crossings to assess the amount of horizontal locomotion exhibited by each animal. A "line-cross" was counted when any part of the rat, except the tail, crossed a line. Rearing was operationally defined as any time the rat raised both front paws from the chamber floor. In P11 rats, rearing was very rare and therefore not scored. However, this behavior was recorded during the P60 test. Statistical analyses Escape latencies and time on the hidden platform were recorded for each trial in the water maze. Group differences in water maze performance were most evident early in training. After the first few trials, rats in all treatment groups moved promptly to the hidden platform with a latency of less than 20 seconds. For this reason, our statistical analyses focused on the initial trials of each session. Animals removed from the maze after not finding the hidden platform in 90 seconds were, nevertheless, assigned a time of 90 seconds for purposes of data analysis. Swim distances, swim speeds, and time spent treading water (stop times) were also calculated for the first 3 swim trials. Towards this end, videotapes were viewed and independently evaluated by raters blind to the experimental condition of the animal. Swim paths were hand-drawn on acetate placed on a video screen during playback of the videotape. The paths were digitized using a light pen providing input to NIH Image software (Bethesda, MD). The lengths of the paths were compared with a calibrated length on the video record in order to calculate the swim distance. An evaluation of the inter-rater reliability of our video scoring methods indicated a high correlation [r(10) = 0.924, p < 0.001]. These methods produced swim distances that were not significantly different [t(9) = 1.02, p > 0.05] between observers. For some of our swim speed analyses, we subtracted out any time that the animal stopped swimming mid-trial, and treaded water at the side of the tank. Dividing the swim distance by the adjusted time to mount the hidden platform produced swim speed. Two observers, blind to the experimental condition of the animals, evaluated each of the tapes of animals locomoting in the open field by counting line crossings and rears. The counts of these 2 observers were then averaged and this data point was used in our statistical analysis. There was high degree of correlation between the ratings of our 2 observers. P11 locomotor test: r(85) = 0.91, p < 0.01; P60 locomotor test: r(72) = 0.90, p < 0.01. Unless otherwise stated, locomotor data for the two different test periods (P11 and P60) and water maze data for the 2 different test periods (P18 and P81) were analyzed via separate repeated-measures, three-way ANOVAs [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19) X Time] with time blocks as the repeated factor and compensation for unequal Ns. We used several rats from each litter and employed statistical corrections in order to avoid spurious inflation of sample size [ 35 ]. Since all the rats in a particular litter received the same drug treatment, we included litter as an independent and nested factor in the analysis. This approach controls for litter effects and offers a direct statistical test of the significance of such effects [ 35 ]. Denenberg [ 36 ] has recommended this procedure to allow the partitioning of litter and treatment effects and thereby allowing investigators to make use of the data from multiple animals in a litter. When significant litter effects were detected, we used the Mean Square (MS) associated with the litters as the error term rather than the MS of the subjects. However, if there was not a statistically significant litter effect, the data were subsequently reanalyzed without this component as part of the general linear model (GLM; software provided by SAS™, SAS Institute, Carey, NC; and, SPSS™ Inc., Chicago, IL). An initial inclusion of subject sex as a factor in our statistical analyses indicated no significant differences between male and female subjects. Therefore, the subsequent analyses reported here were run without this factor. Platform navigation for juvenile rats might be more challenging than the task presented to older rats. A small water maze was used in these studies and the same start-to-platform distance was used for all tests. Still, the P18 rat may have found it significantly more challenging than the P81 rat to traverse this distance. For this reason, we intentionally avoided comparing the water maze data of our juvenile and young-adult rats. Likewise, we did not make statistical comparisons between the locomotor responses of animals run at the 2 different ages since the use of different-sized apparatuses would presumably influence these data. If a repeated-measure ANOVA revealed a significant trial effect (indicating a change over time) a two-way ANOVA [Drug (100 mg/kg ketamine HCl, saline control) X Treatment age (E18, E19)] was run to analyze the group differences during a particular trial. Individual group comparisons were accomplished by using either the Tukey-Kramer Multiple Comparisons Test or t-tests [ 78 ] using the Bonferroni compensation for multiple comparisons. Our previous studies with ketamine-treated fetuses lead us to a priori hypotheses regarding possible behavioral differences between rats treated with ketamine on E18 versus E19 [ 21 ]. When a priori planned comparisons were made, one-tail probabilities were computed. An α = 0.05 was used throughout these analyses presented here. Authors' contributions GAM designed the studies, performed some of the behavioral work, conceptualized the statistical analyses and drafted the manuscript. CK helped design the studies, performed most of the behavioral work, assisted with statistical analyses and the drafting of the manuscript. CM supervised most of the behavioral work, assisted with statistical analyses and the drafting of the manuscript. AS supervised and performed much of the behavioral work. AY performed most of the behavioral work. DL-F assisted with the statistical analyses. EV assisted with the behavioral work. BW assisted with the behavioral work. JMB performed aspects of the behavioral work and assisted with the drafting of the manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC528728.xml
550650	Cardiovascular comorbidities among public health clinic patients with diabetes: the Urban Diabetics Study	Background We sought to determine the frequency and distribution of cardiovascular comorbidities in a large cohort of low-income patients with diabetes who had received primary care for diabetes at municipal health clinics. Methods Outpatient data from the Philadelphia Health Care Centers was linked with hospital discharge data from all Pennsylvania hospitals and death certificates. Results Among 10,095 primary care patients with diabetes, with a mean observation period of 4.6 years (2.8 after diabetes diagnosis), 2,693 (14.3%) were diagnosed with heart disease, including 270 (1.4%) with myocardial infarction and 912 (4.8%) with congestive heart failure. Cerebrovascular disease was diagnosed in 588 patients (3.1%). Over 77% of diabetic patients were diagnosed with hypertension. Incidence rates of new complications ranged from 0.6 per 100 person years for myocardial infarction to 26.5 per 100 person years for hypertension. Non-Hispanic whites had higher rates of myocardial infarction, and Hispanics and Asians had fewer comorbid conditions than African Americans and non-Hispanic whites. Conclusion Cardiovascular comorbidities were common both before and after diabetes diagnosis in this low-income cohort, but not substantially different from mixed-income managed care populations, perhaps as a consequence of access to primary care and pharmacy services.	Background Patients with diabetes are at increased risk of a wide range of complications and comorbidities, which adversely affect quality of life, mortality, and health care services utilization. Cardiovascular diseases are the primary causes of morbidity and mortality among patients with diabetes, yet data on cardiovascular disease among patients with diabetes are limited [ 1 ]. Although microvascular pathologies, including retinopathy and nephropathy, have been shown to be strongly associated with glycemic control, macrovascular complications, including heart disease and cerebrovascular disease, appear to be less responsive to glycemic control, and strategies to reduce them are focussed on controlling other risk factors such as hypertension, hyperlipidemia, smoking, and obesity [ 2 ]. This complex array of risk factors and outcomes makes diabetes care exceptionally demanding both for patients and for health care providers. Data on the prevalence and incidence of macrovascular comorbidities are largely derived either from clinical trials with selected patient populations, self-reported cross-sectional survey data, or managed-care studies of insured patients of higher socioeconomic status. The rising prevalence of diabetes in low-income inner-city populations underscores the need to understand the frequency with which these comorbidities occur in such populations and to assess possible disparities in their occurrence. The Philadelphia Health Care Centers (HCCs) provide primary care and pharmacy services to approximately 100,000 patients annually through eight neighborhood centers and one sexually transmitted disease clinic. Individual patients are not billed for any services provided. Although the HCCs are available to all Philadelphia residents, their patients are almost universally low-income; over 60% have no health insurance. A majority of HCC patients are African American, but there are also substantial numbers of non-Hispanic white, Hispanic, and Asian patients. Diabetes is one of the most common diagnoses among adult HCC patients. Since to our knowledge no previous study has reported the prevalence or incidence of cardiovascular diseases in a comparable, ethnically diverse, low-income diabetic population, we sought to determine the frequency and distribution of cardiovascular comorbidities among HCC patients with diabetes. Methods The Urban Diabetics Study cohort consists of 10,095 patients diagnosed with diabetes in the HCCs between January 1, 1996 and December 31, 2001. All diabetic patients were included except those for whom Social Security numbers were unavailable (less than 6% of all diabetic patients). All HCC visits between March 1, 1993 and December 31, 2001, all hospital discharges from any Pennsylvania hospital between January 1, 1993 and December 31, 2001, and death records have been linked for these patients. The data includes outpatient visits and hospitalizations both before and after the initial diabetes diagnosis, within the study time period. Race/ethnicity was classified based on the outpatient records. Diabetes incidence was calculated based on the admission date of the first hospitalization for which a diabetes diagnosis was recorded or the date of the first HCC outpatient visit for which a diabetes diagnosis was recorded, whichever came first. The cardiovascular comorbidities assessed included any heart disease (ICD9 codes 402 and 410–429, ICD10 codes I5-I9, I11, I13, I20-I27, and I30-I52), myocardial infarction (MI) (ICD9 code 410, ICD10 codes I21-I23), congestive heart failure (CHF) (ICD9 code 428, ICD10 code I50), cerebrovascular disease (ICD9 codes 430–438, ICD10 code I6), and hypertension (ICD9 codes 401–405, ICD10 codes I10-I15). For each comorbid condition we assessed baseline prevalence at or before first diabetes diagnosis, incidence rate per 100 person-years of follow-up time following first diabetes diagnosis among those free of the condition at baseline, and cumulative prevalence, i.e., any occurrence over the course of the observation period. Logistic regression and proportional hazards regression were used to simultaneously assess multiple demographic risk factors, including sex, race/ethnicity, exact age, and year of diabetes incidence. Patients with unknown race/ethnicity (n = 7) were excluded from multivariable analyses. Results Demographic characteristics of the 10,095 patients included are shown in Table 1 . Mean age at diabetes diagnosis was 49.2 years, and mean observation period was 4.59 years, including 1.74 years before and 2.85 years after first known diabetes diagnosis. Among the patients for whom income was ascertained, median income was $7,092; 86% had annual incomes below $15,000. Table 2 shows which of the linked data sources provided diagnoses of each comorbidity. Table 1 Demographic characteristics of Urban Diabetics Study cohort Total African American Hispanic Non-Hispanic White Asian Other Unknown N N N N N N N Total 10095 7243 1119 1056 394 276 7 Females 5671 4230 528 540 224 145 4 Age 0–4 17 12 2 2 1 0 0 5–14 38 28 2 2 4 2 0 15–24 213 149 25 28 8 3 0 25–34 477 359 52 41 11 14 0 35–44 1086 826 89 97 50 23 1 45–54 1614 1214 161 151 50 35 3 55–64 1542 1139 142 146 72 41 2 65–74 508 362 44 55 21 25 1 75–84 149 114 11 16 6 2 0 85+ 30 27 0 2 1 0 0 Year of Diabetes Diagnosis 1996 948 720 79 90 45 14 0 1997 969 714 78 116 40 20 1 1998 817 604 77 88 23 24 1 1999 854 637 92 74 26 25 0 2000 926 699 84 75 35 33 0 2001 1157 856 118 97 55 29 2 Males 4424 3013 591 516 170 131 3 Age 0–4 28 26 0 1 1 0 0 5–14 78 68 7 2 1 0 0 15–24 419 371 20 22 3 3 0 25–34 1174 1015 81 55 15 7 1 35–44 2567 2243 155 113 29 26 1 45–54 3183 2797 163 132 52 38 1 55–64 2807 2467 119 134 45 42 0 65–74 902 800 36 32 20 14 0 75–84 290 254 9 23 3 1 0 85+ 58 54 1 2 1 0 0 Year of Diabetes Diagnosis 1996 732 509 92 101 17 13 0 1997 743 496 96 101 27 22 1 1998 692 486 95 74 21 16 0 1999 638 423 80 84 24 27 0 2000 694 457 103 75 33 25 1 2001 925 642 125 81 48 28 1 Table 2 Data sources for cardiovascular comorbid conditions Outpatient only Hospital only Death Certificate only Outpatient And Hospital Outpatient and Death Certificate Hospital and Death Certificate Outpatient, Hospital, and Death Certificate Heart Disease 1125 700 38 731 9 31 59 Congestive Heart Failure 330 314 6 246 2 7 7 Myocardial Infarction 5 240 13 3 0 9 0 Cerebrovascular Disease 109 333 9 123 0 11 3 Hypertension 5248 218 0 2296 9 3 30 Heart disease Baseline prevalence of any heart disease was 14.8 %, including 4.1% with CHF and 1.0% with MI (Table 3 ). In multiple regression analyses, non-Hispanic white race/ethnicity (compared to the African-American reference group) was associated with substantially higher baseline prevalence of MI (Table 4 ). Hispanic race/ethnicity was associated with lower baseline prevalence of CHF, and Asian race/ethnicity was associated with lower baseline prevalences of MI, CHF, and any heart disease. Age and male sex were positively associated with baseline prevalence of MI, CHF, and any heart disease. Year of diabetes diagnosis was negatively associated with baseline prevalence of MI, CHF, and any heart disease, indicating that baseline prevalences declined between 1996 and 2001. Table 3 Baseline prevalence, incidence, and cumulative prevalence of cardiovascular comorbid conditions Comorbidities Total Cases Cumulative Prevalence Baseline Prevalence At or Before Initial Diabetes Diagnosis Incident Cases After Diabetes Diagnosis Incidence Rate N % N % N % of those at risk per 100 Person-Years Heart Disease 2,693 26.7% 1,495 14.8% 1,198 13.9% 5.36 Congestive Heart Failure 912 9.0% 417 4.1% 495 5.1% 1.85 Myocardial Infarction 270 2.7% 104 1.0% 166 1.7% 0.59 Cerebrovascular Disease 588 5.8% 294 2.9% 294 3.0% 1.07 Hypertension 7,804 77.3% 5,226 51.8% 2,578 52.9% 26.46 Table 4 Associations with baseline prevalence of comorbid conditions Odds Ratio, (95% Confidence Interval) Male White Hispanic Asian Other Age (per 10 years ) Year of Diabetes Incidence Heart Disease 1.24 (1.10–1.39) 1.32 (1.11–1.57) 0.59 (0.48–0.73) 0.45 (0.31–0.65) 0.49 (0.32–0.74) 1.73 (1.66–1.81) 1.02 (0.99–1.05) Congestive Heart Failure 1.33 (1.08–1.63) 0.86 (0.63–1.19) 0.28 (0.17–0.48) 0.37 (0.18–0.75) 0.32 (0.13–0.79) 1.76 (1.63–1.89) 1.06 (1.00–1.12) Myocardial Infarction 1.36 (0.92–2.01) 1.94 (1.19–3.14) 0.52 (0.23–1.21) 0.00 (0.00->9.99) 0.31 (0.04–2.26) 1.47 (1.28–1.70) 1.03 (0.92–1.15) Cerebrovascular Disease 1.46 (1.15–1.85) 0.90 (0.61–1.32) 0.64 (0.41–1.00) 0.56 (0.27–1.14) 0.10 (0.01–0.70) 1.89 (1.73–2.06) 1.01 (0.94–1.08) Hypertension 0.75 (0.69–0.82) 0.69 (0.60–0.79) 0.39 (0.34–0.45) 0.32 (0.26–0.41) 0.30 (0.23–0.40) 1.85 (1.78–1.91) 1.85 (1.78–1.91) Odds ratios are adjusted for other variables shown in the table: sex, race/ethnicity, exact year of age, and year of diabetes incidence. Reference groups for odds ratios are: Females (for Males); African Americans (for White, Hispanic, Asian, and Other); lower age; and earlier year of diabetes diagnosis. Incidence of heart disease, among those who had not been diagnosed with it at the time of their diabetes diagnosis, was 5.36 cases per 100 person-years, with 13.9% of those at risk being diagnosed before the end of the study period (Table 3 ). Incidence of MI was 0.59 per 100 person-years. Incidence of CHF was 1.85 per 100 person-years. In multiple regression analyses (Table 5 ), Non-Hispanic whites, as compared to African-Americans, were at higher risk of MI and all heart disease. Hispanic and Asian patients were at lower risk of CHF. Incidence of heart disease increased slightly with later year of diabetes diagnosis. Age at diabetes diagnosis was associated with higher incidence of all heart disease outcomes, as was male sex. Table 5 Risk factors for incident comorbid conditions Hazard Ratio, (95% Confidence Interval) Male White Hispanic Asian Other Age (per 10 years ) Year of Diabetes Incidence Heart Disease 1.34 (1.19–1.50) 1.26 (1.06–1.50) 1.00 (0.84–1.20) 0.83 (0.62–1.13) 0.63 (0.42–0.95) 1.48 (1.42–1.54) 1.07 (1.03–1.12) Congestive Heart Failure 1.57 (1.31–1.87) 1.10 (0.85–1.43) 0.57 (0.40–0.81) 0.55 (0.31–0.98) 0.99 (0.59–1.67) 1.56 (1.46–1.67) 1.06 (0.99–1.13) Myocardial Infarction 1.71 (1.25–2.32) 1.81 (1.23–2.67) 0.95 (0.56–1.61) 0.47 (0.15–1.49) 0.45 (0.11–1.84) 1.56 (1.40–1.75) 1.01 (0.90–1.15) Cerebrovascular Disease 1.20 (0.96–1.52) 1.08 (0.77–1.51) 0.61 (0.39–0.97) 0.47 (0.21–1.05) 0.56 (0.23–1.35) 1.66 (1.53–1.82) 0.99 (0.90–1.09) Hypertension 1.04 (0.96–1.13) 0.71 (0.62–0.81) 0.67 (0.59–0.75) 0.78 (0.65–0.94) 0.61 (0.49–0.76) 1.32 (1.28–1.36) 1.29 (1.25–1.33) Hazard ratios are adjusted for other variables shown in the table: sex, race/ethnicity, exact year of age, and year of diabetes incidence. Reference groups for hazard ratios are: Females (for Males); African Americans (for White, Hispanic, Asian, and Other); lower age; and earlier year of diabetes diagnosis. Through the entire study period, 2,693 patients (26.7%) were diagnosed with heart disease (Table 3 ). MI was diagnosed in 270 patients (2.7%) and CHF in 912 (9.0%). In multiple regression analyses (Table 6 ), Non-Hispanic white race/ethnicity was associated with higher cumulative prevalence of MI (odds ratio [OR] 1.90) and any heart disease (OR 1.32). Hispanic race/ethnicity was associated with lower cumulative prevalence of CHF and any heart disease (OR 0.78, 95% CI 0.66–0.91). Asian race/ethnicity was associated with lower cumulative prevalences of all heart disease outcomes. Age at baseline and male sex were positively associated with cumulative prevalence of MI, CHF, and any heart disease. Year of diabetes diagnosis was negatively associated with cumulative prevalence of each of the heart disease outcomes. Table 6 Associations with cumulative prevalence of comorbid conditions Associations with Cumulative Prevalence of Comorbidities Odds Ratio, (95% Confidence Interval) Male White Hispanic Asian Other Age (per 10 years) Year of Diabetes Incidence Heart Disease 1.33 (1.21–1.46) 1.32 (1.14–1.53) 0.78 (0.66–0.91) 0.58 (0.45–0.76) 0.52 (0.38–0.72) 1.71 (1.65–1.78) 0.85 (0.82–0.87) Congestive Heart Failure 1.48 (1.28–1.71) 0.99 (0.79–1.23) 0.43 (0.31–0.58) 0.44 (0.28–0.70) 0.66 (0.41–1.05) 1.68 (1.59–1.78) 0.85 (0.82–0.88) Myocardial Infarction 1.56 (1.22–2.00) 1.90 (1.39–2.59) 0.78 (0.50–1.23) 0.27 (0.09–0.85) 0.40 (0.13–1.26) 1.53 (1.39–1.67) 0.81 (0.75–0.87) Cerebrovascular Disease 1.32 (1.11–1.57) 0.99 (0.76–1.29) 0.62 (0.44–0.86) 0.49 (0.28–0.86) 0.31 (0.13–0.70) 1.80 (1.69–1.93) 0.82 (0.78–0.86) Hypertension 0.87 (0.79–0.97) 0.58 (0.49–0.68) 0.41 (0.36–0.48) 0.39 (0.30–0.49) 0.33 (0.25–0.43) 1.85 (1.78–1.93) 0.91 (0.88–0.93) Odds ratios are adjusted for other variables shown in the table: sex, race/ethnicity, exact year of age, and year of diabetes incidence. Reference groups for hazard ratios are: Females (for Males); African Americans (for White, Hispanic, Asian, and Other); lower age; and earlier year of diabetes diagnosis. Cerebrovascular disease Cerebrovascular disease was diagnosed in 588 patients, evenly split between cases diagnosed at baseline and incident cases subsequent to diabetes incidence (Table 3 ). Baseline prevalence of cerebrovascular disease was 2.9%. In multiple regression analyses (Table 4 ), Hispanic, Asian, and "other" race/ethnicities were associated with lower baseline prevalences of cerebrovascular disease. Age at baseline and male sex were positively associated with baseline prevalence of cerebrovascular disease, while year of diabetes diagnosis was negatively associated with baseline prevalence of cerebrovascular disease (OR 0.78, 95% CI 0.74–0.83). The incidence rate for cerebrovascular disease, among those free of it at baseline diabetes diagnosis, was 1.07 per 100 person-years. In multiple regression analyses, only Hispanic race/ethnicity was associated with lower risk. Age was the only variable associated with a higher incidence of cerebrovascular disease. Cumulative prevalence of cerebrovascular disease was 5.8%. Hispanic and Asian race/ethnicities were associated with lower cumulative prevalences of cerebrovascular disease, as was year of diabetes diagnosis. Age at baseline and male sex were positively associated with cumulative prevalence of cerebrovascular disease. Hypertension A majority of patients (51.8%) had a diagnosis of hypertension at baseline (Table 3 ). Non-Hispanic white, Hispanic, Asian, and "other" race/ethnicities were all associated with lower baseline prevalence of hypertension, as was male sex (Table 4 ). Age and year of diabetes diagnosis were associated with higher baseline prevalence. An additional 2,578 patients were diagnosed with hypertension after diabetes diagnosis, for a cumulative prevalence of 7,804 (77.3%). The incidence rate was 26.46 cases per 100 person-years. Non-Hispanic white, Hispanic, Asian, and "other" race/ethnicities were all associated with lower incidence of hypertension than the African-American reference group, while age and later date of diabetes diagnosis were associated with higher incidence. Cumulative prevalence of hypertension was negatively associated with non-Hispanic white, Hispanic, Asian, and "other" race/ethnicities, male sex and year of diabetes diagnosis, and positively associated with age. Discussion Patients in the Urban Diabetics Study cohort, like other diabetic patient populations, faced a substantial burden of cardiovascular comorbidity. Many were affected by comorbid conditions before they were diagnosed with diabetes, including 14.8% with preexisting heart disease, 2.9% with preexisting cerebrovascular disease, and 51.8% with preexisting hypertension. Among those free of these comorbidities at the time of diabetes diagnosis, similar proportions went on to develop them during followup. The diabetic patients served by the Philadelphia HCCs are almost uniformly low-income and uninsured or underinsured. A large majority are African American. Nonetheless, the prevalence and incidence of major cardiovascular complications does not, in general, appear to exceed the rates of these complications in other patient populations, whether assessed in nationally-representative surveys or in insured, predominantly middle class managed care populations. Disparities associated with race/ethnicity were in varying directions. Non-Hispanic whites had lower rates of hypertension than African Americans but higher rates of heart disease, especially MI, while Asians and Hispanics had more favorable outcomes on several measures. As expected, age was positively associated with all cardiovascular comorbidities. Male sex was associated with higher incidence and prevalence of all comorbidities except hypertension. These findings are comparable to those for other diabetic populations not limited to low-income or uninsured patients. The cumulative prevalence of any heart disease in this study population (26.7%) was similar to the 24.5% prevalence of self-reported coronary heart disease among diabetic patients in the nationally representative 1999–2001 National Health Interview Surveys (NHIS), while the cumulative prevalence of cerebrovascular disease in our study (5.8%) was substantially lower than self-reported stroke in the NHIS sample (9.3%) [ 3 ]. However, the age distribution of diabetics in the NHIS sample was substantially older than in this population. If the comparison is restricted to NHIS diabetic patients age 35–64, national prevalences were 31% lower than in our population for heart disease (18.4% vs. 26.7%) and almost identical for cerebrovascular disease (5.9% vs. 5.8%). The overall incidence rate of MI (0.59 per 100 person-years) in the Urban Diabetics cohort was lower than that in the Kaiser Permanente managed care population studied by Karter, et al. [ 4 ]. Again, this may reflect the fact that the Urban Diabetics cohort was younger (mean age 49.2 years vs. 59.8 years). When the overall incidence rate in our population is multiplied by the hazard ratio for 10 years additional age in our study, the result (0.92 per 100 person-years) is almost identical to the age- and sex-adjusted rate for African Americans reported by Karter, et al. (0.91 per 100 person-years). The incidence rate of CHF, on the other hand, was higher in our population than in studies of Kaiser Permanente patients [ 5 ], while that of cerebrovascular disease was similar to the incidence of stroke reported by Karter, et al. Another study conducted with the Kaiser Permanente diabetes patients found evidence of hypertension for 74% of those diagnosed with diabetes [ 6 ], similar to the 77% cumulative prevalence in the Urban Diabetics study. Other studies have reported somewhat divergent findings for heart disease [ 7 , 8 ] and cerebrovascular disease [ 9 - 11 ], but these studies involved either younger type 1 diabetic patients [ 7 ], earlier time periods [ 8 ], an older population [ 9 ], or analyses that excluded some patients with prior histories of cardiovascular disease [ 10 , 11 ]. Several studies have reported high rates of hypertension among diabetic patients, although none as high as the cumulative prevalence in this cohort [ 9 , 11 - 14 ]. Many of our findings on racial/ethnic differences in the incidence of MI, CHF, and cerebrovascular disease among diabetic patients are also similar to those found by Karter, et al. Like them, we found higher risks of MI for non-Hispanic whites than for other groups, and lower risks of CHF and cerebrovascular disease for Hispanics and Asians than for African Americans and non-Hispanic whites. The magnitudes of the differences between African-American and non-Hispanic white rates were broadly similar between the two studies, while rates for Asians and Hispanics were more variable. The 1999–2001 NHIS also found the prevalence of coronary and other heart disease among patients with diabetes (based on self-reported data) highest among non-Hispanic whites and lowest among Hispanics (rates for Asians were not reported) [ 3 ]. Unlike our study, the NHIS analyses found rates of cerebrovascular disease higher among African Americans with diabetes than among either Hispanics or non-Hispanic whites. Among diabetic patients in the Veterans Health Administration, rates of cardiovascular disease were lower for African Americans, Hispanics, and Asians than for non-Hispanic whites [ 9 ]. However, that study was restricted to patients with at least 4 outpatient clinic visits in a 12-month period, and race/ethnicity was not ascertained for 21.4% of the patients in that study, leaving considerable potential for bias in these results. In our study, male sex was associated with higher baseline prevalence of all comorbidities, higher incidence of the heart disease comorbidities, and higher cumulative prevalence of all comorbidities except hypertension. Our findings are broadly consistent with those in other diabetic populations [ 3 , 4 , 15 ]. The similarities of our findings to those from the Kaiser Permanente studies are striking, given that the latter assessed an insured, West Coast cohort in which most Hispanics were Mexican American [ 4 ], while our study population was an overwhelmingly poor, uninsured or underinsured, East Coast cohort in which most Hispanics were Puerto Rican. The national origins of the Asian patients in the two studies probably also differ, as the Philadelphia Asian community includes larger proportions of South and Southeast Asians and smaller proportions of Japanese and Filipino individuals than the Asian communities of northern California [ 16 ]. Among both Hispanics and Asian Americans, national groups vary widely in socioeconomic status and health outcomes [ 17 - 19 ]. Our study is based on administrative records from several sources, each of which may contain errors, and is unlikely to capture as many cardiovascular endpoints than studies with study-specific, patient-level data collection [ 20 , 21 ]. Because the outpatient encounter forms allowed a maximum of four diagnostic codes, there may have been underreporting of some comorbidities, although cardiovascular disease was unlikely to go unrecorded. Outpatient care outside of the eight Philadelphia HCCs and hospitalizations outside of Pennsylvania were not ascertained. The design of the study could therefore undercount comorbid conditions and cardiovascular endpoints for patients who acquired health insurance or moved out of the city and therefore changed outpatient providers. As a test of the sensitivity of the results to migration in and out of the health system, we repeated the analyses, restricting the follow-up time from the dates of the first to the last to outpatient visit; the results were not materially changed. Among the strengths of the study are its inclusion of a wide range of complications, 8+ year longitudinal design, large sample size, and the combination of data sources to enhance endpoint ascertainment. Our design captured diabetes diagnoses and cardiovascular comorbidity and endpoint data from three complementary sources: outpatient visits in any of the eight Philadelphia HCCs, inpatient visits within any hospital in Pennsylvania and death certificate records. Table 2 supports the observation of Kashner and colleagues that the addition of outpatient administrative data resulted in significantly higher estimates of comorbidity prevalence, strongly suggesting that it has value as an additional data source [ 22 ]. By limiting the study to patients with an initial diabetes diagnosis after the first 34 months for which we have data, we were able to ascertain the prevalence of cardiovascular conditions before and at the time of diabetes diagnosis as well as incident conditions diagnosed after diabetes incidence. We are unaware of any study that has presented similar data except for one limited to elderly African Americans and whites in North Carolina, which included only 653 patients with diabetes [ 23 ]. The fact that patients included in the study had no record of a diabetes diagnosis either in the Philadelphia Health Care Centers or in a hospital discharge record between March 1993 and the date of their diabetes diagnosis, which was no earlier than January 1996, should ensure that the great majority of patients included here were incident diabetes cases. There is a strong, well-established association between socioeconomic disadvantage and cardiovascular disease in industrialized populations [ 24 ], including diabetic populations [ 25 ]. The absence of excess cardiovascular disease beyond that experienced in other diabetic patient populations in this disadvantaged cohort suggests that some factor has offset this disadvantage for this cohort. One possibility is that the provision of primary care, other outpatient services to which patients are referred by their primary care physicians, and prescription medications without out-of-pocket costs to all patients in the Philadelphia HCCs removes a significant barrier to effective care. Restricted use of medications due to costs is common [ 26 ] and has been shown to lead to poorer outcomes for several chronic diseases, including cardiovascular diseases [ 27 , 28 ]. This is the only study of which we are aware to report on cardiovascular comorbidity in a large, ethnically diverse cohort of low-income patients with diabetes with near-universal ascertainment of race/ethnicity. It includes substantial populations of low-income Asians, Hispanics, and non-Hispanic whites as well as African Americans, and allows comparisons of the experiences of these groups. The data is longitudinal and includes both outpatient and hospital discharges over an extended period. By looking at comorbid conditions both before and after diabetes incidence, we have provided both a picture of the health status of these patients at the initial presentation of diabetes and estimates of the incidence of additional comorbidities while being treated for diabetes. Conclusions Cardiovascular comorbidities were common both before and after diabetes diagnosis in this low-income cohort, but not substantially different from mixed-income managed care populations. It is possible that access to primary care and pharmacy services without fees to individuals in this public health clinic system prevented the poorer outcomes usually seen for disadvantaged patients. Competing interests The author(s) declare that they have no competing interests. Authors' contributions JMR and DAW conceived the study and obtained the data. JMR designed the study, performed the analyses, and drafted the manuscript. DAW and CNS revised the manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC550650.xml
555853	Comparative performance of the 16S rRNA gene in DNA barcoding of amphibians	Background Identifying species of organisms by short sequences of DNA has been in the center of ongoing discussions under the terms DNA barcoding or DNA taxonomy. A C-terminal fragment of the mitochondrial gene for cytochrome oxidase subunit I ( COI ) has been proposed as universal marker for this purpose among animals. Results Herein we present experimental evidence that the mitochondrial 16S rRNA gene fulfills the requirements for a universal DNA barcoding marker in amphibians. In terms of universality of priming sites and identification of major vertebrate clades the studied 16S fragment is superior to COI . Amplification success was 100% for 16S in a subset of fresh and well-preserved samples of Madagascan frogs, while various combination of COI primers had lower success rates. COI priming sites showed high variability among amphibians both at the level of groups and closely related species, whereas 16S priming sites were highly conserved among vertebrates. Interspecific pairwise 16S divergences in a test group of Madagascan frogs were at a level suitable for assignment of larval stages to species (1–17%), with low degrees of pairwise haplotype divergence within populations (0–1%). Conclusion We strongly advocate the use of 16S rRNA as standard DNA barcoding marker for vertebrates to complement COI , especially if samples a priori could belong to various phylogenetically distant taxa and false negatives would constitute a major problem.	Background The use of short DNA sequences for the standardized identification of organisms has recently gained attention under the terms DNA barcoding or DNA taxonomy [ 1 - 3 ]. Among the promising applications of this method are the assignments of unknown life-history stages to adult organisms [ 4 , 5 ], the large-scale identification of organisms in ecological or genomic studies [ 1 , 6 ] and, most controversially, explorative studies to discover potentially undescribed "candidate" species [ 4 , 7 , 8 ]. Although it is not a fundamentally new technique [ 9 ], DNA barcoding is promising because technical progress has made its large-scale, automated application feasible [ 3 , 6 ] which may accelerate taxonomic progress [ 10 ]. Although not necessarily under the specific concepts of DNA barcoding and DNA taxonomy, the diagnosis and even definition of taxa by their DNA sequences are realities in many fields and organism groups, such as prokaryotes, fungi, and soil invertebrates [ 1 , 6 ]. To use this approach on a large and formalized scale, consensus of the scientific community is essential with respect to the most suitable genes that allow robust and repeatable amplification and sequencing, and that provide unequivocal resolution to identify a broad spectrum of organisms. While D. Tautz and co-workers [ 3 ] proposed the nuclear ribosomal RNA genes for this purpose, P. D. N. Hebert and colleagues have strongly argued in favor of a 5' fragment of the mitochondrial gene for cytochrome oxidase, subunit I ( COI or COXI ) [ 2 , 11 ]. This gene fragment has been shown to provide a sufficient resolution and robustness in some groups of organisms, such as arthropods and, more recently, birds [ 2 , 4 , 7 , 11 ]. A genetic marker suitable for DNA barcoding needs to meet a number of criteria [ 2 ]. First, in the study group, it needs to be sufficiently variable to discriminate among most species, but sufficiently conserved to be less variable within than between species. Second, priming sites need to be sufficiently conserved to permit a reliable amplification without the risk of false negatives when the goal is the analysis of pooled samples, e.g. when the total of invertebrates from a soil sample is to be studied without separating individuals, or of environmental DNA such as subfossil DNA remains from the soil [ 12 , 13 ]. Third, the gene should convey sufficient phylogenetic information to assign species to major taxa using simple phenetic approaches. Fourth, its amplification and sequencing should be as robust as possible, also under variable lab conditions and protocols. Fifth, sequence alignment should be possible also among distantly related taxa. Here we explore the performance of a fragment of the 16S ribosomal RNA gene ( 16S ) in DNA barcoding of amphibians. As a contribution to the discussion about suitable standard markers we provide experimental data on comparative amplification success of 16S and COI in amphibians, empirical data on conservedness of priming sites, and an example from the 16S -based identification of amphibian larval stages. Results Amplification experiments We performed independent amplification experiments with one set of 16S primers and three published sets of COI primers [ 2 , 7 ] focusing on representatives of different frog, salamander and caecilian genera. The experiments were concordant in yielding more reliable and universal amplifications for 16S than COI . In a set of fresh and well-preserved samples from relatively closely related mantellid frogs from Madagascar (Table 1, Additional file 1 ), the 16S amplification success was complete, whereas the three sets of COI primers yielded success rates of only 50–70%. Considering all three primer combinations, there were two species of frogs (10%) that did not amplify for COI at all ( Boophis septentrionalis and B. tephraeomystax ). Priming sites The variability of priming sites was surveyed using nine complete amphibian mitochondrial sequences from Genbank (Fig. 1 ), and 59 mt genomes of fishes, reptiles, birds and mammals (Fig. 2 ). A high variability was encountered for COI . The sequences of some species were largely consistent with the primers: Xenopus had two mutations only at each of the priming regions. However, other sequences were strongly different, with up to seven mutations, all at third codon positions. No particular pattern was recognizable for any major group that would facilitate designing COI primers specific for frogs, salamanders or caecilians. Interestingly the variability among the amphibian sequences available was as large as or larger than among the complete set of vertebrates at many nucleotide positions of COI priming sites (Fig. 2 ), indicating a possible higher than average variability of this gene in amphibians. Figure 1 Variability of priming sites in amphibians. Variability of priming sites for 16S rRNA and COI in amphibians. Figure 2 Variation of priming sites vertebrates. Variation in priming sites of 16S rRNA (a, F-primer; b, R-primer) and COI (c, Bird-F1, LCO1490; d, HCO2198; e, Bird-R1, Bird-R2) fragments studied herein. Values are nucleotide variability as calculated using the DNAsp program. Grey bars show the values for nine amphibians, black bars the values for a set of 59 other vertebrates (see Materials and Methods, and Figs. 3-4). In contrast, the 16S priming sites were remarkably constant both among amphibians and among other vertebrates (Fig. 1 , 2 ). A wider survey of priming sites, i.e., the alternative reverse priming sites used in arthropod and bird studies [ 2 , 7 ], confirmed the high variability of COI in amphibians, and in vertebrates in general (Fig. 2 ). A screening of the first 800 bp of the C-terminal part of the gene in nine amphibians of which complete mitochondrial genes were available did not reveal a single fragment of 20 bp where all nine species would agree in 80% or more of their nucleotides. Recovery of major groups The phenetic neighbor-joining analysis using the 16S fragment produced a tree that contained eight major groupings that conform to or are congruent with the current classification and phylogeny (Fig. 3 ): cartilaginous fishes, salamanders, frogs, turtles, eutherian mammals, mammals, squamates, birds. Of these, the COI tree (Fig. 4 ) recovered only the lineages of cartilaginuous fishes and birds. The COI analysis did not recover any additional major lineage. Figure 3 16S Neighbor-joining tree of selected vertebrate taxa. Neighbor-joining tree of selected vertebrate taxa based on the fragment of the 16SrRNA gene amplified by primers 16SaL and 16SbH. Numbers in black circles indicate major clades that were recovered by this analysis: (1) cartilaginous fishes, (2) salamanders, (3) frogs, (4) turtles, (5) eutherian mammals, (6) mammals, (7) squamates, (8) birds. Figure 4 COI Neighbor-joining tree of selected vertebrate taxa. Neighbor-joining tree of selected vertebrate taxa based on the fragment of the COI gene amplified by primers LCO1490 and HCO2198. Numbers in black circles indicate major clades that were recovered by this analysis, corresponding to the numbering in Supp. material D. Only two of the clades recovered by the 16S analysis are also monophyletic here: (1) cartilaginous fishes, (8) birds. 16S rDNA barcoding of tadpoles From an ongoing project involving the large-scale identification of tadpoles of Madagascan frogs [ 5 ] we here provide data from larval and adult frog species from two sites of high anuran diversity in eastern Madagascar, Andasibe and Ranomafana. These two localities are separated by a geographical distance of ca. 250 km. The results will be presented in more detail elsewhere. We selected target species for which morphological and bioacoustic uniformity suggests that populations from Ranomafana and Andasibe are conspecific. All these species belong to the family Mantellidae. We then analysed haplotypes within and between these populations. In addition we assessed divergences among sibling species of mantellid frogs (Tables 2-4, Additional file 1 ). These were defined as morphologically similar species that are phylogenetically sister to each other, or are in well-defined but phylogenetically poorly resolved clades of 3–5 species. Results revealed a low intrapopulational variation of 0–3% uncorrected pairwise distances in the 16S gene, a surprisingly large differentiation among conspecific populations of 0–5.1%, and a wide range of differentiation among species, ranging from 1–16.5% with a mode at 7–9% (Fig. 5 ). The few species separated by low genetic distances were allopatrically distributed. The interspecific divergence was higher in those species pairs in which syntopic occurrence has been recorded or is likely (2.7–16.5% divergence, mean 8.5%) as compared to those that so far only have been found in allopatry (1.0–12.9%, mean 6.9%). Figure 5 16S inter- and intraspecific genetic variation in Malagasy frogs. Variation in the fragment of the 16S rRNA gene (ca. 550 bp) studied herein, (a) within populations, (b) among conspecific populations and (c) among sibling species of frogs in the family Mantellidae from Madagascar. The values are uncorrected p-distances from pairwise comparisons in the respective category. Only one (mean) value per species was used in (a) and (b), even when multiple individuals were compared. Grey bars in (a) and (b) show the mean values from all possible individual comparisons within a species, black bars are the maximum divergences encountered between two individual sequences. Phylogenetic and phenetic analyses (Bayesian and Neighbor-joining) of these and many additional sequences (to be published elsewhere) mostly grouped sequences of those specimens from Ranomafana and Andasibe that a priori had been considered to be conspecific (exceptions were Mantidactylus boulengeri , not considered in the intraspecific comparisons here, and M. blommersae ). This indicates that cases in which haplotypes of a species are more similar to those of another species than to those of other conspecific individuals or populations, are rare in these frogs. Sharing of identical haplotypes among individuals belonging to different species, in our dataset, was limited to three closely related species pairs of low genetic divergences: Boophis doulioti and B. tephraeomystax , B. goudoti and B . cf. periegetes , Mantella aurantiaca and M. crocea . Depending on the taxonomic scheme employed, our complete data set contains 200–300 species of Madagascan frogs. Hence, haplotype sharing was demonstrated in 2–3% of the total number of species only. To explore the reliability of tadpole identification using the 16S gene we used local BLAST searches against a database containing about 1000 sequences of adult frogs from a wide sampling all over Madagascar. 138 tadpoles from the Andasibe region and 84 tadpoles from the Ranomafana region were compared with adult sequences in the database. In 77% of the cases the highest scores were those from comparisons to adults from the same site as the tadpoles. In most of the unsuccessful comparisons, adult sequences of the corresponding species were not available from the tadpole site (21%). In only 5 cases (2%) conspecific adults collected from a different site than the tadpoles yielded higher BLAST scores although adult sequences from the same site were in the database. Conclusion DNA barcoding in amphibians DNA barcoding has great appeal as a universally applicable tool for identification of species and variants of organisms, possibly even in automated handheld devices [ 14 ]. However, doubtless severe restrictions exist to its universal applicability [ 9 ]. Some taxa, e.g. cichlid fishes of Lake Victoria, have radiated so rapidly that the speciation events have not left any traces in their mitochondrial genomes [ 15 ]; identifying these species genetically will only be possible through the examination of multiple nuclear markers, as it has been done to assess their phylogeny [ 16 ]. Some snails are characterized by a high intraspecific haplotype diversity, which could disable attempts to identify and distinguish among species using such markers [ 17 ]. Haplotype sharing due to incomplete lineage sorting or introgression is also known in amphibians [ 18 ] although it was not common in mantellid frogs in our data set. However, a number of species showed haplotype sharing with other species, or non-monophyletic haplotypes, warranting a more extensive discussion. In Mantidactylus boulengeri , specimens from Andasibe and Ranomafana have similar advertisement calls and (at least superficially) similar morphologies, but their 16S haplotypes were not a monophyletic group (unpublished data). This species belongs to a group of direct-developing frogs that, like the Neotropical Eleutherodactylus [ 19 ] may be characterized by a high rate of cryptic speciation. Further data are necessary to decide whether the populations from Ranomafana and Andasibe are indeed conspecific. In contrast, there is little doubt that the populations of Mantidactylus blommersae from these two sites are conspecific, yet the Ranomafana haplotypes are closer to those of the clearly distinct species M. domerguei . The species pairs where haplotype sharing has been observed (see Results) all appear to be allopatrically to parapatrically distributed and show no or only low differences in advertisement calls, indicating that occasional hybridization along contact zones may be possible [e.g., [ 20 ]]. Haplotypes of each of these species pairs always formed highly supported clusters or clades, and had divergences below 3%, indicating that haplotype sharing in mantellids may only constitute a problem when individuals are to be assigned to such closely related sister species. Although our data show that DNA barcoding in mantellids is a largely valid approach when both reference and test sequences come from the same site, the occurrence of non-monophyletic and highly divergent haplotypes within species characterizes these and other amphibians as a challenging group for this technique. Certainly, DNA barcoding is unable to provide a fully reliable species identification in amphibians, especially if reference sequences do not cover the entire genetic variability and geographic distribution of a species. However, the same is true for any other morphological or bioacoustic identification method. Case studies are needed to estimate more precisely the margin of error of molecular identification of amphibian species. For many approaches, such as the molecular survey of the trade in frog legs for human consumption [ 21 ], the error margins might be acceptable. In contrast, the broad overlap of intraspecific and interspecific divergences (Fig. 5 ) cautions against simplistic diagnoses of presumably new amphibian species by DNA divergences alone. A large proportion of biological and evolutionary species will be missed by inventories that characterize candidate species by DNA divergences above a previously defined threshold. Comparative performance of DNA barcoding markers in amphibians Phenomena of haplotype sharing or non-monophyletic conspecific haplotypes will affect any DNA barcoding approach that uses mitochondrial genes, and are also to be expected with nuclear genes [e.g., [ 22 ]]. Nevertheless, some genes certainly outperform others in terms of discriminatory power and universal applicability, and these characteristics may also vary among organism groups. The mitochondria of plants are characterized by very different evolutionary patterns than those of animals, including frequent translocation of genetic material into and from the nucleus [ 23 ], which limits their use for DNA barcoding purposes. Nuclear ribosomal DNA ( 18S and 28S ), proposed as standard marker [ 3 ], has a high potential in invertebrate DNA barcoding but its high-throughput amplification encounters difficulties in vertebrates. As a consequence, despite the need of consensus on markers for universal applicability of DNA barcoding, the use of different genes in different groups of organisms seems reasonable. It has been hypothesized that universal COI primers may enable amplification of a 5' terminal fragment from representatives of most animal phyla due to their robustness [ 2 ]. The success in DNA barcoding of lepidopterans and birds suggests that this gene fragment can indeed be used as a standard for many higher animal taxa [ 2 , 4 , 7 ]. In our experiments we compared 16S primers commonly used in amphibians to COI primers that had been developed for other vertebrates [ 7 ] or invertebrates [ 2 ]. It may well be possible, with some effort, to design primers that are more successful and consistent in amplifying COI from amphibians. However, our results from mantellid frogs (Table 1, Additonal file 1 ) indicate a very good amplification success of the primers for some species, but failure for other, related species. This and our results on variability of priming sites predict enormous difficulties in designing one pair of primers that will reliably amplify this gene fragment in all vertebrates, all amphibians, or even all representatives of any amphibian order. A set of one forward and three reverse COI primers have been successfully used to amplify and sequence a large number of bird species [ 7 ], but birds are a much younger clade than amphibians with a probably lower mitochondrial variability. A further optimization of COI amplification may also be achieved regarding the PCR protocol. Herein we used standard protocols that optimized annealing temperature only, whereas more complex touchdown protocols have been used for birds and butterflies [ 4 , 7 ]. However, one major requirement for a DNA barcoding marker is its robustness to variable lab conditions. If DNA barcoding is to be applied as a standard in many different labs, primers and genes need to be chosen that amplify reliably under very different conditions and under standard protocols. This clearly applies to 16S , which we have amplified with very different annealing temperatures and PCR conditions in previous exploratory studies (results not shown). Alignment of 16S sequences is complicated by the prevalence of insertions and deletions, and this gene is less variable than COI [ 2 ]. Nevertheless, our results indicate that even using an uncritical automated alignment this gene has a higher potential than COI to assign vertebrate sequences to the level of classes and orders. The 16S gene is a highly conserved mitochondrial marker but mutations are common in some variable regions, corresponding to loops in the ribosomal RNA structure. In amphibians, where many species are relatively old entities [ 24 ], this ensures a sufficient amount of mutations among species. Our results for amphibians, and previous experience with fishes, reptiles and mammals, indicates that 16S is sufficiently variable to unambiguously identify most species. A further mitochondrial gene that has been widely used in amphibian phylogenetic and phylogeographic studies is cytochrome b . This gene can easily be amplified in salamanders and archaeobatrachian frogs using primers that anneal with adjacent tRNA genes. However, neobatrachian frogs (the wide majority of amphibian species) are characterized by rearrangements of the mitochondrial genome [ 25 , 26 ], and cytochrome b in these species borders directly to the control region. Although cytochrome b primers are available that work in many neobatrachians [ 27 , 28 ], they are not fully reliable. According to our own observations in mantellid frogs, these primers may amplify this gene in one species but fail in other closely related species, presumably because of mutations at the priming sites and similar to the COI primers tested here. In contrast, the 16S primer pair used here can be considered as truly universal not only for amphibians but even for vertebrates. This is also reflected by the high number of amphibian 16S sequences in Genbank (2620 hits for 16S vs. 483 hits for COI , as of September 2004). Moreover, the 16S and 12S rRNA genes have been selected as standard markers for phylogeny reconstruction in amphibians [ 29 ], which will lead to a near-complete global dataset of amphibian 16S sequences in the near future. If the development of handheld devices [ 14 ] is envisaged as a realistic goal, then the universality and robustness of primers should be among the most relevant characteristics of a gene for DNA barcoding. When pooled samples containing representatives of various higher vertebrate taxa are to be analysed, the risk of false negatives strongly increases with decreasing universality of primers. As a consequence we recommend the use of 16S as additional standard DNA barcoding marker for vertebrates, especially for but not limited to applications that involve pooled samples. Methods To test for universality of primers and cycling conditions, we performed parallel experiments in three different laboratories (Berkeley, Cologne, Konstanz) using the same primers but different biochemical products and thermocyclers, and slightly different protocols. The selected primers for 16S [ 30 ] amplify a fragment of ca. 550 bp (in amphibians) that has been used in many phylogenetic and phylogeographic studies in this and other vertebrate classes: 16SA-L, 5' - CGC CTG TTT ATC AAA AAC AT - 3'; 16SB-H, 5' - CCG GTC TGA ACT CAG ATC ACG T - 3'. For COI we tested (1) three primers designed for birds [ 7 ] that amplify a 749 bp region near the 5'-terminus of this gene: BirdF1, 5' - TTC TCC AAC CAC AAA GAC ATT GGC AC - 3', BirdR1, 5' - ACG TGG GAG ATA ATT CCA AAT CCT G - 3', and BirdR2, 5' - ACT ACA TGT GAG ATG ATT CCG AAT CCA G - 3'; and (2) one pair of primers designed for arthropods [ 2 ] that amplify a 658 bp fragment in the same region: LCO1490, 5' - GGT CAA CAA ATC ATA AAG ATA TTG G - 3', and HCO2198, 5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3'. Sequences of additional primers for COI that had performed well in mammals and fishes were kindly made available by P. D. N. Hebert (personal communication in 2004) and these primers yielded similar results (not shown). The optimal annealing temperatures for the COI primers were determined using a gradient thermocycler and were found to be 49–50°C; the 16S annealing temperature was 55°C. Successfully amplified fragments were sequenced using various automated sequencers and deposited in Genbank. Accession numbers for the complete data set of adult mantellid sequences used for the assessment of intra- and interspecific divergences (e.g. in Fig. 5 ) are AY847959–AY848683. Accession numbers of the obtained COI sequences are AY883978–AY883995. Nucleotide variability was scored using the software DNAsp [ 31 ] at COI and 16S priming sites of the following complete mitochondrial genomes of nine amphibians and 59 other vertebrates: Cephalochordata: AF098298, Branchiostoma . Myxiniformes: AJ404477, Myxine . Petromyzontiformes: U11880, Petromyzon . Chondrichthyes : AJ310140, Chimaera ; AF106038, Raja ; Y16067, Scyliorhinus ; Y18134, Squalus . Actinopterygii : AY442347, Amia ; AB038556, Anguilla ; AB034824, Coregonus ; M91245, Crossostoma ; AP002944, Gasterosteus ; AB047553, Plecoglossus ; U62532, Polypterus ; U12143, Salmo . Dipnoi : L42813, Protopterus . Coelacanthiformes : U82228, Latimeria . Amphibia, Gymnophiona : AF154051, Typhlonectes . Amphibia, Urodela : AJ584639, Ambystoma ; AJ492192, Andrias ; AF154053, Mertensiella ; AJ419960, Ranodon . Amphibia, Anura : AB127977, Buergeria ; NC_005794, Bufo ; AY158705; Fejervarya ; AB043889, Rana ; M10217, Xenopus . Testudines : AF069423, NC_000886, Chelonia ; Chrysemys ; AF366350, Dogania ; AY687385, Pelodiscus ; AF039066, Pelomedusa . Squamata : NC_005958, Abronia ; AB079613, Cordylus ; AB008539, Dinodon ; AJ278511, Iguana ; AB079597, Leptotyphlops ; AB079242, Sceloporus ; AB080274, Shinisaurus . Crocodilia : AJ404872, Caiman . Aves : AF363031, Anser ; AY074885, Arenaria ; AF090337, Aythya ; AF380305, Buteo ; AB026818, Ciconia ; AF362763, Eudyptula ; AF090338, Falco ; AY235571, Gallus ; AY074886, Haematopus ; AF090339, Rhea ; Y12025, Struthio . Mammalia : X83427, Ornithorhynchus ; Y10524, Macropus ; AJ304826, Vombatus ; AF061340, Artibeus ; U96639, Canis ; AJ222767, Cavia ; AY075116, Dugong ; AB099484, Echinops ; Y19184, Lama ; AJ224821, Loxodonta ; AB042432, Mus ; AJ001562, Myoxus ; AJ001588, Oryctolagus ; AF321050, Pteropus ; AB061527, Sorex ; AF348159, Tarsius ; AF217811, Tupaia ; AF303111, Ursus (for species names, see Genbank under the respective accession numbers). 16S sequences of a large sample of Madagascan frogs were used to build a database in Bioedit [ 32 ]. Tadpole sequences were compared with this database using local BLAST searches [ 33 ] as implemented in Bioedit. The performance of COI and 16S in assigning taxa to inclusive major clades was tested based on gene fragments homologous to those amplified by the primers used herein (see above), extracted from the complete mitochondrial sequences of 68 vertebrate taxa. Sequences were aligned in Sequence Navigator (Applied Biosystems) by a Clustal algorithm with a gap penalty of 50, a gap extend penalty of 10 and a setting of the ktup parameter at 2. PAUP* [ 34 ] was used with the neighbor-joining algorithm and LogDet distances and excluding pairwise comparisons for gapped sites. We chose these simple phenetic methods instead of maximum likelihood or maximum parsimony approaches because they are computationally more demanding and because the aim of DNA barcoding is a robust and fast identification of taxa rather than an accurate determination of their phylogenetic relationships. Authors' contributions MV designed the study and drafted the manuscript. MT performed parts of the PCR experiments and carried out the molecular identifications of tadpoles. AVDM and DRV performed part of the PCR experiments. YC provided results on 16S differentiation among Madagascan frogs. All authors read and approved the final manuscript. Supplementary Material Additional File 1 Summary of results of amplification experiments, and detailed data of inter- and intraspecific divergences in mantellid frogs. Click here for file	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC555853.xml
517725	Strategies for the recovery of active proteins through refolding of bacterial inclusion body proteins	Recent advances in generating active proteins through refolding of bacterial inclusion body proteins are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. In particular, the pros and cons of well-established robust refolding techniques such as direct dilution as well as less common ones such as diafiltration or chromatographic processes including size exclusion chromatography, matrix- or affinity-based techniques and hydrophobic interaction chromatography are discussed. Moreover, the effect of physical variables (temperature and pressure) as well as the presence of buffer additives on the refolding process is elucidated. In particular, the impact of protein stabilizing or destabilizing low- and high-molecular weight additives as well as micellar and liposomal systems on protein refolding is illustrated. Also, techniques mimicking the principles encountered during in vivo folding such as processes based on natural and artificial chaperones and propeptide-assisted protein refolding are presented. Moreover, the special requirements for the generation of disulfide bonded proteins and the specific problems and solutions, which arise during process integration are discussed. Finally, the different strategies are examined regarding their applicability for large-scale production processes or high-throughput screening procedures.	Background Recombinant DNA technology made available several simple techniques for transferring and efficiently expressing desired genes in a foreign cell. Thus, it was thought that unlimited and inexpensive sources of otherwise rare proteins would become accessible. It soon was observed that the host cell had a great influence on the quality and quantity of the produced recombinant protein. For example, recombinant protein production in mammalian cells yields a biologically active protein with all the required posttranslational modifications. However, mammalian cell cultivation is characterized by low volumetric yields of the recombinant protein, long cultivation times and requirements for expensive bioreactors and medium components. All these points have a great impact on the production costs. On the other hand, bacterial cultivation processes are based on inexpensive media in which fast growth and high cell concentrations can be obtained. These high cell concentrations combined with higher production rates of the bacterial expression system result in higher volumetric productivities. However, the production of recombinant proteins in bacteria such as Escherichia coli frequently yields an inactive protein, aggregated in the form of so-called inclusion bodies. Though, producing an inactive target protein in the form of inclusion bodies is an important drawback, it also has several advantages such as the high degree of purity of the target protein in the aggregate fraction and the increased protection from proteolytic degradation compared to the soluble counterpart. Inclusion bodies have long been considered completely inert towards in vivo dissolution; only recently it was shown that proteins can be resolubilized in vivo from inclusion body deposits [ 1 ]. Although inclusion bodies in general consist of inactive proteins, E. coli can be the superior expression system compared to eukaryotic expression systems when the activity of the recombinant protein can be regained through refolding from the produced inclusion bodies. However, one needs to consider that the decision to select a specific expression system frequently is based on more trivial reasons such as staff knowledge and available equipment and facilities of the producing company/institute. A good example to demonstrate the diverse routes that can be used for recombinant protein production is the manufacturing of tissue-type plasminogen activator (tPA). This protein enables the dissolution of blood clots and is used therapeutically for the treatment of myocardial infarction, thrombosis, pulmonary embolism, and strokes. To assure sufficient tPA for such a widespread application, an economic production process is a necessity. From the beginning, both the mammalian as well as the microbial route were explored for the production of tPA [ 2 ]. tPA is a fairly large (527 amino acids) monomeric protein containing 17 disulfide bridges. Because of this complexity, tPA was first produced in E. coli in the form of inclusion bodies while the mammalian expression system yielded an active protein that was secreted into the culture medium. More recently, obtaining active tPA through secretion into the periplasm of E. coli was attempted [ 3 - 5 ]. The early unsatisfactory yields have been improved [ 6 , 7 ] rendering the E. coli secretion system as a future potential alternative route to generate functional tPA. Other recombinant organisms such as yeast [ 8 ], fungi [ 9 ] or insect cells [ 10 ] have not yet been considered as industrial producers for this protein. Initially, the recombinant tPA introduced into the market was obtained from genetically engineered mammalian cells [ 2 ]. At that time, generating biologically active tPA from E. coli produced material was a process with a poor overall yield [ 2 ]. Today, the majority of commercial tPA (alteplase, Activase ® ) is still produced using a mammalian expression system (Genentech: ). In addition, an amino substituted tPA produced by the mammalian expression system with increased half-life (tenecteplase) was developed. Alternatively, a non-glycosylated, truncated tPA (reteplase, Retavase ® ) produced in E. coli in form of inclusion bodies and afterwards refolded to its biologically active form is now on the market (Centocor: ) and apparently gains market share at the cost of the mammalian-derived product(s) (see Genentech 2004 First Quarter Report). Thus, continuous research effort focused on developing new refolding techniques or improving existing ones by including novel refolding aiding agents can make the bacterial inclusion body system an excellent alternative to the mammalian expression system or other expression systems that can directly generate active proteins with a complex disulfide bond structure. The foremost aim in improving protein refolding from E. coli produced inclusion bodies is to increase both the allowed protein concentrations during the refolding process and the final refolding yield. Recent advances in this area are summarized in conjunction with a short overview on inclusion body isolation and solubilization procedures. Moreover, the different techniques are discussed regarding their applicability for large-scale production processes or high-throughput screening procedures. Isolation and solubilization of inclusion bodies A high degree of purification of the recombinant protein can be achieved by inclusion body isolation [for recent reviews on various aspects of inclusion body formation and renaturation of inclusion body proteins please refer also to [ 11 - 18 ]]. Inclusion bodies are in general recovered by low speed centrifugation of bacterial cells mechanically disrupted either by using ultrasonication for small, French press for medium, or high pressure homogenization for large scale. Main protein contaminants in the crude inclusion body fraction are proteins from the cell envelope, the outer membrane proteins [ 19 ]. These proteins are not integral inclusion body contaminants but coprecipitate together with other insoluble cell material during inclusion body recovery. Lysozyme-EDTA treatment before cell homogenization facilitates cell disruption. Addition of detergents such as Triton X-100 and/or low concentrations of chaotropic compounds either prior to mechanical cell breakage or for washing crude inclusion body preparations allow the removal of membrane proteins or other nonspecifically adsorbed cell material [ 11 - 14 ]. After their isolation, inclusion bodies are commonly solubilized by high concentrations of chaotropic agents such as guanidinium hydrochloride or urea. Although expensive, guanidinium hydrochloride is in general preferred due to its superior chaotropic properties. Moreover, urea solutions may contain and spontaneously produce cyanate [ 20 ], which can carbamylate the amino groups of the protein [ 21 ]. In addition, inclusion body solubilization by urea is pH dependent and optimum pH conditions must be determined for each protein [ 22 ]. There are also reports that inclusion bodies can be solubilized at extreme pH in the presence or absence of low concentrations of denaturants [ 23 - 25 ]. However, extreme pH treatments can result in irreversible protein modifications such as deamidation and alkaline desulfuration of cysteine residues [ 26 ]. Finally, inclusion bodies can be solubilized with different types of detergents [ 27 , 28 ], low concentrations of denaturants [ 29 , 30 ], or even by utilization of the aggregation suppressor arginine [ 29 ]. Inclusion body proteins solubilized under these mild conditions can possess a native-like secondary structure [ 28 - 30 ], and may even reveal some biological activity [ 29 , 31 ]. It has also been demonstrated that the utilization of milder solubilization conditions can lead to higher final refolding yields compared to solubilization by high concentrations of guanidinium hydrochloride or urea [ 27 ]. In addition to the solubilizing agent, the presence of low molecular weight thiol reagents such as dithiothreitol (DTT) or 2-mercaptoethanol is generally required. These substances will reduce nonnative inter- and intramolecular disulfide bonds possibly formed by air oxidation during cell disruption and will also keep the cysteines in their reduced state [ 14 , 15 ]. Optimum conditions for disruption of existing disulfide bonds are found at mild alkaline pH since the nucleophilic attack on the disulfide bond is carried out by the thiolate anion. Residual concentrations of reducing substances can negatively affect the refolding process, thus, they are frequently removed ( e.g . by dialysis) before starting the refolding procedure. As an alternative, immobilized reducing agents ( e.g . DTT; VectraPrime™, Biovectra) could simplify reducing agent removal by centrifugation after the solubilization process. Finally, the pH must be reduced before the removal of the reducing agent from the solution containing the solubilized protein to prevent the formation of undesired disulfide bonds. Principles of refolding solubilized and unfolded proteins Correct refolding versus aggregation In general, the methods used for inclusion body solubilization result in a soluble protein that is devoid of its native conformation. This protein must then be transferred into conditions that allow the formation of the native structure ( e.g . low denaturant concentration). Moreover, appropriate redox conditions have to be established when the protein contains disulfide bonds in the native state. When proper conditions for refolding are identified, the refolding process can require a few seconds or several days. During this period, the correct refolding pathway competes, often in disadvantage, with misfolding and aggregation of the target protein (Figure 1 ). Protein refolding involves intramolecular interactions and follows first order kinetics [ 32 - 35 ]. Protein aggregation, however, involves intermolecular interactions and, thus, is a kinetic process of second or higher order, which is favored at high protein concentrations [ 32 - 35 ]. In fact, refolding yields commonly decrease with increasing initial concentrations of the unfolded protein independent of the refolding method applied [ 35 - 40 ]. Aggregates are formed by nonnative intermolecular hydrophobic interactions between protein folding intermediates, which have not yet buried their hydrophobic amino acid stretches (Figure 1 ). When the refolding process is beyond these aggregation-prone intermediates, the productive folding pathway is favored and aggregation does not occur. Therefore, prevention of hydrophobic intermolecular interaction during the first steps of refolding is crucial to allow successful renaturation at high protein concentrations. Only recently a non-empirical method for predicting the fate of proteins during the refolding process was proposed [ 41 ]. It is based on the second viral coefficient, which indicates the magnitude of protein interaction under certain refolding conditions, and thus its tendency to aggregate. However, though being soluble in the refolding buffer is essential for a protein molecule to refold, it does not ensure that it will fold into the native form. Are further purification steps required after solubilization of inclusion bodies? The recombinant target protein represents in general the major fraction of the inclusion body proteins. Therefore, refolding attempts can be undertaken directly after solubilization of the inclusion bodies. Some reports, however, claim higher refolding yields when the solubilized inclusion body proteins are purified prior to the refolding attempt [ 36 , 39 , 42 , 43 ]. Additional purification has been recommended when the protein of interest represents less than 2–5% of the total cell protein [ 26 ] or less than 2/3 of the total inclusion body protein [ 42 ]. The type of contaminants can also be crucial for the success of the refolding process. For example, typical non-proteinaceous contaminants of inclusion body preparations did not affect refolding yields of lysozyme, while proteinaceous contaminants, which have a high tendency towards aggregation significantly reduced refolding yields [ 42 ]. Further purification prior to the refolding attempt does not seem to be required, even at low target protein concentrations, when the solubilized inclusion body proteins are subjected to refolding conditions during size exclusion chromatography where refolding and purification can occur simultaneously [ 44 ]. All pros and cons of any further purification step have to be carefully evaluated as they cause potential protein loss and additional production costs. Techniques for protein refolding Direct dilution The simplest refolding procedure is to dilute the concentrated protein-denaturant solution into a refolding buffer that allows the formation of the native structure of the protein. Most frequently, the final protein concentration after dilution is in the 1–10 μg/ml range in order to favor the productive refolding instead of the unproductive aggregation pathway. Though ideal at laboratory scale, this technique has serious drawbacks during scale-up as huge refolding vessels and additional cost-intensive concentration steps are required after renaturation. A major improvement of this technique was the development of a method where the solubilized, denatured protein is added in pulses or continuously into the refolding buffer [ 37 , 40 , 45 - 47 ]. This technique still keeps the simplicity of the direct dilution method while considerably increasing the final concentration of the refolded protein. Prerequisite is an appropriate knowledge of the folding kinetics of the target protein. The addition of the concentrated protein-denaturant solution should occur at rates slower than the rate-determining folding step of the target protein, thereby avoiding the accumulation of aggregation-prone folding intermediates [ 37 , 46 ]. For pulse addition it has been recommended that 80% of the maximum refolding yield should be reached before adding the next pulse [ 14 ]. Other factors to be considered are the increasing residual concentration of the denaturant with each pulse, which should not surpass concentrations that affect the refolding of the protein, and the amount of protein added per pulse, which should be optimized in batch experiments to minimize aggregation [ 14 ]. Membrane controlled denaturant removal Another technique to transfer the solubilized and unfolded protein to conditions allowing the formation of the native structure is the utilization of dialysis and diafiltration systems for denaturant removal [ e.g . [ 48 - 51 ]]. In contrast to the direct dilution method, the change from denaturing to native buffer conditions occurs gradually. Thus, the protein passes through different regimes of denaturant concentrations, where folding intermediates that are prone to aggregation may become populated. Most often, these techniques cause more aggregation during refolding compared to the direct dilution method [ e.g . [ 52 ]]. Additionally, refolding yields can be negatively affected by non-specific adsorption of protein to the membrane. However, for some proteins and with the appropriate denaturant removal rates, adapted to the requirements of the target protein, high refolding yields at high protein concentrations can be obtained [ 50 - 53 ]. A fairly simple device was recently introduced allowing continuous or pulse refolding in a similar way as in the direct dilution method [ 54 ]. Chromatographic methods for protein refolding Protein refolding based on size exclusion chromatography Buffer exchange for denaturant removal can also be carried out by using size exclusion chromatography (SEC). Most frequently, the denaturant-protein solution is injected into a column previously equilibrated with the refolding buffer [ 44 , 55 - 58 ]. Subsequent elution with the refolding buffer results in a refolded protein in the eluate fraction with a considerably higher concentration compared to concentrations that can be reached by the simple dilution technique [ 44 , 56 , 58 ]. Protein refolding may be completed in the column or for proteins with slow folding kinetics the final folding steps may occur in the eluate fraction [ 44 ]. Aggregate formation is supposed to be reduced either by physical separation of aggregation-prone folding intermediates in the porous structures of the gel [ 56 ] or, more likely, by resolubilization of formed aggregates through the delayed running front of the denaturant, which gives the solubilized aggregates another opportunity to refold [ 14 ]. For proteins, which exhibit superior refolding yields during gradual denaturant removal, such as lysozyme, elution during SEC is preferably performed by using a decreasing denaturant gradient [ 38 , 52 , 59 ]. In specific cases, the denaturant removal can be accompanied with other changes in the buffer composition ( i.e . pH) for further optimization of refolding conditions [ 52 ]. An additional advantage of this chromatographic method is the concomitant purification of the target protein during the refolding process [ 44 ]. Furthermore, some recent applications have shown the feasibility of using SEC for continuous processes of protein refolding [ 60 , 61 ]. Also, SEC in combination with the use of an annular chromatography system can be coupled to an ultrafiltration and recycling unit for reinjection of resolubilized aggregates, which may form during the refolding process [ 60 ]. Some parameters for refolding using SEC are of key importance. For example, protein aggregation during sample injection can cause low refolding yields [ 62 ]; injecting the sample followed by an additional small volume of denaturant solution solves this problem [ 44 , 52 , 62 ]. Also, optimum results can only be reached when the properties of the chromatographic resin allow efficient separation of the renatured target protein from different folding intermediates, misfolded protein, and aggregates that might form during the refolding process [ 59 , 63 , 64 ]. In general, lower refolding yields are obtained by injecting the denatured protein at high concentrations [ 38 , 58 , 60 , 61 , 64 ] and/or by elution at high rates [ 52 , 62 , 64 ]. Both conditions result in poor separation among different folding intermediates thereby boosting protein precipitation. Matrix-assisted protein refolding Attaching the solubilized and unfolded protein to a solid support prior to changing from denaturing to native buffer conditions is another approach to avoid the unwanted intermolecular interaction between aggregation-prone folding intermediates. Binding of the solubilized and unfolded protein to the matrix requires the formation of a stable protein-matrix complex withstanding the presence of chaotropic agents. However, after changing to native buffer conditions, the detachment of the refolded target protein from the matrix should easily be accomplished. Several combinations of binding motives and matrices have been employed for binding the unfolded protein to the solid support. For example, proteins with a natural occurring charged patch in the unfolded chain, which binds to ion exchange resins [ 59 , 65 - 67 ], or proteins containing artificially engineered peptide tags such as the hexahistidine tag, which binds to immobilized metal ions [ 59 , 68 - 70 ], or N- or C-terminal hexaarginine tags binding to a polyanionic support [ 71 ], or protein fusions with denaturant-resistant binding domains, such as a glutathione S-transferase fragment, which binds to an anion exchange matrix [ 72 ] or the cellulose binding domain of the cellulose degrading multienzyme complex of the thermophilic bacterium Clostridium thermocellum , which binds to a cellulose matrix [ 73 ], have been employed. After binding, the matrix-protein complex is brought to refolding conditions by any of the above-mentioned techniques such as dilution [ 71 ], dialysis [ 68 , 73 ], or buffer exchange through chromatography [ 59 , 66 , 69 , 70 , 72 ]. Finally, the refolded protein can be detached from the matrix, e.g . in the case hexahistidine-tagged proteins by elution with EDTA [ 69 ] or imidazole [ 59 , 70 ] or by buffers with high ionic strength in the case of proteins bound by ionic interactions [ 59 , 65 , 66 , 71 , 72 ]. Due to the selective binding, matrix-assisted refolding can combine the renaturation of the target protein along with its purification from host cell protein contaminants [ 69 , 70 , 72 ]. Refolding using hydrophobic interaction chromatography Hydrophobic interaction chromatography (HIC) has also been successfully used for protein refolding with concomitant removal of contaminating proteins during the renaturation process [ 74 - 78 ]. Unfolded proteins are applied to the column at high salt concentrations and refolded and eluted with a decreasing salt gradient. In contrast to the above-mentioned chromatographic methods there is no requirement for typical refolding aiding agents such as arginine during the in-column refolding process. Moreover, refolding of the disulfide containing protein proinsulin was even obtained in the absence of a redox system in the mobile phase [ 76 ]. It has been proposed that refolding is facilitated during HIC because unfolded proteins adsorb at high salt concentrations to the hydrophobic matrix and, thus, are not prone to aggregation. Additionally, hydrophobic regions of the protein that adsorb to the HIC matrix form microdomains around which native structure elements can form. During migration through the column, the protein will pass through several steps of adsorption and desorption, controlled by the salt concentration and hydrophobicity of the intermediate(s), resulting finally in the formation of the native structure [ 75 ]. Physical and chemical features improving protein refolding yields Apart from any of the above-mentioned techniques for protein refolding, there are physical and chemical variables that have a great impact on the final yield of biologically active protein. For example, temperature as well as the composition of the refolding buffer are important variables influencing the final refolding yield. Physical variables aiding protein refolding The most important physical variable influencing the refolding yield is the temperature [ 40 , 45 , 50 , 51 ]. Temperature has a dual effect on the refolding process. On one side, it influences the speed of folding and on the other it influences the propensity towards aggregation of folding intermediates with exposed hydrophobic patches. Also, there is limited temperature range in which each protein is thermodynamically stable in a given buffer system [ 79 ]. In general, low temperatures support the productive folding pathway as hydrophobic aggregation is suppressed. However, low temperatures also slow down the folding rates, thus increasing the time required for renaturation [ 51 ]. For refolding attempts of a new protein, 15°C has been proposed as a good starting point [ 14 ]. Pressure was identified as another important physical variable affecting protein structure as well as protein refolding processes [ 80 ]. It was shown that high pressure up to 3 kbar can disrupt oligomeric protein structures [ 80 ] and can dissolve protein aggregates and inclusion bodies [ 81 , 82 ]. The disassembled protein monomers retain native-like secondary structure up to 5 kbar [ 80 ]. After gradual depressurization, they can reach their native state even at high protein concentrations, because folding intermediates prone to aggregate at atmospheric pressure are prevented from aggregation by high pressure [ 81 - 83 ]. Chemicals aiding protein refolding Certainly, L-arginine is nowadays the most commonly used refolding aiding agent [ 14 ]. It impedes aggregate formation by enhancing the solubility of folding intermediates, presumably by shielding hydrophobic regions of partially folded chains. In addition, it has been shown that numerous other low molecular weight additives such as detergents, protein-stabilizing agents such as glycerol or even low residual concentrations of denaturants improve refolding yields by suppressing aggregation [ 14 ]. In addition, high-molecular weight additives such as polyethylene glycol were used successfully for enhancing protein refolding yields [ 84 ]. More recently, low-molecular weight non-detergent zwitterionic agents such as sulfobetaines, substituted pyridines and pyrroles and acid substituted aminocyclohexanes have been employed successfully for protein renaturation [ 40 , 85 - 87 ]. Moreover, polymers with temperature-dependent hydrophobicity were effectively applied for protein refolding at higher temperatures [ 88 , 89 ]. The benefit of each of these refolding aiding agents for a given renaturation system has to be elucidated experimentally, as they are not equally advantageous for all proteins. The mechanisms of interactions of these refolding aiding agents with the folding intermediates remain often obscure although it is clear that all these substances suppress aggregation in favor of the productive folding pathway [ 90 ]. Micelles and liposomes as protein refolding aiding systems Detergents [ 91 , 92 ] and phospholipids [ 93 , 94 ], in the form of micelles and liposomes [ 95 ], respectively, as well as mixed micelle systems formed by phospholipids and detergents [ 92 , 95 , 96 ] have shown potential to aid protein refolding. Most likely, illegitimate hydrophobic interactions between folding intermediates are suppressed by transient nonpolar interactions between the protein and the micelle or liposome [ 91 - 93 ]. Additional transient polar interactions in mixed micelles are supposed to be responsible for higher refolding yields compared to only detergent-based micelle systems [ 92 , 96 ]. Moreover, liposomes linked covalently to chromatographic resins have potential to combine renaturation and separation of the refolded target protein [ 93 , 94 ]. Reversed micelles, formed when an aqueous detergent solution is mixed with an organic solvent, can also facilitate protein refolding by avoiding aggregate formation [ 97 ]. The denatured protein, once transferred to this solution, tries to avoid the organic phase, and, after reaching the hydrophilic core of the reversed micelle, can refold as a single molecule [ 97 ]. Recently, it was demonstrated that protein precipitates can be solubilized by direct addition into the reversed micellar system allowing refolding with high yields at high protein concentrations [ 98 - 100 ]. Yet, direct solubilization of inclusion bodies in reversed micellar systems has not been reported. In addition, recovery of refolded protein from these micellar structures is not easily accomplished [ 97 , 99 ]. Chemical and biological protein refolding aiding agents mimicking in vivo folding conditions Natural chaperones Chaperones are a group of proteins conserved in all kingdoms, which play a key role in assisting in vivo protein folding and protecting cellular proteins from different types of environmental stress by suppressing protein aggregation. For example, the major E. coli chaperonin GroEL is involved in the in vivo folding of 10% of all newly synthesized proteins at normal growing conditions, and of 30% under stress conditions [ 101 ]. GroEL assists protein folding by a first capturing step of aggregation-prone folding intermediates [ 102 ]. The release of the folding-competent form is then accomplished in an ATP-dependent fashion through the action of the cochaperonin GroES [ 102 ]. Natural chaperones have also been applied successfully to refold various proteins in vitro [ 103 ]. However, their routine application is limited by their cost, the relatively high chaperone concentration required (at least equimolar to the target protein) and the need for their removal after the refolding procedure [ 103 , 104 ]. Some procedures have tried to overcome these limitations by utilizing immobilized and reusable (mini)-chaperone systems [ 104 - 106 ]. Nevertheless, chaperone-based refolding processes are not robust enough for large-scale processes [ 14 ]. Artificial chaperones A further development of the detergent-based micellar system mimics the two-step mechanism of chaperone-assisted protein folding. The capturing step is performed by diluting the denatured protein into a detergent solution, which prevents protein aggregation through the formation of mixed protein-detergent micelles [ 107 - 110 ]. Aqueous solutions of hydrogel nanoparticles ( e.g . self-assembly of hydrophobized polysaccharides such as cholesterol-bearing pullulan) have been also used for the capturing step [ 111 ]. The release of the folding-competent protein is subsequently initiated by the addition of cyclodextrins [ 107 - 112 ]. They are added in excess to the capturing agent and strip the detergent from the protein-detergent micelles through the formation of a tight detergent-cyclodextrin complex. Long cyclodextrin polymers as striping agent were reported to result in higher refolding yields compared to monomeric cyclodextrins [ 113 ]. Also, rapid addition of soluble cyclodextrins is thought to result in higher refolding yields compared to slow addition [ 108 , 109 ] or the utilization of immobilized cyclodextrins [ 108 , 109 ]. However, at least for α-glucosidase similar refolding yields were reported by stripping the detergent either with soluble or immobilized cyclodextrins [ 114 ]. The utilization of these cyclodextrin polymer beads allows simple removal of the cyclodextrin-detergent complex by centrifugation and, moreover, these beads can be used in expanded-bed columns in semicontinuous refolding processes [ 114 ]. Liquid paraffin as pseudolipid bilayer membrane In vivo , many proteins are transported through bilayered membranes in an extended and partially unfolded form either simultaneously or after their synthesis [ 115 ]. A rather peculiar protein refolding procedure mimicking the effect of a bilayered membrane was carried out in a three-phase liquid system built up in a centrifugation tube [ 116 ]. The upper phase contained an organic solution, which was separated from the aqueous refolding buffer by liquid paraffin. The protein, in an aggregated and denatured form, was added to the organic phase and forced to pass through the paraffin film into the refolding buffer by centrifugation. This procedure was successfully applied for the refolding of aggregated and denatured preparations of the model proteins RNase A and BSA. Template-assisted protein folding Several proteins are synthesized in their natural environment with amino-terminal propeptides usually located between a signal sequence and the mature part of the protein. In vivo , these propeptides are known to play a key role in assisting the correct folding of the mature part of the protein [ 117 ]. In vitro studies have demonstrated that they can facilitate the refolding in cis , when the denatured mature protein is still linked to its propeptide prior to the transfer into the refolding buffer, or in trans by including the isolated propeptide into the refolding buffer [ 118 - 120 ]. This propeptide assisted protein refolding can be exploited for the renaturation of inclusion body proteins either by synthesizing the mature part linked to its propeptide, thus allowing later facilitated refolding [ 121 , 122 ], or by synthesizing the mature protein and then including the appropriate propeptide into the refolding buffer [ 123 ]. Another method of template-assisted protein refolding exploits the specific binding properties of monoclonal antibodies to the target protein to reduce the time required for protein refolding and to enhance the final refolding yield [ 124 , 125 ]. This procedure does not work with all antibodies and depends on the availability of specific antibody clones. Thus, it represents more a proof-of-principle rather than a practical approach to generate active proteins through refolding of inclusion body proteins. Proteins containing disulfide bonds: special requirements In general, solubilization of inclusion body proteins by chaotropic agents is carried out in the presence of reducing agents such as dithiothreitol or β-mercaptoethanol to allow the disruption of nonnative disulfide bonds. Following solubilization, naturally disulfide-bonded proteins have to be refolded under conditions, which permit the formation of their native disulfide bonds. In the simplest way, free cysteine residues can be oxidized by molecular oxygen, a redox reaction catalyzed by Cu 2+ ions [ 126 , 127 ]. Though a cheap option, air oxidation is slow, often results in mismatched disulfides, and is not suitable for disulfide-bonded proteins, which also have free cysteines [ 26 ]. Disulfide bonds are more efficiently formed when a mixture of low molecular weight thiols ( e.g . glutathione) in their reduced and oxidized state is added to the refolding buffer [ 126 , 128 ]. Best conditions for refolding of disulfide-bonded proteins are commonly established when the reduced form is present in excess and the pH is slightly alkaline. These conditions allow rapid disulfide exchange reactions until the protein reaches the most stable disulfide-bonded configuration, in general the native state of the protein [ 26 , 126 , 128 - 130 ]. Recently, a novel generation of aromatic thiols was developed which have lower p K a values as the aliphatic thiols thus enabling disulfide-bond formation at lower pH values [ 131 , 132 ]. These thiol reagents might be useful for the refolding of proteins with limited stability at alkaline conditions. Also, an immobilized disulfide-reshuffling system based on thiol-carrying latex particles has recently been successfully applied for the refolding of RNase A [ 133 , 134 ]. Naturally disulfide-bonded proteins in their reduced states are often very unstable and exhibit a high tendency towards aggregation, especially during the early stages of refolding [ 128 ]. These problems can be overcome by modifying the reduced thiol groups in the unfolded protein, either by S-sulfonation [ 39 , 128 , 135 , 136 ] or by transforming the free cysteines into mixed disulfides with the oxidized form of a thiol reagent ( e.g . glutathione) [ 128 , 137 ]. These chemical modifications introduce numerous charged residues into the protein, which prevent the intermolecular interactions responsible for aggregation. The chemically modified protein is then transferred to refolding conditions. Correct disulfide bond formation for S-sulfonated proteins is initiated by supplementing the refolding buffer with the appropriate redox system [ 39 , 128 , 135 , 136 ], or, for proteins with mixed disulfides by adding trace amounts of the reduced form of the thiol reagent [ 128 , 137 ]. Improvements of refolding yields of disulfide-bonded proteins have also been achieved by using protein disulfide isomerase (PDI) in combination with a redox system. PDI is a folding catalyst that assists disulfide bond formation in vivo [ 138 ] and was successfully implemented for aiding disulfide bond formation during in vitro protein refolding [ 139 , 140 ]. In some cases PDI did not show significant effects on the refolding yield but significantly increased the refolding rate [ 141 ]. However, residual concentrations of chaotropic agents in the refolding buffer, especially guanidinium hydrochloride, can drastically reduce PDI activity [ 142 ]. Traces of small peptides containing the active site of PDI [ 143 ] and chemically synthesized dithiol molecules mimicking PDI function [ 144 - 146 ] have also shown potential to increase the in vitro refolding yields generally obtained with the common redox systems [ 143 , 146 ]. Process integration Published refolding processes are often composed of numerous and cumbersome steps, both downstream ( e.g . cell disruption, inclusion body isolation and purification by several centrifugation and washing steps followed by a final solubilization procedure) and upstream of the renaturation process ( e.g . removal of aggregates and misfolded protein and final purification of the correctly refolded target protein). Scale-up problems can arise when some of these steps are not transferable to larger scale processes. As an alternative to the common downstream process, inclusion bodies can directly be solubilized from chemically treated E. coli cells [ 147 - 150 ] or in combination with mechanical treatments [ 72 ]. Even more, inclusion body solubilization directly from cells in the cultivation broth is feasible as was shown for periplasmic [ 147 ] as well as for cytoplasmic inclusion bodies [ 151 ]. A high degree of purification, removal of cell debris and E. coli host cell proteins, can be achieved by selective extraction of inclusion body proteins combined with diafiltration [ 148 ], aqueous two-phase extraction [ 147 ] or selective capture by either expanded bed chromatography [ 72 , 149 , 151 ] or by attachment to magnetic particles recoverable in high gradient magnetic fields [ 152 ]. Major difficulties often arise by the increase of broth viscosity due to release of DNA after chemical treatment requiring its selective removal e.g . by precipitation through spermidine addition [ 149 , 151 ] or preferably by treatment with DNA-degrading enzymes [ 153 ]. Afterwards, the prepurified and solubilized target protein can be subjected to refolding conditions using any of the above-mentioned methods. Moreover, there are reports on integrated processes where solubilization of the target protein from chemically treated cells is followed by a chromatographic process in which the capturing step and removal of E. coli contaminants is followed directly and in the same operation unit by refolding and subsequent purification [ 72 , 74 ]. Utilization of refolding methods based on chromatographic processes is additionally advantageous as they combine refolding with an at least partial purification of the target protein [ 44 , 69 - 72 ]. In addition, aggregates formed during the refolding process can also be removed through chromatographic processes as they have a different retention time compared to the correctly folded protein [ 56 , 64 , 67 ]. Finally, chromatographic processes can be performed continuously [ 60 , 61 ] with the possibility to recycle aggregates formed during the refolding process thus leading to processes with refolding yields up to 100% [ 60 ]. Perspectives After the first enthusiasm about protein production using recombinant microorganisms, it was promptly understood that obtaining an active form of the desired protein was not a simple task. Many proteins form nonnative precipitates in form of inclusion bodies when synthesized in bacteria and there is no universal refolding recipe for the generation of native protein from solubilized inclusion bodies. For any given protein, the best refolding conditions still have to be determined empirically. Among a lot of experience and "a good feeling for the best way", the use of experimental design methodologies [ 154 , 155 ] and further improvements in predicting the likelihood of aggregation [ 41 , 156 ] may increase the speed for finding the optimal refolding conditions for a given protein. Also, less-established and new techniques as well as new refolding aiding additives may become more widely used in the near future. However, these techniques or new protein refolding aiding substances await rigorous testing for refolding of not only easy-going model proteins such as RNase A but also for more recalcitrant inclusion body proteins. Moreover, refolding strategies also have to be adapted to the required quantity and final use of the refolded protein. For therapeutic proteins needed in great quantities more effort can be undertaken to identify the best refolding conditions leading to high yields of the correctly folded protein. For a protein where just a few milligrams are required for biochemical and/or structural studies process optimization with respect to high yields is not such a necessity. Also, special demands for high throughput refolding screening arising from structural genomic projects require robust strategies that will lead to monodisperse refolded protein samples [ 157 ]. In this case, the direct dilution method in combination with variations in temperature and buffer composition is still the best approach. Altogether, new strategies need to increase the robustness of refolding processes and/or decrease the costs to find acceptance for broader applications.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC517725.xml
546191	Research protocol: EB-GIS4HEALTH UK – foundation evidence base and ontology-based framework of modular, reusable models for UK/NHS health and healthcare GIS applications	EB-GIS4HEALTH UK aims at building a UK-oriented foundation evidence base and modular conceptual models for GIS applications and programmes in health and healthcare to improve the currently poor GIS state of affairs within the NHS; help the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, and requirements; and also foster the much-needed NHS-academia GIS collaboration. The project will focus on diabetes and dental care, which together account for about 11% of the annual NHS budget, and are thus important topics where GIS can help optimising resource utilisation and outcomes. Virtual e-focus groups will ensure all UK/NHS health GIS stakeholders are represented. The models will be built using Protégé ontology editor based on the best evidence pooled in the project's evidence base (from critical literature reviews and e-focus groups). We will disseminate our evidence base, GIS models, and documentation through the project's Web server. The models will be human-readable in different ways to inform NHS GIS implementers, and it will be possible to also use them to generate the necessary template databases (and even to develop "intelligent" health GIS solutions using software agents) for running the modelled applications. Our products and experience in this project will be transferable to address other national health topics based on the same principles. Our ultimate goal is to provide the NHS with practical, vendor-neutral, modular workflow models, and ready-to-use, evidence-based frameworks for developing successful GIS business plans and implementing GIS to address various health issues. NHS organisations adopting such frameworks will achieve a common understanding of spatial data and processes, which will enable them to efficiently and effectively share, compare, and integrate their data silos and results for more informed planning and better outcomes.	" Geocoding (street address matching or assignment of latitude and longitude) will be the basis for data linkage and analysis in the 21st century. The versatility of GIS supports the exploration of spatial relationships, patterns, and trends that otherwise would go unnoticed ." – US Healthy People 2010 Objectives (item 23-3 – ) Introduction Geography matters – the need for an evidence-based, spatio-temporal approach to public health Geography plays a major role in understanding the dynamics of health, and the causes and spread of disease [ 1 ]. The classic public health triad composed of man, agent/vehicle and environment emphasises the importance of geographic location (environment or space where we live) in health and disease. Interactions within this triad can also change with time. Today's health planners aim at developing health policy and services that address geographic and social inequalities in health, and therefore should benefit from evidence-based approaches that can be used to investigate spatial aspects of health policy and practice, and evaluate geographic equity (or inequity) in health service provision [ 2 ]. On geo-information and the need for applications to support the decision maker According to the US Federal Geographic Data Committee (FGDC), geographic location is a key feature of 80–90% of all government data [ 3 ]. The same can be also said about government data in other countries, including data generated by the health sector in the UK. This locational or spatial reference is a "main key" in the transformation of data into information, and for linking and integrating many health and other datasets from disparate sources covering same and contiguous locations [ 4 ]. Unlike other resources like employees or funds, spatial data do not suffer any wear and tear from repeated use. On the contrary, reusing data increases the possibilities for improving the content quality of data collections and gaining new insights by linking and exploring the relationships between the different datasets that makeup the big picture [ 4 ]. This implies the need to develop applications and not just focus on data. An overemphasis on data acquisition, without health sector-linked applications, will not provide any momentum for further development. Visualisation, modelling and analysing activities will be the focus of value-added services in the coming years [ 4 , 5 ]. Methods must be identified and/or developed to process our spatial data assets to produce meaningful, bottom-line conclusions that can support the decision maker rather than mere bunches of facts. According to Openshaw [ 5 ], the ideal methods should be safe and usable by people with no higher degrees in statistical or spatial sciences. The methods should also respond to user needs on the ground, be highly automated, explicitly handle spatial data imprecision, and produce self-evident results that can be mapped and communicated to non-experts. On GIS and their health and healthcare applications In 2003, the US National Library of Medicine added the term "Geographic Information Systems" (GIS) to its controlled vocabulary thesaurus known as MeSH (Medical Subject Headings – see ), a step reflecting the importance and growing use of GIS in health and healthcare research and practices. The US FGDC defines GIS as "computer systems for the input, storage, maintenance, management, retrieval, analysis, synthesis, and output of geographic or location-based information. In common usage by organisations, GIS include hardware, software, and data. GIS also imply the people and procedures involved in GIS operation" (cited in [ 6 ]). The inclusion of "procedures" as part of the above definition is essential for GIS applications in a public health context, given the need to link the science and methods of geographic information science, spatio-temporal statistics, medical geography, epidemiology and public health to GIS output to avoid producing invalid or misleading results [ 6 ]. GIS offer a very rich toolbox of methods and technologies that goes far beyond the mere production of simple maps (or digital cartography). From a community health perspective, GIS could potentially act as powerful evidence-based practice tools for early problem detection and solving. When properly used, GIS can be used to: inform and educate (professionals and the public); empower decision-making at all levels; help in planning and tweaking clinically and cost-effective actions, in predicting outcomes before making any financial commitments and ascribing priorities in a climate of finite resources; change practices; and continually monitor and analyse performance and changes, as well as sentinel events. Traditionally, two broad types of GIS applications can be distinguished which also reflect the two traditions in health geography (geography of disease and geography of healthcare systems), namely health outcomes and epidemiology applications, and healthcare delivery applications. There are also studies at the interface (overlap) between epidemiological and healthcare delivery applications, for example in relation to healthcare commissioning and needs assessment [ 1 , 7 , 8 ]. On current issues limiting a wide-scale, optimum adoption of GIS in the UK NHS and many other healthcare systems around the world The use of GIS in the UK National Health Service (NHS) can still be considered as an emerging technology (in this respect the NHS is not anyway better or more advanced than many other healthcare systems in less developed countries). Despite all their potentials, GIS remain very much under-utilised in the UK NHS in mostly low-level, non-strategic tasks, and in a largely fragmented and uncoordinated way. Spatial data and GIS are still not mentioned in any main UK health information strategy or policy document [ 7 , 8 ]. In striking contrast to this, the US National Health Information Infrastructure Strategy document (also known as "Information for Health") refers explicitly to GIS and real-time health and disease monitoring and states that "public health will need to include in its toolkit integrated data systems; high-quality community-level data; tools to identify significant health trends in real-time data streams; and geographic information systems" (see ). GIS are also explicitly included in the National Electronic Disease Surveillance System (NEDSS) specifications and systems architecture of the US Centres for Disease Control and Prevention (CDC – ). Although multiple novel spatial statistical and GIS methods are potentially available, we still need to unambiguously determine which method(s) and data specifically should be used by practitioners for each specific health condition of interest, and whether the proposed methods are cost-effective and scalable in the context of UK/NHS settings [ 8 ]. Moreover, several researchers have highlighted a gap between academic health-related applications of GIS and their everyday use within the NHS. Research undertaken in academia has certainly highlighted the benefits of spatial statistics and GIS approaches in mapping disease and in healthcare planning, but still needs to respond to NHS needs on the ground [ 7 , 8 ]. On the other hand, it is not uncommon for GIS research to include very practical and useful gems, but these often remain confined to the closed circles of researchers and hidden from the larger communities of GIS professionals and users. The best, current evidence derived from GIS research still needs to be embedded (and regularly updated) in everyday practice and all professional training programmes [ 8 ]. In a recent major review paper by this author, the reasons behind the under-utilisation of spatial information and GIS in the NHS, as well as the causes of the gap between academic GIS research and the current everyday use of GIS in the NHS were investigated, and remedial recommendations were made [ 8 ]. The reader is referred to this paper for further details. Overview and significance of the proposed research Research question How could GIS be beneficial in optimal ways in the context of UK/NHS settings and needs? (Or in other words, how to harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, and requirements, and to optimise NHS resource utilisation and improve health outcomes.) Any answer(s) or proposed solutions must be based on the best current evidence in the health GIS field. The proposed research This author proposes to build, consolidate and disseminate a comprehensive evidence base for GIS applications in health and healthcare in the context of UK/NHS settings, and an associated set of evidence-based GIS programme and application models (EB-GIS4HEALTH UK). We will learn from national projects running in the US (a leading country in health GIS) and elsewhere, while ensuring that EB-GIS4HEALTH UK's output properly fits the UK health and healthcare agenda. A systematic and critical review and consolidation are needed of the evidence for GIS for specific preventable, mitigable and treatable health conditions. A good model is the CDC "Guide to Community Preventive Services" [ 9 ]. Topics identified in this guide include alcohol abuse, cancer, diabetes, mental health, motor vehicle occupant injury, oral health, physical activity, sexual behaviour, social environment, tobacco product use, vaccine preventable diseases, and violence. The guide has started building an excellent evidence base, but this does not cover GIS methods and applications (Figure 1 ). Figure 1 Key to "Strength of Evidence". Key to "Strength of Evidence" as displayed within the CDC Community Guide . We propose to address a subset of the topics in the CDC Community Guide (diabetes and dental care – see below) during the three-year period of our project by conducting a focused review of GIS literature on the chosen topics, and then categorising the "nature of the scientific evidence" documenting whether GIS add any value to our understanding and management of the reviewed topics and/or the evidence that it would be feasible and cost-effective for the respective UK NHS and public health programmes tackling the reviewed topics to adopt GIS. Areas requiring further research will be also highlighted. A good example that comes to mind in this context is the 73-page "GIS for cancer" handbook titled "Using Geographic Information Systems Technology in the Collection, Analysis, and Presentation of Cancer Registry Data: A Handbook of Basic Practices" that was published by the North American Association of Central Cancer Registries . However, it should be noted that this particular handbook was conceived with the US healthcare system in mind and is thus more suited to US settings, though still useful as a model to follow. In reviewing GIS literature for the above mentioned purposes, this author appreciates the fact that the set of definitions and criteria for reviewing evidence as used in the CDC Community Guide is not directly usable for reviewing currently available GIS literature due to the nature of the latter; a modified set of definitions and criteria will first need to be developed. Furthermore, we will organise virtual e-focus groups that bring together UK programme administrators, practitioners and the public to complement the expected gaps and deficiencies in current GIS literature. The desired GIS information outputs and ways of using them within the NHS will be determined for both diabetes and dental care. Datasets (inputs) and the appropriate processing methods required to reach the desired outputs will be identified driven by the best available evidence (from the literature and e-focus groups), and vendor-neutral GIS programme and application models or ontologies will be created accordingly. Any limitations of these applications and any associated possible data/analysis problems or errors will be also highlighted, along with techniques for recognising and reducing their negative impact on result interpretation and any drawn conclusions. We will also launch EB-GIS4HEALTH UK Web server to disseminate our results and reach out to the wide NHS audience and the public. The project's value and potentials In a recent study by Higgs et al , a substantial proportion of respondents from health authorities (90%) and trusts (74%) stated that a dedicated Web site giving advice on GIS matters for NHS organisations would be helpful in promoting and disseminating good practice examples of GIS use in healthcare [ 10 ]. EB-GIS4HEALTH UK Web server will provide this kind of information and much more (for the topics covered during the duration of this project). In this way, EB-GIS4HEALTH UK will be also (partially) addressing the problems of "insufficient guidance" and "lack of awareness of the value of GIS to the NHS" that have been identified in different studies and reviews among the main factors hindering the wider use of GIS within the NHS [ 7 , 8 , 10 ]. EB-GIS4HEALTH UK will establish links between real-world NHS practice and the growing body of health and healthcare GIS research produced by the academia and research communities to ensure quality GIS practice and innovative applications are developed and implemented in the UK health service. By putting strong emphasis on real-world, practical GIS scenarios in a UK context, and by being based on the current best evidence, EB-GIS4HEALTH UK will also provide a much needed contribution to future national GIS training and raising awareness campaigns. Such evidence-based training and raising awareness activities have been strongly recommended by different researchers to improve the current poor GIS state of affairs within the NHS [ 8 ]. Raising awareness activities are also vital given the need to build business cases for the development of GIS within NHS organisations and to show the capabilities and "business benefits" of GIS to directors [ 10 ] (Figure 2 ). EB-GIS4HEALTH UK will act as a vehicle to reach out to policy and strategy makers in the UK health sector and will provide part of the evidence and "proof of concept and benefits" required to gain their support and long-term funding for realising the wider and ultimate vision of a national spatial data and information infrastructure and associated multivariate, GIS-enabled health surveillance services to run alongside and become coupled to the nation-wide integrated electronic health and social care records [ 8 ]. Figure 2 Why build an evidence base? Why an evidence base is needed (Modified from ). EB-GIS4HEALTH UK will provide semantically and procedurally consistent health and healthcare GIS application frameworks. This will help individual NHS organisations adopting such frameworks achieve a common understanding of data and processes (shared semantics), which in turn will enable them to efficiently and effectively share, compare, and integrate their data silos and results at local, regional and national levels for more informed planning, national comparisons, joined-up working, and better outcomes. The foundation for a wider vision EB-GIS4HEALTH UK also forms the basis of our vision of a next-generation intelligent tools specifically designed for the UK public health and NHS, and seamlessly weaved into everyday workflows and decision-making processes to enable users to focus and spend the larger part of their work time on what they want to achieve rather than on learning and overcoming the limitations of tools they are supposed to use to achieve their goals. These tools are part of our wider vision of a "national spatial data and information infrastructure and associated multivariate, GIS-enabled health surveillance services processing real-time or near-real-time data streams rather than just retrospective data" mentioned above and in [ 8 ]. Such tools must be able to convey reasonable conclusions (rather than just bunches of facts on thematic, coloured maps). The ideal tools also need to be fault-tolerant and capable of analysing and presenting assembled data in ways that facilitate only appropriate interpretations of integrated data. This can be achieved by using some form of user friendly, "intelligent", goal-oriented health GIS wizards based on robust statistical and epidemiological methods, so that only valid results and maps are produced, even when users attempt to select inappropriate settings for a particular analysis. The tools are also best designed and built to work in modular and nested fashions, so that they may be reused, linked and combined in different ways as needed to serve different scenarios and compound situations with little or no modifications (of the tools) [ 8 ]. This vision definitely calls for a sound evidence base and comprehensive conceptual blueprints (the proposed EB-GIS4HEALTH UK evidence-based models) to drive the envisaged tools and wizards. Why diabetes and dental care An incremental approach has been widely recommended in the literature for programmes with a national vision like EB-GIS4HEALTH UK. Rather than addressing all the spectrum of health and healthcare topics that are amenable to GIS processing in one project, we selected only two health conditions to start with. Diabetes and dental care were chosen because of their importance and the huge burden they place on the NHS, and the facts that they affect various age and socio-economic groups and involve a wide spectrum of preventive and curative interventions, which make it possible to apply the GIS models we are proposing to develop for these two topics to many other health and healthcare topics with little modification. Diabetes has a significant impact on the UK health and social services. Around five per cent of total NHS resources and up to ten per cent of hospital in-patient resources are used for the care of people with diabetes [ 11 ]. It has been estimated that diabetes accounts for some nine per cent (approximately £5.2 billion in 2000) of the annual NHS budget [ 12 ] (similar figures could be expected in many other countries). Moreover, in the UK, significant inequalities exist in the risk of developing diabetes, in access to health services and the quality of those services, and in health outcomes, particularly with regard to Type 2 diabetics. Reducing health inequalities is a core strand of The NHS Plan and, as the diabetes NSF (National Service Framework) is implemented, particular regard will need to be given to identifying the need for services, including unmet need; planning and delivering services on the basis of need, including reaching those who may not currently be accessing services or are accessing them late; ensuring the active involvement of users in service development; ensuring services are appropriate to individuals' needs, such as ethnicity, language, culture, religion, gender, disability, age and location; performance monitoring; and measuring and monitoring the health inequalities gap to ensure it is narrowing [ 11 ]. In all these areas, GIS can assist in analysing the socio-demographic makeup of service areas, and in planning and monitoring interventions and programmes to address these inequalities in the most efficient and effective ways, making sure NHS funds are properly allocated to areas most in need [ 13 , 14 ]. Regarding dental care, the cost of General Dental Services to the government in the year to March 2002 was £1.12 billion (about two per cent of the annual NHS budget of £65 billion in 2002) [ 15 ]. Under the recent Health and Social Care (Community Health and Standards) Act 2003, from April 2005 commissioning and contracting for NHS dentistry will devolve from the Department of Health to PCTs (Primary Care Trusts) [ 16 ]. Moreover, in September 2003, it was announced that £65.2 million will be made available to improve dental care for NHS patients (see ). Of this latter sum, £35 million will be allocated to enable PCTs to improve access, choice and quality for patients, and £30 million will be directed to information technology to integrate dentistry within the national information technology programme. (The latter has yet to recognise the many potentials of spatial information and GIS for the NHS.) Profiling service areas and needs assessment, dental healthcare commissioning, and improving/ensuring equitable access to dental services are all areas where GIS can make a positive difference. GIS have significant potential in examining spatial patterns in dental health, and in analysing patterns of registration and utilisation of dental services for different sectors of the community. GIS can reveal gaps in dental health provision, and thus help target resources and programmes to particular areas of needs. GIS can be also used to analyse the composition and spatial distribution of the dental workforce, and to inform the development and monitor the execution of programmes for attracting dental care professionals to work in under-served areas [ 2 , 17 , 18 ]. The project's beneficiaries These include: (1) the NHS and public health services in England (and the UK) – EB-GIS4HEALTH UK aims at adding the missing spatial information dimension to NHS organisations, and informing the development of successful GIS business plans for the health conditions under consideration; (2) the recently established NHSU, the corporate university for the NHS with Special Health Authority status – , could also benefit from EB-GIS4HEALTH UK as a foundation resource to provide evidence-based training programmes to NHS staff in "GIS for health and healthcare" and foster the much-needed NHS-academia collaboration. NHSU currently does not have programmes covering this important area. As an aside, one might add that when the Director of the Medical Informatics programme that the US CDC runs for its new staff conducted a poll of 40 students on the area they most wanted more information about, top of their list was GIS (Gerard Rushton, Department of Geography, University of Iowa, personal communication – December 2003); and (3) the UK citizenry and communities who will be empowered to become more active partners in their healthcare, and will also benefit on the long run from improved health services and outcomes as a result of the introduction of well-founded spatial information management within the NHS through EB-GIS4HEALTH UK and other synergistic/follow-on projects in the future. Aim, objectives and methods Aim EB-GIS4HEALTH UK aims at building a foundation evidence base and conceptual models for GIS applications in health and healthcare in the context of UK/NHS settings to (i) improve the currently poor GIS state of affairs within the NHS; (ii) help the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, requirements, and NSFs (National Service Frameworks); and also (iii) foster the much-needed NHS-academia GIS collaboration. Objectives 1. Organise e-focus groups of representatives of all stakeholders of UK/NHS health GIS to inform the development of all EB-GIS4HEALTH UK products. 2. Review the literature and current UK Public Health/NHS data flows and practices of relevance, and build an evidence base of GIS applications in diabetes and dental care (the two topics chosen for this project) in the context of UK/NHS settings. 3. Build modular GIS programme and application models for diabetes and dental care tailored to UK/NHS settings (driven by the evidence identified through objectives 1 and 2). 4. Run a Web server to disseminate the project's evidence base and resultant GIS programme and application models for diabetes and dental care to the wide NHS audience and the public. 5. Conduct a small-scale formative evaluation of EB-GIS4HEALTH UK server use and potential impact on NHS resource utilisation and improving health outcomes. Methods I. Process: gathering the evidence; product: building EB-GIS4HEALTH UK evidence base for diabetes and dental care Gaining insight about UK/NHS settings and formulating UK oriented questions We will gain insight about current Public Health/NHS (in England) data assets, flows and processes of relevance to EB-GIS4HEALTH UK through our NHS collaborators, and by identifying key contacts and organising virtual e-focus groups (see below). This insight will help us come up with a set of "localised" (UK oriented) questions that we will have to answer through our literature review and further e-focus group rounds. Developing a search strategy for locating the evidence and criteria for reviewing it We will formulate a search strategy to locate potential resources for our evidence base. This will also cover the so-called grey literature. Our search strategy will span MEDLINE/PubMed and other journal resources not listed in PubMed, e.g., Cartography and Geographic Information Science , Transactions in GIS , International Journal of Geographical Information Science , CDC Public Health GIS News and Information , ESRI's HealthyGIS and ArcUser Online ( and ), etc. We will also hand search a range of textbooks on GIS applications in health and healthcare. We will also review the extensive online documentation of flagship UK and foreign projects and initiatives of relevance to our proposed research. These include, among others, the UK Department of Health NSFs ; the US CDC National Public Health Performance Standards Programme (NPHPSP – ); the US Primary Care Service Area Project (PCSA – ) and the related Dartmouth Atlas project ; the "Mapping A Shared Vision of Hope: The American Indian/Alaska Native (AI/AN) GIS Diabetes Atlas", which was developed to assist in the analysis, design, and evaluation of AI/AN diabetes intervention and prevention programmes (Shirley Baros, Earth Data Analysis Centre, University of New Mexico, personal e-mail communication – January 2004); Georgia Medical Care Foundation Diabetes Quality Indicators project ; and CDC's data systems for oral health . We will review studies that used GIS or spatial methods in diabetes and dental care (e.g., [ 2 , 13 , 14 , 17 - 19 ]), and studies describing GIS or spatial methods that can be used in diabetes and dental care. The soundness and robustness of all reviewed literature and methods will be assessed and quality of the evidence determined. The conventional set of definitions and criteria for reviewing the evidence in medical literature by distinguishing between case-control, prospective cohort studies, and so forth is not directly usable for reviewing health GIS literature. Most GIS literature is in the form of application/methodology reports and expert opinions/reviews; a modified set of criteria needs to be agreed upon that recognises the different nature of GIS literature and its technical aspects. One thing to look at is whether a given method/approach is successfully replicated in more than one study. This sort of "consensus of opinions" could be an indicator of good evidence quality. Another issue to cover in our reviews is whether a given piece of evidence is suitable to UK/NHS settings and datasets. The virtual e-focus groups We will organise virtual e-focus groups of UK public health and GIS/informatics programme administrators (including our NHS collaborators), practitioners and representatives of the public to (1) inform the project about NHS settings; (2) define and refine the key questions that decision makers would want to be able to answer with GIS for diabetes and dental care, and think explicitly about what data and methods should be used to answer those questions; (3) discuss the reviewed literature and guide us to any relevant literature we may have missed for our evidence base; (4) complement our review of the evidence by the group members' own experiences and consensus of opinions, especially in areas where the literature is lacking; and (5) provide iterative feedback on EB-GIS4HEALTH UK models, as these are developed and refined. The virtual e-focus groups will ensure that representatives of all stakeholders of UK/NHS health GIS are involved in formulating EB-GIS4HEALTH UK products, a very important ingredient of success [ 8 , 20 ]. The e-focus groups will take place online using threaded discussion technology on EB-GIS4HEALTH UK Web server. Simple audio-conferencing over the Internet, e-mail/e-mail list, and/or telephone may be also used if necessary. The project's team will moderate/facilitate and guide/coordinate the discussions by adopting a kind of Delphi process and/or "cluster analysis" approach (where group members list their ideas, opinions and own experiences, then these are sorted and compiled into "themes" by the moderators and presented back to the group, and further rounds of knowledge distillation and aggregation are carried out as appropriate). The evidence base/metadatabase A metadatabase of reviewed resources classified according to topics and applications will be created and populated. This will form EB-GIS4HEALTH UK searchable online evidence base (see below). The design of resource records in this metadatabase will be guided by the spirit of the matrix method for conducting and organising literature reviews [ 21 ]. A classification system will be developed to easily sort and categorise the reviewed evidence. This could be an adapted version of Hu et al 's set of categories they have recently used in a small-scale GIS literature review, with the following top classes/sub-classes among others: data collection method(s) (e.g., field survey, disease surveillance system input, etc.); spatial data analysis method(s) (e.g., visualisation, exploratory, spatio-temporal modelling, etc.); study scale (e.g., country/state, city/town, suburb/district, etc.); resolution/geographic unit of analysis (areas, points, others); study purpose (e.g., identify disease risk factors, disease prediction, resource allocation, etc.); GIS software used; bias of study design/limitations; etc. (Wenbiao Hu and colleagues, Queensland University of Technology, Australia, unpublished report – February 2004). The metadatabase will include fields to record the strength of evidence and bottom-line summaries of the reviewed articles. EB-GIS4HEALTH UK models and their documentation will have cross-links to the underpinning evidence in the online metadatabase. Suitable input from the virtual e-focus groups will be also included in the evidence base. Resources and findings of insufficient evidence quality will still be documented in the evidence base (with a poor evidence quality rating attached to them), but will not be used in developing EB-GIS4HEALTH UK models. II. Process: translate evidence of acceptable strength into recommendations/modular GIS models; product: modular, conceptual GIS programme and application workflow models for diabetes and dental care Developing a general conceptual framework We will develop a general conceptual framework for EB-GIS4HEALTH UK models as outlined below, guided by Briggs' indicators methodology for environmental health hazard mapping [ 22 , 23 ], the US National Association of County and City Health Officials (NACCHO) core and extended health indicators, which form part of their Community Health Status Assessment (CHSA) Toolbox [ 24 ], and the Health Data Model (HDM), a project at the University of California at Santa Barbara (UCSB) to develop a US-oriented data model for health using proprietary ESRI software . EB-GIS4HEALTH UK will take the concepts and methodologies of these projects one step further by developing evidence-based, vendor-neutral modular GIS programme and application models rather than mere data models or vendor-specific solutions. The project's two types of models EB-GIS4HEALTH UK will feature two interrelated types of models: application models and programme models (Figure 3 ). Figure 3 The project's two types of models. EB-GIS4HEALTH UK features two interrelated types of models: (1) application models; and (2) programme models, each comprising at least two linked application models. Programme models can also have links with/feed into each other (not shown in this figure). An application model comprises (1) data and/or input from other models to be processed; (2) processing methods and tools (methods of geographic information science, spatio-temporal biostatistics, medical geography, epidemiology, health services and public health practice); (3) desired information outputs and output visualisation methods; as well as (4) all valid interpretations/inferences that can be made from the model's output and ways of using them within the NHS. The best current evidence (from the literature and virtual e-focus groups) will be used to formulate all EB-GIS4HEALTH UK models, and will form an integral part of their documentation. Application types For any single broad health/healthcare topic covered in EB-GIS4HEALTH UK (related to diabetes and dental care), modelled applications can include all or some of the following (among other possibilities): (1) monitoring and measuring NHS performance (many aspects could be measured and analysed such as improvements in the health of the general population, accessibility and utilisation of services, and outcomes of NHS care); (2) surveillance/monitoring/early detection of health and disease patterns and trends in populations; (3) profiling target populations and health service catchment areas; (4) selection of appropriate target groups for public health interventions, optimising these interventions to match target group profiles, and measuring intervention success; and (5) needs assessment and optimising healthcare system planning and responses, including new healthcare facility siting and improving hospital bed availability. EB-GIS4HEALTH UK models will take into account the influence of non-medical determinants (e.g., income, occupation, and environment) on population health status, qualitatively relate these determinants to health outcomes, and consider their implications on healthcare service planning and delivery [ 8 , 20 ]. For each application model, an ontology (conceptual data and process/workflow model) will be built to capture the properties of, and relationships between the different datasets involved in the application in question, as well as the various data elements (and their relationships) within individual datasets. The ontology will also capture the interactions of application data with all involved processing methods. This will help make explicit all application data requirements and processes, and facilitate data and application integration. A programme model is also an ontology that addresses a single broad health/healthcare topic, and comprises at least two application models linked together and interacting with each other in predetermined and purposeful ways towards a broader goal than that covered by a single application model. Application models may have uses in more than one programme (either unchanged or with slight modifications to suit different programmes, e.g., different input datasets). The project's ontology editor EB-GIS4HEALTH UK ontologies (the models) will be built using Protégé , a free ontology modelling tool from Stanford Medical Informatics with which this author has long experience [ 25 , 26 ]. Protégé features an extensive library of free plugins and applications that can extend the tool's basic functionality in many useful ways , including a Java-based Web application for sharing Protégé ontologies over the Web . Protégé is not alien to geographic information science; last year (2003) for example, a geographic information metadata (ISO 19115) ontology was developed at Drexel University, US, using Protégé ( – Figure 4 ). Figure 4 Protégé screenshot of the geographic information metadata (ISO 19115) ontology. Protégé screenshot of the geographic information metadata (ISO 19115) ontology developed at Drexel University, US, and available for downloading from . The ontology is distributed in OWL Web Ontology Language format, which is supported by Protégé. OWL is now also an official World Wide Web Consortium (W3C) Recommendation (see ). Anatomy of a model Metadata A model's ontology/documentation will specify the model's version/date and release history, the programme(s) (for application models)/topic(s)/sub-topic(s) to which the model relates, the model's rationale and role, any alternative or related models/model sets (programmes), links to the underpinning evidence (from the literature/the project's evidence base), and a listing of all agencies/NHS bodies involved in the model's process(es) (and their exact roles). Inputs Data involved in the modelled applications will be identified and assessed regarding availability, quality, characteristics, and constraints in terms of the model in question. Whenever this is possible, EB-GIS4HEALTH UK models will strive to refer to and model readily available data already collected for other purposes, since implementing new data collection processes can have prohibitive costs, and healthcare workers have repeatedly demonstrated poor compliance with additional data collection and administrative tasks [ 27 ]. For data that are not available but are "required" we will investigate and suggest the most efficient and effective way(s) for collecting and maintaining such datasets. During the course of the project, we will be identifying and communicating with the custodians of various datasets in the UK health sector and related sectors, as appropriate, to check the availability of required datasets to the UK NHS, note any constraints related to their release and use (e.g., privacy issues), and flag any need to review policies related to the release of such data. The results of all these data-related investigations will be included in the documentation of EB-GIS4HEALTH UK models. Processing/methods The spatial and related methods involved in processing the model's input to produce the desired outputs will be described in detail in the model. A reusable toolbox of "generic" methods will be identified to make this process easier for all the models we develop, e.g., "pattern spotters and testers" and "relationship seekers and provers" [ 5 ]. All methods used in EB-GIS4HEALTH UK models will be based on documented evidence of acceptable strength, and will be used in ways that allow only valid analyses and visualisations of data. Outputs and their interpretation The model's information output(s), any units of measurements used in presenting outputs, and any specified output visualisation methods will be also described in detail. The area across which the model can be used (scale of application or aggregation level) will be determined. Finally, the ways in which the model's output may be interpreted (in relation to the topic(s)/sub-topic(s) it covers) and linked to other EB-GIS4HEALTH UK models/programmes will be described. This includes determining what inferences can be made from apparent trends or patterns in the model's output and how such inferences can be used within the NHS, and any constraints on the interpretation of this output, due for example to data limitations or complexities in the relationships implied by the model. Data and methodological problems and limitations are not uncommon and a wide range of them has been well documented in the literature and must be anticipated and cared for [ 8 ]. Techniques for recognising and reducing their negative impact on conclusions drawn from spatial analysis will be investigated and also incorporated into the models. For a programme model, the component application models will be listed and their relationships and interactions within the programme also documented (Figure 3 ). What's next EB-GIS4HEALTH UK knowledge-based models (ontologies) in Protégé will not only be human-readable and visualisable in a variety of ways (to inform NHS GIS service developers and implementers), but it will be also possible to use the models to generate the necessary template databases for running the modelled applications. Furthermore, the resulting ontologies could form the basis for the development of goal-oriented, user friendly and fault tolerant health GIS wizards and solutions in the future using software agents technology [ 28 , 29 ], since we are also representing workflows and methods in our ontologies and not just data. The goal for EB-GIS4HEALTH UK application and programme models is to provide practical, vendor-neutral modular workflow models and ready-to-use, evidence-based frameworks for implementing GIS to address various health and healthcare topics within the NHS. NHS organisations adopting such frameworks will be able achieve a common understanding of data and processes (shared semantics), which in turn will enable them to efficiently and effectively share, compare, and integrate their data silos and results at all levels. III. Process: disseminate the project's evidence base and resultant GIS models for diabetes and dental care to the wide NHS audience and the public; product: EB-GIS4HEALTH UK Web server We will launch EB-GIS4HEALTH UK public Web server (with it's own domain name) to disseminate our evidence base, GIS models and associated documentation/metadata, as well as other project documentation and recommendations. The server will also host the virtual e-focus groups. The server will run one of the many free content management and discussion board platforms available today to manage and make searchable all of the project's online components. IV. Process: formative evaluation of EB-GIS4HEALTH UK online service; product: evaluation report We will advertise EB-GIS4HEALTH UK online service among target groups (NHS and academia) and the general public, and conduct a small-scale formative evaluation of its use and user-perceived utility of the project using an online user questionnaire, in addition to analysis of EB-GIS4HEALTH UK server transaction logs. This formative evaluation study will be carried during the third year of the project (after the online publication of the project's completed evidence base and associated GIS models for diabetes and dental care) with the goal to inform and guide any further development of EB-GIS4HEALTH UK or similar projects. The study will evaluate the potential impacts of EB-GIS4HEALTH UK on NHS resource utilisation and improving health outcomes, among other things, by surveying participants opinions. Evaluation results will be also available from EB-GIS4HEALTH UK public Web server. Methodological and ethical issues Research into ontology-driven geographic information systems (ODGIS) is very rapidly growing due to the many potential and unique advantages that ODGIS promise [ 30 - 36 ]. We anticipate that the ontological representation of health-related GIS/spatial methods will be one of the most challenging, but also most rewarding parts of our project (the description of data and metadata within EB-GIS4HEALTH UK models will be much easier and more straightforward by comparison). A useful discussion of how GIS methods could be described is presented in [ 36 ]. This author also advises the use of Tomlinson's methodology to plan for the successful deployment of GIS within the NHS, but only at a later stage [ 8 , 37 , 38 ]. Tomlinson's methodology seems a bit "lacking" when it comes to the health sector and that's where EB-GIS4HEALTH UK can come to help by providing a sound foundation upon which the methodology can be successfully applied at a later stage. For example, the "functions" (to use Tomlinson's terminology) and software used for health GIS analyses are a superset of those used in other sectors (e.g., functions provided by tools like and ). We conduct many complex analyses in health and healthcare to answer much more complicated questions (compared to other sectors), and there are many problematic issues surrounding our analyses like, for example, data confidentiality and Jacquez's famous "gee whiz" effect [ 8 ]. But more importantly, NHS users are currently not fully aware of all useful spatio-temporal analysis possibilities available to them (the academia/research-real-world practice/NHS split mentioned earlier), and as such will need a foundation project like EB-GIS4HEALTH UK to help them identify these many possibilities and uses (or "information products" to again use Tomlinson's terminology) that go far beyond the mere production of simple shaded maps to further empower their decision making processes. EB-GIS4HEALTH UK does not raise any ethical issues (in some of the applications to be modelled, the requirement for personal identifiable information will be referred to and modelled, but no actual real data will be used in the models). Public engagement in science Besides being one of the main project beneficiaries, the UK citizenry (and indeed anyone connected to the World Wide Web) will have full access to EB-GIS4HEALTH UK public reports and fully documented products on the project's public Web server. Representatives of the public will be also involved in the project's e-focus groups (see above). Lay audience short summaries of the project's progress and expected benefits in a jargon-free language will be also published on EB-GIS4HEALTH UK public Web server (see Table 1 for an example). Another valid possibility for EB-GIS4HEALTH UK would be to offer a public lecture/day on "the importance of location in health and healthcare, the project's nature and its value". The lecture could perhaps follow the successful model of GIS days . Table 1 EB-GIS4HEALTH UK example English, jargon-free summary for the lay (educated) audience EB-GIS4HEALTH UK example English, jargon-free summary for the lay audience Factors affecting health vary with location and over time. Geographic Information Systems (GIS) can help us better understand the geography and interactions of health-related events, exposures, and public health/healthcare resources, and also use this understanding to develop optimised prevention and intervention strategies and programmes. A wide range of GIS applications in health and healthcare have been described in the literature that could benefit public health decision makers, practitioners and the public. These GIS applications range from assisting in the early detection of a bioterrorist attack, to understanding and acting on the complex relationships between the environment, socio-economic factors and health, to healthcare needs assessment and the optimum siting of an appropriate new healthcare facility in a given community, and even route optimisation for ambulance vehicles and healthcare professionals doing home visits. However, despite all their potentials, GIS remain very much under-utilised in the NHS in mostly non-strategic tasks, and in a largely fragmented and uncoordinated way. Geographic data and GIS are still not mentioned in any main UK health information strategy or policy document, in striking contrast to the corresponding US strategy documents and specifications, which explicitly mention GIS. EB-GIS4HEALTH UK aims at helping the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, and requirements. Virtual e-focus groups that include representatives of the public will inform the development of all EB-GIS4HEALTH UK products. These include a sound evidence base of GIS methods and applications that are relevant to UK practices and settings, and an associated set of evidence-based conceptual models or blueprints for developing successful GIS business plans and implementing GIS to address various health issues within the NHS. The project will focus on diabetes and dental care, which together account for about 11% of the annual NHS budget, and are thus important topics where GIS can help optimising resource utilisation and outcomes. However, products and experience gained in this project will be transferable to address other national health topics based on the same principles. EB-GIS4HEALTH UK ultimate beneficiaries are the UK citizenry and communities who will be empowered to become more active partners in their healthcare, and will also benefit on the long run from improved health services and outcomes, and reduced health inequalities as a result of the introduction of well-founded geographic information management within the NHS through this and other synergistic/follow-on projects. Commercial exploitation Most major GIS vendors and solution providers have been targeting the UK health sector for many years now (e.g., and ), but this has never resulted in any coherent national strategic adoption of GIS within the NHS. This is due to the fact that a successful national strategic implementation of GIS in the health sector is dependent upon many other very important ingredients besides the acquisition of hardware and software systems, and core digital geo-datasets [ 8 ]. This author believes that these GIS vendors and solution providers will be definitely interested in the output of EB-GIS4HEALTH UK, which should provide them (and the NHS) with a convincing application-oriented framework for tailoring their services to suit the requirements of a wide-scale strategic adoption of GIS by the UK health sector. Concluding remarks Healthcare systems work differently around the world and this has knock-on effect on health GIS. When it comes to UK health GIS, we still need to go back to the design board, and to link/integrate what research has to offer into workflow models and "recipes" that are both usable and useful in everyday practice in this country (England) . All national health GIS stakeholders must be involved in this process, including representatives of the general public (the ultimate beneficiaries). EB-GIS4HEALTH UK is just the start towards this goal. An incremental approach has been widely recommended in the literature for programmes with a national vision like EB-GIS4HEALTH UK. The experience gained at the end of this project will hopefully be transferable to further develop the covered topics (diabetes and dental care – in subsequent follow-on projects), and for addressing other national health and healthcare topics not initially covered in this project, based on the same principles. It must be emphasised that the evidence base and conceptual GIS workflow models we are proposing to build in this project are not per se our ultimate goals. The actual purpose of this design/modelling exercise and its ultimate goals are optimising NHS resource utilisation and improving health outcomes by paving the way to the incorporation of the missing spatial information dimension in NHS organisations. This paper presented an overview of a project we are proposing to carry out here in England, where the author is based. However, the same concepts, principles and approaches described in this paper can be also applied in other countries, especially where financial constraints are not a major issue. The task might not be as easy as replacing "UK" in this proposal with another country name, but the author believes that the paper has provided enough details and set an example to enable "quick starting" similar health GIS foundation projects in other countries. Our proposal also fits very well into the spirit of Mark Musen's philosophical paper on medical informatics as an academic discipline, in which he argued that "informatics involves the construction of ontologies that define the concepts relevant to different aspects of human experience and the elucidation of problem-solving methods that can solve specific computational tasks" [ 39 ]. Notes A detailed breakdown of human and other resources required to complete this project with a timeline of the main EB-GIS4HEALTH UK execution tasks distributed over a suggested three-year duration of the project are not provided in this manuscript, but are directly available from the author. A slightly abridged version of this proposal has been submitted to, and very favourably reviewed by the UK Medical Research Council (MRC) during 2004, and received a final ALPHA-C banding (i.e., "work which is nationally competitive and will make valuable contributions to addressing important scientific and/or policy questions" – ). As one of the reviewers rightly noted, "the key aspect to this proposal is that it deals with an area that cannot be neatly fitted into a single category. It crosses over into a number of specialisms – health informatics, geographic information science (GIS), health, geography, public health, policy and practice, epidemiology, etc. It is therefore important to bear in mind that if the proposal is judged by criteria generally applicable to only one or two of these specialist areas, it might be found deficient and it would be quite inappropriate to do this".	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546191.xml
544962	Recent developments in national Aboriginal and Torres Strait Islander health strategy	In this paper I will describe some of the sentinel events in Aboriginal and Torres Strait Islander health policy and strategy during 2003 and the early part of 2004. This will involve discussion on the: • National Strategic Framework in Aboriginal and Torres Strait Islander Health • National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009 • National Aboriginal and Torres Strait Islander Health Performance Framework • The roll-out of the Primary Health Care Access Program • The National Aboriginal and Torres Strait Islander Social Survey and the National Indigenous Health Survey These developments are consistent with a policy agenda that has evolved, in general terms, since the release of the National Aboriginal Health Strategy in 1989. However, I will also consider significant developments in the broader context for Aboriginal and Torres Strait Islander affairs, particularly the decision made in early 2004 by the Howard government to abolish the Aboriginal and Torres Strait Islander Commission (ATSIC). While the key events and developments that are reported in this paper elaborate on an agenda that has been developing for more than a decade, the decision to abolish ATSIC is likely to have a revolutionary impact on the future development of Aboriginal health strategy.	Introduction Following the lead of the National Aboriginal Health Strategy (NAHS) [ 1 ], national strategy in this field has focussed on health sector reform and the development of inter-sectoral strategies to improve Indigenous health outcomes. In 1995, the health portfolio assumed responsibility for the management of the Australian government's Aboriginal health program from the Aboriginal and Torres Strait Islander Commission (ATSIC). Since that time, mechanisms have been established that provide a platform for collaborative, inter-governmental planning, engaging with both the Aboriginal community sector and the non-health sectors of government [ 2 - 4 ]. The key elements of this national planning framework include: • Framework Agreements in Aboriginal and Torres Strait Islander Health (multi-party agreements between the Australian government; State and Territory governments; the Aboriginal and Torres Strait Islander Commission and the Aboriginal community controlled health sector); • Joint Planning Forums (established at a jurisdictional level with responsibility for the developing State and regional Aboriginal and Torres Strait Islander health plans). The NAHS has been the guiding framework for action in this field since it was endorsed in 1989. Consequently, it was significant that the Australian Health Ministers Conference endorsed its successor on the 31 st of July 2003, the "National Strategic Framework for Aboriginal and Torres Strait Islander Health" (hereafter, the "National Framework"). Agreement has also been recently brokered that details strategies for Indigenous social and emotional well-being, which is one of the Key Result Areas for the "National Framework". Significant progress has also been made in the development of a national performance management framework for Aboriginal and Torres Strait Islander health that aligns with the "National Framework". The agenda in Aboriginal and Torres Strait Islander health strategy that was adopted by the health portfolio post 1995 had focussed on reform priorities focussed on the development of [ 3 ]: • The capacity of primary health services to respond to Aboriginal and Torres Strait Islander health need (with a particular focus on financing and workforce); • disease and risk strategies that aimed to improve Aboriginal and Torres Strait Islander health outcomes; • the evidence base for policy and practice in this sector (through strategic research and improvements in the quality of health and related data). With respect to primary care capacity, the roll-out of the Primary Health Care Access Program (PHCAP) continues to be one of the central planks of this agenda and I will provide an overview of recent progress. Significant progress has also been made over the last couple of years in the development of the Australian Bureau of Statistics Indigenous Survey program, which promises to enhance the information available to assist decision-making within the sector. I will provide a report on the recent developments in the roll-out of this program. While the recent developments in Aboriginal and Torres Strait Islander health policy and strategy represent an evolution of a health reform agenda that has been developing for more than a decade, the abolition of ATSIC points to a much more revolutionary change in the broader institutional and programmatic context for Aboriginal affairs. ATSIC had play a critical role in integrating Australian government programs in Indigenous affairs and providing an institutional structure that facilitated Aboriginal and Torres Strait Islander input into policy and program development. ATSIC, for instance, continued to play a role in health strategy following the transfer of specific health program responsibilities in 1995. It retained, for instance, responsibility for the delivery of environmental health service. A memorandum of understanding was developed between the Department of Health and Human Services and ATSIC to support collaboration between the sectors [ 6 ]. Consequently, the decision to abolish the ATSIC and radically overhaul of the administration of Commonwealth programs in Aboriginal Affairs has potential implications for national health strategy. These are discussed later in this paper. Discussion National Strategic Framework in Aboriginal and Torres Strait Islander Health The National Aboriginal and Torres Strait Islander Health Council (NATSIHC) oversaw the development of the "National Framework". However, this process was stalled by political conflict between the key stakeholders. In December 2000, Council members representing the National Aboriginal Community Controlled Health Organisation (NACCHO) resigned in protest over a consultation draft of the 'National Framework'. NACCHO, which is the peak body representing the Aboriginal community controlled health sector, was concerned with [ 7 ]: The way the Draft is written distances Aboriginal and Torres Strait Islander people, undermines the concept of Aboriginal community control of primary health care service delivery and diminished structures which NACCHO believe are still useful. The document's tone and language is wrong in a number of ways... Following further negotiation, NACCHO withdrew its resignation, and the NATISHC was reconstituted with revised membership and Terms of Reference [ 8 ]. Despite this successful outcome, this ruction in the relationship with NACCHO illustrates the tenuous nature of partnership structures and processes in this sector – an issue that I will return when discussing the issues that may potentially flow on following the abolition of ATSIC. The agreed "National Framework" consists of two documents: • The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Framework for action by Governments", which sets out a five- to ten-year reform agenda in 9 key result areas [ 5 ]. • The "National Strategic Framework for Aboriginal and Torres Strait Islander Health – Context", which outlines the rationale for the Framework and its context [ 9 ]. There are nine Key Result Areas set out in the Framework including [ 5 ]: • Community controlled primary health care: building community capacity so that individuals and communities can better address and manage their own health needs. • Health system delivery framework: improving the responsiveness of the mainstream health system to Indigenous Australians and developing stronger partnerships between mainstream and Indigenous-specific services. • A competent health workforce: improving the training, supply, recruitment and retention of appropriately skilled health professionals, health service managers and policy officers in both mainstream and Indigenous-specific health services. • Emotional and social well-being: improving outcomes with respect to mental health, suicide, family violence, substance misuse and male health (through non-health sectors strategies). • Environmental health: improving the delivery of safe housing, water, sewerage and waste disposal. • Wider strategies that impact on health: undertaking action in portfolios outside the health sector and implementing health gain strategies in the areas of education, employment transport, food and nutrition, custodial health, aged and disability services, recreation and exercise. • Data, research and evidence: aiming to improve the quality of information about how well the health sector is meeting the needs of Indigenous Australians. • Resources and finance: aiming to provide an optimal level of resources for Indigenous health commensurate with levels of need, costs of delivering services and community capacity to deliver health outcomes. • Accountability: both to communities and to governments for the delivery and effectiveness of health services. The "National Framework" was endorsed as a plan to guide all Australian governments in a coordinated, collaborative and multi-sectoral approach to achieving Aboriginal and Torres Strait Islander health gain over the next decade. It does not have a specific funding program attached to its implementation, although arguably, the roll-out of the Primary Health Care Access Program (described later) will provide additional capacity to the implementation of the "National Framework". It is also possible that the National Framework will guide the allocation of any new resources made available through the joint planning processes established under the Framework Agreements. To further these ends, it is significant that the "National Framework" was endorsed through each government's cabinet process, providing a whole-of-government commitment to its implementation in each State and Territory and at the Commonwealth level. Each jurisdiction is developing its implementation plan against which it will report annually on progress and outcomes in health portfolios and biennially on whole of government progress. The plans will identify the specific strategies and timeframes for each action area. The National Aboriginal and Torres Strait Islander Health Council will develop a plan for an independent mid-term and final evaluation. The National Strategic Framework for Aboriginal and Torres Strait Islander Peoples Mental Health and Social and Emotional Well Being 2004–2009 The "Social and Emotional Well Being Framework (SEWB Framework)" [ 10 ] is based on Aboriginal health values that emphasise the need for a holistic and 'whole of life' approach to achieving the conditions for well-being. Although this framework encompasses the traditional field of mental health, these issues are situated in an approach that also addresses the emotional and social well-being of Indigenous Australians and their communities. The nine guiding principles for the "SEWB Framework" were been extracted from "Ways Forward" [ 11 ], an earlier strategy that established the importance of this holistic approach in this area of health. In supporting this holistic approach the "SEWB Framework" articulates strategies that support self-determination and culturally valid understandings of health. It further recognises the impact of trauma, grief, loss, discrimination and human rights issues on the social and emotional well being of Aboriginal and Torres Strait Islander communities. In 2003 the Social Health Reference Group (SHRG) (established to oversee its development) conducted extensive consultations on a draft framework document. Since then the 'SEWB Framework' has endorsed by the NATSIHC and the National Mental Health Working Group in November 2003, and the Standing Committee on Aboriginal and Torres Strait Islander Health in December 2003. It was anticipated that the final 'SEWB Framework' document would be endorsed out of session by the Australian Health Ministers Advisory Council by the middle of 2004. The Aboriginal and Torres Strait Islander Health Performance Framework The development of the Aboriginal and Torres Strait Islander Health Performance Framework has built on the foundations of earlier work which has established the key elements of this framework, including the: • national performance indicators in Aboriginal and Torres Strait Islander health for the Australian Health Ministers Advisory Council [ 3 ]; • service activity reporting for Aboriginal community controlled health services [ 12 ]; • Australian government health portfolio indicators [ 13 ]; and • the reporting against key indicators of Aboriginal and Torres Strait Islander disadvantage for the Council of Australian Governments [ 14 ]. It is intended that the Aboriginal and Torres Strait Islander Health Performance Framework will both integrate those government performance reporting processes that have already been developed; streamline reporting processes in Indigenous health and, ensure the strategic management of policy relevant and quality information in published reports (such as the National Health Performance Committee, the Productivity Commission's Report of Government Services and the Indigenous Disadvantage Report) [ 15 ]. As starting point, the National Health Performance Committee Framework, which has already been endorsed by the Australian Health Ministers Conference will be used as a guide to the relevant measurement domains for Aboriginal and Torres Strait Islander specific framework [ 15 ]. It is also intended that the Aboriginal and Torres Strait Islander Health Performance Framework will support the implementation of the 'National Framework' by: • mapping the relationship between the Key Result areas of the 'National Framework' and key domains of health performance (effectiveness, safety, responsiveness etc); and • identifying priorities for data development and improvement based on priorities of the 'National Framework'. Roll-out of the Primary Health Care Access Program The Primary Health Care Access Program (PHCAP) was introduced in the 1999–2000 Federal Budget to improve access to primary health care for Aboriginal and Torres Strait Islander people. PHCAP achieves this by funding increased primary health care provision, such as additional general practitioners, nurses, Aboriginal Health Workers, and through preventive and health promotional activities, such as diabetes education and management. Funds are also used for supports to service provision such as capital works and equipment. The program also aims to work with existing health services to ensure they are responsive to the needs of Aboriginal and Torres Strait Islander people. On average across Australia, PHCAP aims to bring the level of Commonwealth funding for Indigenous primary health care to three times the average MBS usage for all Australians. The key objectives of PHCAP are [ 3 ]: • Increased availability of appropriate primary health care services where they are currently inadequate; • Local health systems that better meet the needs of Aboriginal and Torres Strait Islander people; and • Individuals and communities that are empowered to take greater responsibility for their own health. Services can be provided through a mix of arrangements, including Indigenous specific, mainstream or a combination of these. Funding can also be used to support mechanisms to assist service providers to deliver better services and enable individuals and communities to become more involved in improving their health. Up until March 2004, new and additional services have been funded in Central Australia (5 regions), Queensland (3 regions) and South Australia (4 regions) through PHCAP, as well as continued funding of services provided through the former Aboriginal Coordinated Care Trials (Yael Cass, Office for Aboriginal and Torres Strait Islander Health, Australian Department of Health and Ageing, personal communication). During 2003 a more streamlined approach to the management of PHCAP rollout was developed [ 4 , 16 ], resulting in more than 200 proposals to improve access to primary care being developed in each state and territory. This was in discussions with members of the state or territory forum or partnership, and drawing on regional plans and other work that has been undertaken over the last several years. From these proposals, $11.8 million in funding was approved on 14 March 2004 for: • additional health professional and support staff, for example, over 20 more health professional positions in the Kimberley region of WA; • capital works for health clinic upgrades and the construction of staff housing in remote communities; • minor capital purchases such as medical equipment; and • one-off health promotional activities and health board support and training. Longer term strategies around enhancement of local service systems, to ensure they are more accessible for Aboriginal and Torres Strait Islander people, and ensuring the commitment by state/territory governments to at least maintain their funding commitments, will continue to be pursued. While the PHCAP program has provided a significant injection of resources into what is generally considered an inadequately funded primary heath care system, the amount made available through this program still does not meet its programmatic benchmarks and targets [ 4 ]. Roll-out of the Australian Bureau of Statistics Indigenous Survey Program One of the development priorities established by the heath portfolio when it took responsibility for the administration of the Aboriginal health program was to develop the evidence base to support policy reform and the development of health service capacity [ 3 ]. The National Health Survey, undertaken by the Australian Bureau of Statistics with funding support from the Australian Department of Health and Ageing, collects information about the health status, use of health services and facilities, socio-economic status and health-related aspects of the lifestyle of Australians. The Indigenous component of this survey aims to benchmark information on a range of health issues and enable comparisons between the health characteristics of Indigenous and non-Indigenous Australians and to allow trends in the health of Indigenous Australians to be monitored over time. The Indigenous Health Survey that was run in 2001, collected data from approximately 3,500 individuals which was reportable at the national level [ 17 ]. In 2004, the Indigenous Health Survey will collect information from 11,000 Indigenous participants in order to be able to provide statistics at the national and State/Territory levels, and some geographical areas. It will also, for the first time, provide information on mental health. It is anticipated that the data collected will be reported in 2005. In parallel with the health survey program the Australian Bureau of Statistics collected data for the 2002 National Aboriginal and Torres Strait Islander Social Survey from August 2002 to April 2003 [ 18 ]. It is planned to repeat this survey a six yearly intervals. A summary of findings has been published that covers topics such as family and culture, health, education work, income and housing law and just and transport. A revolution in program administration in Aboriginal Affairs On 20 April 2004, the Prime Minister, John Howard and the Minister for Aboriginal Affairs, Senator Amanda Vanstone, announced the intention of the Australian government to abolish the ATSIC. ATSIC had been established in 1989 when the program responsibilities of the Commonwealth Department of Aboriginal Affairs and the Aboriginal Development Corporation were merged into a structure that enable the regional allocation of resources through elected regional councils. The board of Commissioners, elected by ATSIC regional councils, was responsible for national policy development and the oversight of national programs. At the Commonwealth level, ATSIC had the lead agency responsible for the administration of a range of programs such as: community development and employment (CDEP); housing and infrastructure; cultural heritage, broadcasting services; legal services; native title, land rights and the Indigenous land fund, etc. The agency also played a critical role in co-ordinating and integrating the Aboriginal strategy across the different government program areas. ATSIC and the health portfolio had collaborated in the implementation of health infrastructure priority projects [ 19 ]. The Memorandum of Understanding developed between the two portfolios enabled collaborative planning across a range of programs that impacted on Indigenous health outcomes. The advantage of this institutional arrangement for cross sectoral strategy is that these programs might otherwise have been dispersed across a number of different government departments or instrumentalities. Further, ATSIC provide a structure for engaging Indigenous participation that broader than the sector specific mechanisms. ATSIC played a pivotal institutional role in the development of 'whole of government' strategies across the Australian government. It was in effect the only institutional mechanism (with the exception of time limited inter-departmental committees) that enabled this. This was until the Council of Australian Governments (COAG) resolved (in 2000 and 2002) to trial, in up to 10 regions across the country, innovative administrative arrangements, developed in partnership with Indigenous communities, which aimed to provide "more flexible programs and services based on priorities agreed with communities" [ 20 ]. From its first term in 1996, the Howard Coalition government had a conflictual relationship with the Commission. However, government confidence in the ATSIC Board deteriorated significantly under the chairmanship of Geoff Clark (first elected chairperson in 1999) to the extent that the Minister for Indigenous Affairs suspended him on the ground of misbehaviour (under section 40 of the ATSIC Act 1989) [ 21 ]. A review of ATSIC was undertaken during the period December 2002–October 2003. It recommended that ATSIC should be retained as the primary vehicle for representing the aspirations of Aboriginal people to all levels of government and that its existing program responsibilities should also be retained pending a determination of its role in the context of [a] broader examination of service delivery [ 22 ]. The review also recommended a comprehensive program of reform primarily focussed at strengthening the capacity of regional councils and improving the relationships between ATSIC and the Australian government and between ATSIC's elected and administrative arms. Prior to the completion of the review the Coalition government moved to structurally separate ATSIC into an elected arm (ATSIC) and an executive agency, Aboriginal and Torres Strait Islander Services (ATSIS). ATSIS retained, under Ministerial delegation, program administrative responsibilities. The Federal cabinet, nevertheless, resolved to a more radical agenda than outlined in the review findings, and announced its intention to abolish ATSIC, its regional councils and the mainstreaming of the administration of all the programs for which ATSIC had been responsible. It is proposed that the elected advisory structures will be replaced by a government appointed national council. It is also proposed that Indigenous specific program dollars will be quarantined and a whole of government approach is to be developed for the delivery of Indigenous specific funding. The key elements of this reform package have been positioned within the broader context of a government commitment to reforms aimed at producing "'joined up' government and the 'seamless' delivery of programmes" [ 23 ]. This new framework for Indigenous policy and program administration also include the establishment of a: • Ministerial Taskforce: which would operate as a cabinet committee, provide collaborative leadership at a government level and set strategic directions. • Secretaries group: which would support Ministerial decision-making, coordinate across government agencies, and oversight annual reporting. • National Indigenous Council: in which the Minister would appointment Indigenous leaders in health, education, employment, law and justice to provide advice and monitor performance. The proposed mechanisms and structures that would be established to deliver this 'joined-up' framework including regional partnership agreements and community shared responsibility agreements (detailing mutual obligations). It is also proposed to establish Indigenous co-ordination centres which will provide a single shopfront in regional and remote Australia for indigenous specific programs, lead the negotiation of regional partnerships an shared responsibility agreements but maintain line responsibility to mainstream departments. The impact of this radical reform agenda to national Aboriginal health strategy is difficult to predict. One the one hand the actual changes to the administration of the health program is insignificant (leaving aside some potentially critical issues in budget development). A mainstream department has administered this program since 1995. One the other hand, the implementation of this reform agenda could have potentially very significant consequences for the development of inter-sectoral strategies in Indigenous health. This depends on the success in implementing the new mechanisms, and on their effectiveness. Furthermore, the political consequences of this radical agenda on the relationship between the Australian government and Aboriginal and Torres Strait Islander peoples have yet to really become clear. Specific partnership arrangements that have been developed within the health sector are tenuous – as is evident by the politics in the development of the "National Framework". These partnerships are critical to successful implementation of strategy in Indigenous health. Consequently, deterioration in the broader relationship between Indigenous Australians and the Australian government may have significant negative consequences for the partnership processes specific to the health sector. Even though 2003 was a year in which policy and strategy in Indigenous health made no or new radical departures, it was a year of considerable tumult in relations between the Australian government and Indigenous peoples. The ramifications of this are only now beginning to unfold. Abbreviations ATSIC Aboriginal and Torres Strait Islander Commission NAHS National Aboriginal Health Strategy NATSICH National Aboriginal and Torres Strait Islander Health Council NACCHO National Aboriginal Community Controlled Health Organisation PHCAP Primary Health Care Access Program Competing interests The authors declare that they have no competing interests.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC544962.xml
528853	Granzyme B; the chalk-mark of a cytotoxic lymphocyte	During cytotoxic lymphocyte (CL) mediated killing of target cells, granzyme B is released from the CL into the immune synapse. Recent studies have found that ELISPOT-detection of granzyme B correlated well with conventional assays for CL mediated killing. In this way, the released granzyme B can be used to mark the spot where a target cell was murdered. We discuss the benefits and potential limitations of using this assay to measure CL mediated killing of target cells.	Introduction Cytotoxic Lymphocytes (CLs) eliminate virally infected cells or tumour cells either by activating death receptors or by delivering cytotoxic granule proteins (granule exocytosis) to the target cell [ 1 , 2 ]. The ability of a virus or a tumour cell to evade detection or survive an attack by CLs is likely to result in a more aggressive disease. The ability to measure specific killing of target cells by CLs is therefore of great interest to clinicians and researchers alike. Any assay for CL-induced death involves mixed cultures of target and effector cells and must include some means of distinguishing between the two. The current approach is to measure the release of a label, such as 51 Cr or, more recently calcein-AM [ 3 ], that has been preloaded into the target cells. Radioactivity limits the utility of 51 Cr and, although this type of assay is presumed to measure rupture of the plasma membrane (cell lysis), it is not formally known what is being measured. Discussion Various alternative assays have been developed to assay CL-induced killing of target cells [ 4 - 10 ], however 51 Cr remains the gold standard. Recently, Shafer-Weaver et al and others have utilized an interesting strategy aimed at measuring the functions of effector cells rather than death of the target cell [ 9 , 11 ]. During granule-mediated killing, granule enzymes (granzymes) are transferred to the target cell [ 2 , 12 ]. In the target cell granzyme B, can initiate target cell death by apoptosis [ 13 , 14 ]. Shafer-Weaver et al ., [ 11 ] demonstrated that detection of granzyme B by ELISPOT correlated well with 51 Cr release during antigen specific target cell death induced by cytotoxic T-lymphocytes and now report utility of this assay for measuring MHC non-restricted killing by natural killer cells [ 15 ]. Following incubation of CL with their targets, Shafer-Weaver et al ., measured granzyme B by ELISPOT and found that the number of SPOTS correlated well with results obtained by the 51 Cr release assay. Unlike the 51 Cr release assay, this ELISPOT assay measures a specific and well-characterized event that occurs following target recognition. Assessing granzyme B by ELISPOT appears superior to other markers, such as IFNγ, because it assays a molecule that directly participates in CL mediated killing. Furthermore, the assay is non-radioactive and under the experimental parameters reported, it appears possible to detect cytolytic activity using fewer cells than are required for 51 Cr release. This assay appears to provide an effective alternative method for assessing CL-mediated cell death, however, users should be aware of possible limitations. The assay measures granzyme B release, not cell death. Frequently, the two will be closely correlated, but under certain circumstances using granzyme B release as a marker could lead either to an under or over estimate of target cell death. For example, perforin-deficient CLs are unable to kill targets [ 16 , 17 ], yet they may release granzyme B in the same way as wild type cells -leading to a false positive result. Alternatively, cells lacking, or expressing small amounts of granzyme B may retain the ability to kill targets by means of other granule components or through death receptor mediated pathways leading to an underestimate of cytotoxic activity [ 18 ]. In addition, a CL may degranulate normally, but certain targets may be inherently resistant to their effects [ 19 ]. Thus, to be certain that degranulation is inducing target cell death, chromium release assays should be performed alongside the granzyme B ELISPOT. The limits of detection of this assay are not clear. It is not known whether the granzyme B released at a single death-inducing synapse are sufficient to produce a spot or whether a CL must degranulate several times, possibly killing multiple targets, to facilitate detection. Even if one spot reflects degranulation by one CL and is directly equivalent to one target cell death, it remains possible that CLs expressing granzyme B below the level of detection by ELISPOT may express sufficient granzyme B to kill their targets. These are difficult issues to address, but the correlation between 51 Cr and granzyme B ELISPOT shown under the conditions used by Shafer-Weaver et al [ 15 ] suggests that the levels of detection of the assay are likely to be broadly equivalent to those required for cell death. It is however too difficult to directly compare these two assays. For example, 316 spots were detected in an assay using 50,000 target cells and 10,000 effectors (Table 1). This is equivalent to 0.6 +/- 0.1 % (as the number of spots must be related to the number of targets for comparison with 51 Cr). Increasing the effectors generated too many spots to count. Therefore an experiment optimised for 51 Cr assay, (0–70% release as reported in Table 1), will only have a dynamic range of between 0 and 0.6% using the ELISPOT assay. In contrast, an assay optimized for ELISPOT is likely to be off scale in a 51 Cr release assay. These data suggest that a small amount of killing (e.g in a sample with low level killing) may easily generate a positive result by ELISPOT. It is therefore likely that stringent titration of both effectors and targets over a narrow range will be essential. Conclusion The granzyme B-ELISPOT introduces a new assay for measuring CL mediated toxicity that will have a widespread utility in experimental systems where granzyme B is present in the effector cell and the target is susceptible to CL mediated killing. However, no assay used in isolation can be the answer to everyone's prayers and the granzyme B ELISPOT, like all others, has limitations. There is no doubt that this assay measures triggering of degranulation, but it does not directly address the question of cell death. Therefore it is likely that the greatest utility of this assay will be found by using it in combination with other existing measures of cytotoxic activity. It may also be extremely valuable as a quick reference to determine whether killing can occur in an assay with defined targets and effectors. Abbreviations CL, cytotoxic lymphocytes; ELISPOT, enzyme linked immunospot; Cr, Chromium; Competing Interests The authors declare that they have no competing interests. Author's contributions All authors contributed to the ideas, discussion and preparation of this manuscript.	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC528853.xml
546185	Measuring depression in nursing home residents with the MDS and GDS: an observational psychometric study	Background The objective of this study was to examine the Minimum Data Set (MDS) and Geriatric Depression Scale (GDS) as measures of depression among nursing home residents. Methods The data for this study were baseline, pre-intervention assessment data from a research study involving nine nursing homes and 704 residents in Massachusetts. Trained research nurses assessed residents using the MDS and the GDS 15-item version. Demographic, psychiatric, and cognitive data were obtained using the MDS. Level of depression was operationalized as: (1) a sum of the MDS Depression items; (2) the MDS Depression Rating Scale; (3) the 15-item GDS; and (4) the five-item GDS. We compared missing data, floor effects, means, internal consistency reliability, scale score correlation, and ability to identify residents with conspicuous depression (chart diagnosis or use of antidepressant) across cognitive impairment strata. Results The GDS and MDS Depression scales were uncorrelated. Nevertheless, both MDS and GDS measures demonstrated adequate internal consistency reliability. The MDS suggested greater depression among those with cognitive impairment, whereas the GDS suggested a more severe depression among those with better cognitive functioning. The GDS was limited by missing data; the DRS by a larger floor effect. The DRS was more strongly correlated with conspicuous depression, but only among those with cognitive impairment. Conclusions The MDS Depression items and GDS identify different elements of depression. This may be due to differences in the manifest symptom content and/or the self-report nature of the GDS versus the observer-rated MDS. Our findings suggest that the GDS and the MDS are not interchangeable measures of depression.	Background Depression is common among residents of nursing homes [ 1 ]. Of the many instruments used to identify depression in the elderly, the Geriatric Depression Scale (GDS)[ 2 ] is probably the most widely used in research settings. The original version comprises 30 items, whereas subsequent versions have been proposed with 15, 12 and later five items [ 3 - 5 ]. None of the items query somatic complaints, rather, questions inquire about the respondent's perspective on their life over the previous week. The Minimum Data Set (MDS), created in response to the US Omnibus Budget Reconciliation Act of 1987, aims to provide a uniform, standardized, and comprehensive assessment of residents in nursing homes [ 6 ]. The MDS has undergone several revisions since its inception, and is currently undergoing another revision process. Section E of MDS version 2.0 assesses 16 depression symptoms that capture verbal and non-verbal indicators of depressed mood or anxiety as perceived by nursing home staff. A summary scale, the Depression Rating Scale (DRS), uses a subset of seven of these symptoms and has been shown to be a reliable and valid measure of depression among nursing home residents [ 7 ]. The MDS DRS is therefore, by nature of its ubiquity, potentially the most widely available depression assessment instrument for older adults in nursing home settings. This study compares the measurement properties of two measures of geriatric depression: the GDS and the MDS. The GDS is evaluated via a 15 and five item form. The MDS is evaluated by the items located in Section E in total and as a subset included in the Depression Rating Scale. Methods Participants were nursing home residents living in nine Massachusetts nursing facilities participating in a research study between July 1994 and March 1998. Details of the study are described elsewhere [ 8 ]. All data used in this study reflect baseline and pre-intervention observations. The sample is considered representative of persons living in nursing homes in Eastern Massachusetts. Potential participants consented individually or by proxy to participate in a research study, using a protocol approved by the local Institutional Review Board. Exclusion criteria included: 1) a terminal prognosis, 2) a projected stay of less than 90 days, or 3) health complications that prohibited contact. Seven hundred four (n = 704) residents and/or their proxies participated in the baseline assessment, representing 67% of those eligible (14% of screened residents were ineligible). Seventy-seven percent of residents were women, 95% were White, non-Hispanic, and the median age was 86 years (interquartile range, 79–91 years). About half (54%) of residents had a chart diagnosis of dementia. Assessment of depression Residents were evaluated with the Minimum Data Set (MDS) Resident Assessment Instrument version 2.0 [ 6 ] as well as a 15-item version of the Geriatric Depression Scale (GDS-15) [ 2 , 3 , 9 ]. Trained research nurses collected the observations. For this analysis, we also considered a 5-item version of the GDS (GDS-5) [ 5 ]. MDS data collected and used in this analysis included demographic and clinical characteristics, level of cognitive and communicative functioning, and symptoms of depression. We used a simple sum of all symptoms in MDS section E1, referred to as E1SUM, as one MDS-based measure of depression. We also used a subset of seven of these MDS symptoms to code the MDS Depression Rating Scale (DRS) [ 7 ]. We also examined the depression symptom scales with regard to indicators of clinically recognized depression: chart diagnoses of depression and recent use of antidepressant medication. These data were also obtained by research nurses using structured chart review forms keyed to data elements collected with the MDS. MDS assessors are instructed to note the presence of a disorder related to the resident's current functional, cognitive, and behavioural status, medical treatments, and risk of death [ 10 ]. Among the disorders assessed is depression. The MDS manual is not specific with regard to clinical or diagnostic criteria for indicating depression diagnoses. The MDS also includes assessment of psychotropic medication use in the seven days preceding the assessment, including antidepressant use. In our analyses, we compared residents receiving any antidepressant with those receiving no antidepressant. Assessment of cognitive impairment We stratified the sample into two groups based on the severity of cognitive impairment. The impaired group comprised residents who were comatose (MDS 2.0 item B1 = 1), and/or with a short-term memory problem (B2a = 1) and those who only rarely/never make themselves understood (C4 = 3). All other residents were assigned to the "cognitively intact" group. This decision rule matches a screening rule for the MDS versus GDS depression symptom assessment proposed in the US Centers for Medicare & Medicaid Services' (CMS) working draft of the MDS version 3.0 [ 11 ]. Analytic approach We compared sample statistics and psychometric properties for each of the four depression scales across strata defined by cognitive impairment. We evaluated missing data by examining the proportion of residents with complete data on all assessment items, and also by the proportion of residents with complete data on a majority of items in the scale. For all other analyses we substituted missing values with the mean of the resident's non-missing items if a majority of the scale items were not missing. We examined means, standard deviations (SD), proportion of residents scoring at the floor, the internal consistency reliability (coefficient alpha [ 12 ], examined with and without a row-wise mean substitution rule for missing item responses) and the correlation among the depression scales. Finally, we examined the relationship of the scale scores to clinical indicators of depression: chart diagnoses of depression and a record of antidepressant use. We compared the mean of the scale scores across each of four cells formed by crossing antidepressant use and depression diagnoses. These comparisons were also made within cognitive impairment strata. Within strata, cell means were standardized with respect to the mean and standard deviation of the group of residents that neither received antidepressants nor had a chart diagnosis of depression. In this way, cell mean differences can be interpreted on an effect size metric [ 13 ]. All analyses were conducted with STATA software (College Station, Texas). Results Sample statistics and missing data Table 1 presents the sample statistics and psychometric properties for the comparison of the GDS and the MDS depression assessment instruments, stratified by level of cognitive impairment. Approximately 70% of residents were classified as cognitively impaired. Among the cognitively impaired, a majority had missing values for the GDS-derived scales (i.e., the GDS-15 and the GDS-5). A substantial proportion (about one in six) of residents were missing data on all GDS items. However, for the GDS-5, about 1 in 20 of the residents without cognitive impairment were missing data on all GDS items. On the other hand, the presence of missing data on the MDS-derived scales (i.e., E1SUM and DRS) was essentially nil in both cognitive impairment groups. About a third of residents had no missing values on the GDS-15 and more than half had missing data on the GDS-5. Table 1 Sample statistics and psychometric properties of Geriatric Depression Scale and MDS Depression Rating Scale. Sample statistic or psychometric property Cognitively Impaired Group (n = 495) Cognitively Intact Group (n = 209) All Participants (N = 704) Number (%) with missing data on all items GDS-15 81 (16%) 35 (17%) 116 (16%) GDS-5 110 (22%) 5 (2%) 115 (16%)* E1SUM 3 (1%) 2 (1%) 5 (1%) DRS 0 (0%) 0 (0%) 0 (0%) Number (%) with complete data on all items GDS-15 139 (28%) 111 (53%) 250 (36%)* GDS-5 223 (45%) 157 (75%) 380 (54%)* E1SUM 492 (99%) 207 (99%) 699 (99%) DRS 494 (100%) 209 (100%) 703 (100%) Number (%) with majority of scale items not missing GDS-15 357 (72%) 203 (97%) 560 (80%)* GDS-5 352 (71%) 200 (96%) 552 (78%)* E1SUM 495 (100%) 209 (100%) 704 (100%) DRS 495 (100%) 209 (100%) 704 (100%) Mean* (SD) GDS-15 4.7 (3.5) 4.9 (3.4) 4.8 (3.5) GDS-5 2.0 (1.5) 2.1 (1.6) 1.5 (1.6) E1SUM 3.9 (3.8) 2.7 (3.4) 3.6 (3.7)* DRS 1.9 (2.1) 1.8 (2.3) 1.9 (2.2) Proportion at floor GDS-15 0.081 0.053 0.072 GDS-5 0.166 0.167 0.166 E1SUM 0.206 0.349 0.249* DRS 0.356 0.397 0.368 Alpha – internal consistency reliability – row-wise complete cases only GDS-15 0.799 0.781 0.791 GDS-5 0.609 0.597 0.602 E1SUM 0.695 0.738 0.708 DRS 0.542 0.672 0.583* Alpha – internal consistency reliability – row-wise mean substitution for missing data † GDS-15 0.825 0.798 0.814 GDS-5 0.663 0.634 0.651 E1SUM 0.695 0.738 0.708 DRS 0.542 0.672 0.583* Correlation coefficients (GDS-5, GDS-15) 0.858 0.852 0.856 (DRS, GDS-15) 0.073 0.098 0.080 (DRS, GDS-5) 0.065 0.064 0.062 (E1SUM, GDS-15) 0.068 0.096 0.072 (E1SUM, GDS-5) 0.055 0.058 0.049 (E1SUM, DRS) 0.850 0.940 0.865* Abbreviations: GDS-15, 15-item Geriatric Depression Scale; GDS-5, 5-item Geriatric Depression Scale; DRS, Minimum Data Set (MDS) Depression Rating Scale; E1SUM denotes the sum of all mood and anxiety items in section E1 of the MDS; SD, standard deviation * For each respondent, if a majority of the items were not missing, any missing item was replaced with the mean of the non-missing items. † Missing item responses replaced with mean score for all respondents 'non-missing' on that item. On the other hand, about half of the cognitively intact group had no missing GDS-15 scores and three-quarters had no missing GDS-5 scores. Using an item-level missing data mean substitution rule, predicated on a resident having at least a majority of items present, the frequency of missing data for scale scores diminished for the GDS-derived scales. However, the impact of missing data remains an important problem for GDS-derived measures among the cognitively impaired: between one quarter and one third of the cognitively impaired still had missing data. All noted differences in the frequency of missing data describe large effect sizes (in Cohen's effect size taxonomy [ 13 ]) and are statistically significant (P < .001). The very high frequency of missing data for the GDS encouraged us to examine the frequency of missing data at the item level. We present this information in Table 2 , limiting the sample to those missing at least one but not all GDS items. The item with the greatest frequency of missing data was item 15 ("do you think that most people are better off than you are") for both the cognitively impaired group and those with better cognitive functioning. The item with the fewest missing values was item 5 among the cognitively impaired group ("are you in good spirits most of the time?") and item 1 among those without cognitive impairment ("are you basically satisfied with your life?"). Although the proportion with missing data on each item differed significantly across cognitive impairment strata for only one item (item 9), many of the differences across groups depict medium or larger effect sizes (items 2, 9, 10, 12). Table 2 Proportion of residents with missing data on individual items of the Geriatric Depression Scale. Limited to residents missing at least one but less than all 15 items. Item descriptions Cognitively Impaired Group (n = 251) Cognitively Intact Group (n = 93) All Participants (N = 344) 1. Are you basically satisfied with your life?* 37 (15%) 6 (6%) 43 (13%) 2. Have you dropped many of your activities and interests? 68 (27%) 10 (11%) 78 (23%) 3. Do you feel that your life is empty? 57 (23%) 16 (17%) 73 (21%) 4. Do you often get bored?* 47 (19%) 14 (15%) 61 (18%) 5. Are you in good spirits most of the time? 30 (12%) 9 (10%) 39 (11%) 6. Are you afraid that something bad is going to happen? 44 (18%) 13 (14%) 57 (17%) 7. Do you feel happy most of the time? 35 (14%) 12 (13%) 47 (14%) 8. Do you often feel helpless?* 55 (22%) 20 (22%) 75 (22%) 9. Do you prefer to stay in your room, rather than go out and doing new things?* 88 (35%) 11 (12%) 99 (29%) 10. Do you feel you have more problems with memory than most? 80 (32%) 11 (12%) 91 (26%) 11. Do you think that it is wonderful to be alive? 48 (19%) 14 (15%) 62 (18%) 12. Do you feel pretty worthless they way you are now?* 78 (31%) 14 (15%) 92 (27%) 13. Do you feel full of energy? 64 (25%) 14 (15%) 78 (23%) 14. Do you feel that your situation is hopeless? 74 (29%) 16 (17%) 90 (26%) 15. Do you think that most people are better off than you? 110 (44%) 31 (33%) 141 (41%) * included in GDS-5 Mean level of depression The means for the GDS- and MDS-derived scales were different between the cognitively intact and impaired groups (Table 1 ). Of note, the cognitively impaired group received higher scores on both the GDS-15 and GDS-5, indicating higher levels of depression. While these differences were small [ 13 ] they describe statistically significant differences (P < .05). Conversely, the cognitively intact residents within both the DRS and E1SUM had higher depression ratings. The difference between cognitive impairment groups was trivial[ 13 ] and not statistically different for the GDS (P = .79), but of moderate size[ 13 ] and statistically significant for E1SUM (P < .001). Floor effect Figure 1 illustrates several characteristics of the GDS-15 and the DRS, including the floor effect. Both instruments produce distributions with a high proportion of respondents scoring zero. For the GDS-derived measures as well as the DRS, the proportion scoring at the floor is similar for both cognitive impairment groups. The difference in the proportion scoring at the floor on the E1SUM measure is moderately different between the cognitively intact and cognitively impaired groups (P < 0.01). Figure 1 Scatter plot of 15-item Geriatric Depression Scale (GDS) and Depression Rating Scale (DRS) scores among 560 Nursing Home Residents in Nine Massachusetts Nursing Homes. Internal consistency reliability The GDS-15 had the highest internal consistency reliability coefficient, and the lowest was observed for the DRS. However, this difference does not substantially exceed that expected due to the greater scale length of the GDS-15. Using the Spearman-Brown prophecy formula [ 14 ], the reliability of the DRS for the total sample would be 0.72 if it had 15 similar items (instead of 7), which is closer to that observed for the GDS-15 (for residents with complete data). There was no statistically significant difference in the internal consistency reliability across cognitive impairment groups (using the variance ratio test[ 15 ]) for the GDS-derived scales and the E1SUM. However, the differences across cognitive impairment strata for the DRS were statistically significant (P < .01). We also note that the estimated internal consistency reliability can be artificially inflated by using a row-wise (i.e., person-wise) mean substitution rule for missing data. This artificial inflation affects GDS-derived scales but not DRS-derived scales. Correlation of DRS and GDS The correlation between the GDS- and DRS- derived measures were not statistically different from zero. All of the differences in correlation coefficients are similar among the cognitively impaired and cognitively intact and not statistically different, except for the correlation of the E1SUM and DRS (P < .001). Relation of DRS and GDS to indicators of conspicuous depression The comparison of mean depression symptom scale scores among residents that received antidepressants and/or had a chart diagnosis of depression, within and across cognitive impairment strata, is reported in Table 3 . For the GDS-15, within both cognitive impairment groups, there are trivial marginal differences in the mean score for either a depression diagnosis or record of antidepressant use. For the GDS-5, the mean score is somewhat higher for residents with a diagnosis or record of antidepressant use. These differences correspond to small to moderate effect size differences [ 13 ]. The marginal differences are not statistically significant within cognitive impairment strata, but for the total sample the pattern of means are essentially the same and are statistically significant for a depression diagnosis (P = .02) and approach significance for antidepressant use (P = .05). Table 3 Mean depression scale score as a function of the presence of diagnosis of depression or receiving antidepressants. Means are standardized to the mean and variance of the scale score for the group without a diagnosis and who did not receive an antidepressant. Cognitively Impaired Group (N = 495) Cognitively Intact Group (N = 209) Total (N = 704) Depression Diagnosis Depression Diagnosis Depression Diagnosis GDS-15 No Yes Total No Yes Total No Yes Total Antidepressant Use No 0.00 0.14 0.02 0.00 .47 0.06 0.00 0.26 0.03 (203) (26) (229) (116) (16) (132) (319) (42) (361) Yes 0.07 0.43 0.31 0.29 0.36 0.09 0.16 0.41 0.32 (42) (86) (128) (30) (41) (71) (72) (127) (199) Total 0.01 0.36 0.12 0.06 0.39 0.15 0.03 0.37 0.13 (245) (112) (357) (146) (57) (203) (391) (169) (560) GDS-5 Antidepressant Use No 0.00 0.05 0.01 0.00 0.51 0.06 0.00 0.23 0.03 (199) (23) (222) (115) (16) (131) (314) (39) (353) Yes 0.00 0.31 0.21 0.23 0.28 0.26 0.09 0.30 0.22 (44) (86) (130) (30) (39) (69) (74) (125) (199) Total 0.00 0.26 0.08 0.05 0.35 0.13 0.02 0.29 0.10 (243) (109) (352) (145) (55) (200) (388) (164) (552) E1SUM Antidepressant Use No 0.00 0.43 0.05 0.00 0.23 0.03 0.00 0.37 0.05 (295) (39) (334) (119) (18) (137) (414) (57) (471) Yes 0.60 0.55 0.57 0.48 -.06 0.18 0.55 0.37 0.44 (62) (99) (161) (31) (41) (72) (93) (140) (233) Total 0.11 0.52 0.22 0.10 0.04 0.08 0.10 0.37 0.18 (357) (138) (495) (150) (59) (209) (507) (197) (704) DRS Antidepressant Use No 0.00 0.40 0.05 0.00 0.16 0.02 0.00 0.32 0.04 (294) (39) (333) (119) (18) (137) (413) (57) (470) Yes 0.59 0.59 0.59 0.51 -0.15 0.13 0.56 0.33 0.42 (62) (99) (161) (31) (41) (72) (93) (140) (233) Total 0.10 0.54 0.22 0.11 -0.06 0.06 0.10 0.33 0.17 (356) (138) (494) (150) (59) (209) (506) (197) (703) Abbreviations: GDS-15, 15-item Geriatric Depression Scale; GDS-5, 5-item Geriatric Depression Scale; DRS, Minimum Data Set (MDS) Depression Rating Scale; E1SUM denotes the sum of all mood and anxiety items in section E1 of the MDS For the MDS-derived depression scales, the differences in means associated with antidepressant use and depression diagnoses are much more dramatic than for the GDS-derived scales, but only among the cognitively impaired. For both the E1SUM and DRS, the marginal differences for depression diagnoses and antidepressant use describe moderate to large effect sizes. These differences are statistically significant (both P < .001). On the other hand, among those residents not identified as cognitively impaired, the marginal differences associated with antidepressant use and a depression diagnosis were trivial to small and not statistically significant. Discussion In this study, we found that MDS- and GDS-derived depression measures were not correlated with one another, but were apparently adequately reliable measures of their intended construct. Thus, we infer that the MDS and the GDS measure different aspects of nursing home residents' depression. Each scale has specific strengths and limitations. The practical utility of the GDS is undermined by a very high frequency of missing data. The proportion of GDS responses missing differs greatly by level of cognitive functioning. The floor effect limits both instruments. While the internal consistency reliability is apparently greater for the GDS, this may simply be due to the greater number of items on the GDS. We observed a weak relationship between GDS-5 scale scores and clinical indicators of depression (diagnoses, antidepressant use), but the strong association between MDS-derived scales and clinical indicators was only seen among cognitively impaired residents. Other investigators have found a low correlation between the DRS and the GDS and other instruments for assessing depression, but these findings vary according to data collection strategies. For example, Anderson et al found a low correlation between the MDS DRS abstracted from residents' charts and symptom data collected with the GDS (r = .13) and the Hamilton Depression Rating Scale (HDRS; r = 0.24) using research interviewers [ 16 ]. Similarly, Hendrix et al[ 17 ] found a lack of correspondence between MDS depression symptoms and depression classified using a cut-point on the Cornell Scale for Depression in Dementia (CSDD). Hendrix and colleagues attributed the low agreement of CSDD and MDS to different data collection strategies. In their study, the CSDD was collected by primary caregivers, while the MDS was abstracted from the chart, and these authors suggest that the nurse administrators that completed the MDS did not consult the primary caregivers and the resident in completing the MDS depression items. Contrast with these findings a recent study by Ruckdeschel and colleagues [ 18 ], who converted the MDS items into a self-report assessment device and reported a very high correlation with depression symptom data collected with the GDS (r = 0.71). In our study, the assessment methods followed more closely how they were designed to be used. The GDS was used as a direct interview, and the MDS was used as instructed in the MDS manual [ 6 ], and included review of the chart, semi-structured interview with the resident, direct caregivers, and available family members or key informants, in order to arrive at final symptom ratings. MDS- and GDS-derived measures were comparably reliable after adjustments for test length, but were nevertheless not very highly correlated. Therefore, whatever differentiates the dimensions assessed by the two devices, it is probably an influence beyond the level of assessor training and the rigor of the evaluation. The fact that the MDS- and GDS-derived scales do not correlate implies that the two scales evaluate different aspects of a resident's depression. For positive MDS depression symptom ratings, residents must visibly act by making negative statements, be easily angered, and display unrealistic fears to trigger MDS symptoms. The GDS asks residents if they are satisfied with their life, feel helpless or worthless, and are often bored. It is conceivable that GDS-derived measures capture a brooding mental set, reflective of a dysphoric personality trait or adjustment disorder (for example in response to a recent change in living situation) rather than the presence of major depression. The lack of correlation between MDS depression measures has implications for proposed revisions to the MDS. Until more is known about the phenomenology and clinical validity of syndromes measured by these and other depression measures used in long-term care settings, adding self-report of symptoms of depression to the MDS is supported by our findings. We note that both CMS's draft revision of the MDS[ 11 ] and new versions of assessment instruments developed for other care settings by inter RAI include a provision for self-assessment of depression [ 19 ]. However, our findings may have further implications for CMS's revision of the MDS. The current draft of the revision calls for a sub-set of MDS section E items for those who are cognitively impaired, and direct questioning with the GDS-5 for those who are cognitively intact. Our findings suggest a more conservative approach might be to use both for all residents, or the MDS for all residents and the GDS for all residents who can communicate regardless of cognitive level. Two key findings underlie this recommendation. First, we find that the GDS and MDS are not complementary, but orthogonal. Second, we find no evidence for compromised measurement properties of the GDS among those with cognitive impairment. Conclusions The Geriatric Depression Scale and Minimum Data Set mood items measure different aspects of the depression syndrome among nursing home residents. The two measures cannot be treated as exchangeable or equivalent, and each has it's own strengths and limitations. The GDS uses self-report, but as a consequence suffers from a high frequency of missing data. The MDS relies upon informant ratings and therefore provides information about most residents. Although the GDS has higher internal consistency reliability than MDS, this is not beyond that expected due to greater scale length. The MDS measures were more strongly related to antidepressant use and record of a diagnosis of depression than were GDS measures. These results highlight the fact that more psychometric research is needed to better understand and improve the measurement of depression among frail nursing home residents. List of abbreviations used GDS Geriatric Depression Scale MDS Minimum Data Set DRS Depression Rating Scale CSDD Cornell Scale for Depression in Dementia HDRS Hamilton Depression Rating Scale Competing interests No financial competing interests. TR, JH, GIC, BEF and JNM are fellows of InterRAI, a non-profit group dedicated to improving the health care for persons who are elderly, frail or disabled. InterRAI owns the copyright to the MDS for nursing homes and many other care settings. InterRAI offers free licenses for the use of assessment forms of which it owns the copyright to. Therefore, there are no financial competing interests for members of the writing group. For more information please visit . Authors' contributions MK and RNJ conceived of the study. MK drafted the manuscript. RNJ performed the analyses and provided critical review of and completed the manuscript. JNM obtained the data, participated in the analysis and provided critical review of the manuscript. JH, MS, GIC and BEF provided critical review of the analyses and manuscript. All authors read and approved the final manuscript. Pre-publication history The pre-publication history for this paper can be accessed here:	/Users/keerthanasridhar/biomedlm/data/PMC000xxxxxx/PMC546185.xml

Subsets and Splits

No community queries yet

The top public SQL queries from the community will appear here once available.