Drosophila Lnk is the single ancestral orthologue of a highly conserved family of structurally-related intracellular adaptor proteins, the SH2B proteins. As adaptors, they lack catalytic activity but contain several protein–protein interaction domains, thus playing a critical role in signal transduction from receptor tyrosine kinases to form protein networks. Physiological studies of SH2B function in mammals have produced conflicting data. However, a recent study in Drosophila has shown that Lnk is an important regulator of the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway during growth, functioning in parallel to the insulin receptor substrate, Chico. As this pathway also has an evolutionary conserved role in the determination of organism lifespan, we investigated whether Lnk is required for normal lifespan in Drosophila. Phenotypic analysis of mutants for Lnk revealed that loss of Lnk function results in increased lifespan and improved survival under conditions of oxidative stress and starvation. Starvation resistance was found to be associated with increased metabolic stores of carbohydrates and lipids indicative of impaired metabolism. Biochemical and genetic data suggest that Lnk functions in both the IIS and Ras/Mitogen activated protein Kinase (MapK) signaling pathways. Microarray studies support this model, showing transcriptional feedback onto genes in both pathways as well as indicating global changes in both lipid and carbohydrate metabolism. Finally, our data also suggest that Lnk itself may be a direct target of the IIS responsive transcription factor, dFoxo, and that dFoxo may repress Lnk expression. We therefore describe novel functions for a member of the SH2B protein family and provide the first evidence for potential mechanisms of SH2B regulation. Our findings suggest that IIS signaling in Drosophila may require the activity of a second intracellular adaptor, thereby yielding fundamental new insights into the functioning and role of the IIS pathway in ageing and metabolism. Drosophila identified the SH2B family adaptor protein, Lnk, as an important regulator of the IIS pathway during organismal growth. In this study, we show that Lnk is also required to determine normal lifespan in Drosophila, as mutations that disrupt Lnk activity result in increased lifespan. In addition, these mutants show improved survival under conditions of stress and metabolic disregulation. Furthermore, we show that the expression of Lnk is regulated by the IIS responsive transcription factor, dFoxo. Our data therefore provide new mechanistic insights into the role of the IIS pathway in ageing.Many human populations are experiencing increased life expectancy, and as populations age the incidence of age-related diseases becomes more prevalent. The identification of single gene mutations that extend lifespan in invertebrate model organisms has revealed that several cellular signaling pathways, including the insulin/insulin-like growth factor (IGF)-1 signaling (IIS) pathway, play a crucial role in modulating the ageing process across multiple species. Thus, studies carried out in yeast, worms, and flies have revealed evolutionarily conserved mechanisms of ageing, which are likely to be relevant to mammals, including humans. A recent study in  SH2B proteins are a recently identified family of intracellular adaptor proteins that transduce signals downstream of a number of receptor tyrosine kinases (RTKs). These include the receptors for insulin, insulin-like growth factor-1, Janus kinase 2 (Jak2), platelet derived growth factor, fibroblast growth factor and nerve growth factor In vitro binding assays have identified interactions between SH2B proteins and a number of other intracellular adaptor proteins including the insulin receptor substrates IRS1 and IRS2, Grb2, Shc and c-Cbl Biochemical studies have demonstrated that SH2B proteins bind via their SH2 domains to phosphotyrosine residues within the intracellular tails of several activated RTKs thereby contributing to receptor activation Mammalian SH2B family members are widely expressed in a number of tissues suggesting that they may share some overlapping, redundant functions Drosophila melanogaster encodes a single SH2B homologue (Lnk) that shares a similar domain structure to its mammalian counterparts, with 36% sequence identity to human SH2B proteins in its PH-domain and 74% sequence identity in its PTB domain as well as containing a highly conserved c-Cbl binding motif. Furthermore, most of the basic metabolic and signaling pathways that maintain homeostasis are conserved in the fly providing an ideal context for in vivo studies of SH2B biological function.Understanding the physiological functions of SH2B proteins in mammals has therefore been complicated by the presence of multiple SH2B isoforms and conflicting data from genetic analyses. The genome of Drosophila Lnk is a key regulator of cell growth and proliferation during development Lnk produce phenotypes reminiscent of reduced IIS signalling such as growth reduction, developmental delay and female sterility. Genetic epistasis experiments indicated that Lnk functions downstream of the Drosophila Insulin Receptor (dInR) and upstream of PI3K in IIS-mediated growth control. Genetic epistasis suggested that Lnk may play a similar role as the insulin receptor substrate, Chico, in the activation of PI3K upon dInR stimulation during growth C. elegans, Drosophila and mouse can increase lifespan in all three organisms, demonstrating that the IIS pathway has evolutionary conserved roles in the determination of adult lifespan. In Drosophila, the effects of insulin receptor activity on lifespan determination are mediated via the Chico/PI3K/forkhead transcription factor Lnk mutant flies exhibit increased lifespan as well as improved survival under conditions of oxidative stress and starvation. We also show that Lnk loss-of-function results in increased stored energy reserves associated with transcriptional changes in genes involved in both lipid and carbohydrate metabolism. Biochemical and genetic data indicate that Lnk functions within both the IIS and Ras/MapK signaling cascades and is itself a direct target for transcriptional regulation by the dFoxo transcription factor.Recent evidence has shown that Lnk were recently isolated in a genetic screen looking for new regulators of growth in flies as Lnk loss-of-function clones were found to cause cell-autonomous growth inhibition in the developing eye Lnk: d07478Lnk containing a P-element insertion within the first intron of the Lnk locus and Del29Lnk, a small deletion generated by FLP-FRT recombination between two pBAC elements that removes the first two exons of Lnk including the predicted translational start site w strain. We then assayed heterozygotes and homozygotes of both alleles for longevity. After backcrossing into the 1118w genetic background, heterozygosity for either d07478Lnk or Del29Lnk did not result in any significant differences in lifespan in either males or females  and the egg laying defects observed in Del29Lnk homozygous females were fully rescued in the presence of a Lnk genomic rescue construct  and glycogen in the fat body, the insect equivalent of the mammalian liver and white adipose tissue. We observed significantly elevated levels of both TAG and glycogen in whole-fly extracts of both males and females when we compared controls . These eonstruct  Despite controls  suggestiLnk mutant males and females when compared to controls (Lnk genomic rescue construct (Lnk mutants and controls (data not shown). The total volume of hemolymph in an adult fly is extremely small (approximately 0.1 µl) and so the contribution of hemolymph trehalose to the total trehalose content can be regarded as negligible. Thus, the increase in trehalose content in whole fly extracts is almost certainly caused by increased tissue trehalose stores. Insect hemolymph also contains circulating glucose which is obtained from the diet and again, we found no significant differences in circulating glucose levels in Lnk mutants compared to controls (data not shown).In addition to glycogen, adult insects possess a second metabolic pool of carbohydrate in the form of the disaccharide trehalose which is a major sugar in the fat body, thorax muscles and hemolymph and is rapidly consumed during certain energy-requiring activities such as flight. We found that whole-body levels of trehalose were also significantly increased in controls  and agaionstruct . HoweverLnk mutants to controls. FlyAtlas, a microarray-based atlas of adult gene expression in multiple Drosophila tissues , we analysed our dataset using Catmap analysis. Catmap assigns significance to functional categories based on their representation within a ranked list of differentially expressed genes. This generated a list of GO terms associated with genes that show altered expression in controls . The majLnk mutants and controls with 1768 genes showing increased expression and 715 genes with decreased expression  between pression . We compLnk mutants compared to controls. The mammalian SH2B proteins have been shown biochemically to function as intracellular adaptors for the mammalian insulin receptor and recent genetic data from Drosophila has shown that Lnk may play a similar role to chico during IIS-mediated growth control. We therefore compared our data set to a comprehensive list of transcripts that function in the IIS pathway in Drosophila , ches-1-like (CG12690), eIF-4E (CG4035) and CG9009  (p = 0.784)  . In cont= 0.784) . Taken tLnk itself may be regulated by dFoxo activity. We therefore looked for perfect matches to the mouse Foxo1/Foxo4 consensus binding site (RWWAACA) within 3Kb upstream of the Lnk translational start site and identified eight putative dFoxo binding sites in the Lnk promoter. To determine if dFoxo is indeed bound at the Lnk promoter, we performed chromatin immunoprecipitation (ChIP) using a specific dFoxo antibody Lnk promoter to two negative control genomic regions: a region within the U6 snRNA promoter and a region 3′ to the Lnk locus just downstream of the last exon  during IIS-mediated growth control. Here, we have characterised a critical role for Lnk in the regulation of lifespan, stress responses and cellular metabolism. Our results support a model in which Lnk functions as an intracellular adaptor for transduction of the IIS and Ras/MapK signaling cascades to mediate these physiological processes.Our understanding of the physiological roles of the SH2B family of intracellular adaptors has been complicated by the presence of multiple family members in mammals. Furthermore, phenotypic analysis of genetic knockouts in mice has produced contradictory results. Recent genetic evidence has described a role for the single ancestral SH2B protein in Drosophila Lnk produce phenotypes reminiscent of reduced IIS during development including impaired growth, developmental delay and female sterility. Genetic epistasis experiments placed Lnk downstream of dInR and upstream of PI3K at the same level as Chico, the single fly insulin receptor substrate. Mutations in both chico and Lnk produce similar phenotypes and display similar reductions in IIS activity. Furthermore, flies homozygous mutant for both genes are lethal suggesting that they may be functionally redundant. The precise mechanisms whereby mammalian SH2B proteins transduce intracellular signaling from the insulin receptor remain unclear although like the IRS proteins, they have been shown to bind to multiple downstream mediators such as PI3K and Grb2 Drosophila Lnk lacks a consensus binding site for PI3K which is present in Chico so it is unlikely that they regulate similar downstream mechanisms.A recent genetic study has shown that mutations in 1chico, a strong loss-of-function allele of chico, show increased lifespan Lnk homozygotes also show increased lifespan although no obvious effects on lifespan were observed in heterozygous animals. Interestingly, the effects of Lnk mutation on lifespan extension were similar in both males and females, which is uncommon in Drosophila, even for IIS mutants. This data therefore suggests that as during growth regulation, signaling via the activated dInR during lifespan determination may require a second intracellular adaptor in addition to the insulin receptor substrate, Chico, and provides the first evidence of a role for SH2B proteins in lifespan determination.The IIS pathway has an evolutionary conserved role in the determination of adult lifespan mediated by the Chico/PI3K/dFoxo branch of the IIS cascade. Previous studies have shown that flies either homozygous or heterozygous for Lnk mutation on male fertility as measured by offspring production. As male homozygous mutants were also long-lived, this suggests that the extended lifespan of Lnk mutant females is not simply due to reduced fecundity. Genetic knockouts of SH2B1 in mice also show infertility due to impaired signal transduction from the IGF-1 receptor resulting in poor gonad development Lnk mutants are probably due to sex-specific differences in Lnk transcript expression as microarray analyses of Drosophila gene expression has shown that Lnk transcripts are enriched within the female ovary but not in the male testis or accessory glands , a number of dFoxo target genes did show changes in expression. Thus, split-ends (CG18497), ches-1-like (CG12690), eIF-4E (CG4035) and CG9009 all showed upregulated expression in our microarray data set. We also observed increased expression of two well-characterised dFoxo target genes, 4eBP and dInR, by quantitative RT-PCR. Taken together, these data suggest that dFoxo activity may be increased in Lnk mutant animals.A comparison of the transcriptomes of 4eBP and dInR between different body parts. Thus, for 4eBP we observed a 1.1-fold increase in expression in head RNA extracts compared to a 3.8-fold increase in RNA extracts from bodies. Similarly, for dInR we observed a 1.5-fold increase in expression in head RNA extracts compared to a 2.6-fold increase in body RNA extracts. These data suggest that different tissues may exhibit differences in the magnitude of the transcriptional response to Lnk loss of function. As our microarray experiments were performed on RNA isolated from adult heads only, this may explain why 4eBP and dInR were not identified in the microarray data set as microarray analysis of gene expression is generally regarded as less sensitive than qRT-PCR especially when changes in expression are small.Interestingly, we observed a marked difference in the magnitude of increased expression of both Lnk loss of function suggest that transcriptional feedback back onto multiple components of the pathway may play an important regulatory role in IIS signal transduction. Previous studies have shown that dInR is itself a direct target of dFoxo so that when IIS levels are low, activated dFoxo increases dInR expression. In this study, we have shown that dFoxo also binds to the Lnk promoter in vivo suggesting that Lnk itself may be a direct target of dFoxo. dFoxo activity may also regulate transcription of IIS genes under basal conditions. Previous studies have shown that dFoxo is required for the basal expression of the dilp3 ligand Lnk transcript expression increases suggesting that dFoxo activity is normally required for Lnk repression. Thus, regulation by dFoxo may involve both positive and negative effects on gene expression.The observations that upstream components of the IIS pathway show transcriptional upregulation in response to Drosophila PI3K is required for maximal PI3K activity during growth and female egg laying linking Ras/MapK and IIS during growth and development in DrosophilaLnk inhibits insulin-stimulated Erk phosphorylation in insect cells. We cannot exclude the possibility that Lnk may play an adaptor function for Ras signaling downstream of other RTKs in addition to the insulin receptor. However, it should be noted that Lnk RNAi knockdown has no effect on Spitz-stimulated Erk phosphorylation via activation of the Drosophila EGF receptor Our microarray data set also contained a number of differentially expressed genes that function within the Ras/MapK signal transduction pathway. Previous studies have shown that the Ras binding domain of Lnk mutants contain elevated levels of both lipid and carbohydrate stores. Consistent with their increased metabolic stores, Lnk mutants also showed increased survival under starvation conditions. Transcriptome analysis revealed gene expression changes in a number of components of metabolic regulation in Lnk mutants compared to controls. Thus, we observed reduced expression of several enzymes that function in the glycolytic pathway and upregulation of genes that function in glycogen synthesis. In addition, several genes in the mitochondrial β-oxidation pathway were downregulated whereas genes involved in the regulation of lipid storage showed increased expression. Taken together, these changes in gene expression are consistent with an overall inhibition of catabolic processes and upregulation of pathways that regulate the synthesis and storage of carbohydrates and lipids.Despite their small body size, Drosophila PPAR homolog has yet to identified but the closest Drosophila relative is the orphan receptor, E75 Lnk mutant flies and there is no evidence to date that a leptin-like hormone exists in Drosophila. A functional Jak has been identified encoded by the hopscotch (hop) gene that has a well characterised role in hematopoesis in flies. We did not observe any obvious hematopoetic defects in Lnk mutants and Lnk was not found to genetically interact with any of the core JAK/STAT pathway components (data not shown). Our data therefore suggests that the increased adiposity in Lnk mutant flies is unlikely to be mediated by increased feeding or by defects in Jak signaling. In fact, our data suggest that the ancestral function of Lnk in Drosophila is to regulate carbohydrate and fat storage by regulating gene expression of several key metabolic regulatory pathways.Studies on the metabolic defects of SH2B knockouts in mice have proved inconsistent. One group has shown that genetic deletion of SH2B1 impairs adipogenesis by downregulating adipogenic gene expression including PPARγ resulting in mice with decreased fat mass Drosophila Lnk and so it will be of interest to determine whether the interaction with c-Cbl is important for Lnk function especially during lifespan regulation.In mammalian cells, SH2B proteins have been shown to have dual functions during insulin signaling transduction by both activating and inhibiting downstream intracellular signaling events. Phosphorylation of SH2B2 by the activated insulin receptor creates a binding site for the proto-oncogene product c-Cbl. This promotes the ubiquitination of tyrosine kinase receptors by functioning as a RING-type E2-dependent ubiquitin protein ligase facilitating either endocytosis or proteasomal degradation of the receptor 1118w and d07478Lnk were obtained from the Bloomington Drosophila Stock Centre. yw, dfoxo21a/TM3 and dfoxo25c/TM3 were a gift from the Hafen lab dfoxo21a and dfoxo25c alleles were backcrossed for at least 6 generations into the yw background before use. The Del29Lnk deletion was generated using the pBAC insertions e01414Lnk and f02642Lnk  according to published protocols CG17370 to the beginning of the first exon of CG5913 was used for the genomic rescue construct. This was inserted by means of ΦC31 mediated integration into a landing site on the second chromosome at 51D white Dahomey (Dahw) stock was derived by incorporation of the 1118w deletion into the outbred Dahomey background by successive backcrossing. Both 1118w and Dahw stocks were negative for the endosymbiont Wolbachia as determined by PCR using primers specific to Wolbachia genomic DNA. Lnk mutants were backcrossed for at least 8 generations into both 1118w and Dahw genetic backgrounds before phenotypic analyses. Stocks were maintained and all experiments were conducted at 25°C on a 12h∶12h light:dark cycle at constant humidity using standard sugar/yeast/agar (SYA) medium Body weights of individual male and female 7-day old flies (n = 10 for each genotype) were measured using a precision balance. Wing areas, cell numbers and cell sizes were measured as previously described For female fecundity tests, female flies were housed with males for 48 hours post-eclosion and then separated into vials at a density of 5 or 10 females per vial. Eggs were collected over two 24-hour periods per week for 4 weeks. The number of eggs laid per vial at each time point was counted. For male fertility tests, individual 3 day old males were mated to 30 virgin females, 3 to 5 days of age. Matings were observed and then females were separated from the males and housed in vials at a density of 3 females per vial. Eggs were collected and counted over four consecutive 24 hour periods.For lifespan experiments, flies were maintained in vials at a density of 10 flies per vial on standard SYA medium. Flies were transferred to new vials three times per week.For all stress assays, flies were reared and housed as for lifespan experiments. For oxidative stress assays, 4-day old flies were first starved for 5 hours on 1% agar and then transferred onto 5% sucrose/agar containing 5% hydrogen peroxide. For starvation experiments, 7-day old flies were transferred to 1% agar.Feeding rates of flies were measured using a proboscis-extension assay in undisturbed conditions as previously described Drosophila S2 cell culture, dsRNA treatment and insulin treatment were as described in Hemolymph was collected and pooled from either 5 third instar larvae or 12 3-day old adult female flies. Glucose and trehalose levels were measured using the Glucose Infinity Reagent (ThermoScientific) as described in Glycogen content of 7-day old adult males was measured as described in actin5C. Three or four independent RNA extractions were used for each genotype. Primer sequences are available upon request.Total RNA was extracted from 10 whole adult flies, 10 adult bodies or 25 adult heads per genotype using standard Trizol (Invitrogen) protocols. cDNA was prepared using oligod(T) primer and Superscript II reverse transcriptase according to the manufacturer's protocol (Invitrogen). Quantitative RT-PCR was performed using the PRISM 7000 sequence-detection system and Power SYBR® Green PCR Master Mix (ABI). Relative quantities of transcripts were determined using the relative standard curve method and normalized to Lnk transcripts are widely expressed but are particularly enriched within the central nervous system and as Lnk mutant ovaries show significant structural differences compared to controls, we restricted our microarray expression analysis to isolated heads.Whole organism microarray experiments are generally only useful for detecting concerted changes of expression of widely expressed genes and most tissues will be under-represented in the array signal from a whole fly. Further complications arise from whole organism arrays when there are significant structural differences between treatments. Drosophila melanogaster genome release version 5.4. Promiscuous  and orphan (no probes in the probe set map to any known or predicted gene in the genome) probe sets were excluded from further analysis. FlyBase gene ids were mapped to Gene Ontology (GO) ids (version 1.107).Raw data (cel files) were processed to correct for probe-sequence biases, and R's implementation of the Affymetrix's MicroArray Suite 5.0 software was used to determine present target transcripts For functional analysis using all expressed genes, we used the Wilcoxon rank sum test implemented in Catmap Sequence analysis was performed using Regulatory Sequence Analysis Tools Chromatin immunoprecipitations were carried out essentially as described by Statistical analyses were performed using JMP software . Log rank tests were performed on lifespan and stress survival curves. Other data were tested for normality using the Shapiro-Wilk W test on studentised residuals and where appropriate log-transformed. One-way analyses of variance (ANOVA) and planned comparisons of means were made using Tukey-Kramer HSD test.Figure S1Lnk mutant flies. (A) Survival of male flies fed 5% hydrogen peroxide. Median survival times are 2.6 days for 1118w , 3.7 days for d07478Lnk  and 4.1 days for Del29Lnk . (B) Survival of female flies fed 5% hydrogen peroxide. Median survival times are 3.5 days for 1118w , 4.6 days for d07478Lnk  and 5.6 days for Del29Lnk . (C) Survival of male flies under starvation conditions. Median survival times are as follows: 2.3 days for 1118w , 3.6 days for d07478Lnk  and 3.9 days for Del29Lnk . (D) Survival of female flies under starvation conditions. Median survival times are 3.6 days for 1118w , 5.5 days for d07478Lnk  and 5.5 days for Del29Lnk .Stress resistance of (0.63 MB TIF)Click here for additional data file.Figure S2Lnk mutant flies. Feeding observations of 7 day old flies housed on standard food at a density of 5 flies per vial. Flies were left undisturbed for at least 15 hours before observations were started. Data are presented as the proportion of feeding events/possible feeding events ± SEM. No significant differences were observed in the feeding behavior of Lnk mutant flies compared to controls .Feeding behaviour of (0.19 MB TIF)Click here for additional data file.Table S1Lnk mutant microarrays. Catmap analysis was used to identify functional categories associated with genes that show altered expression within Lnk mutants compared to controls. For brevity, the full hierarchy of the significant Gene Ontology (GO) categories has not been shown.Functional Categories that are significantly down- or upregulated in (1.11 MB TIF)Click here for additional data file.Table S2Lnk mutants relative to controls. RNA was extracted from the heads of 7-day old females homozygous for the Del29Lnk allele, along with RNA from age-matched controls. RNA was labelled and hybridised to Affymetrix Drosophila 2.0 microarrays. All experiments were conducted in quadruplicate to facilitate statistical analysis. The raw data files were background corrected and normalised by loess normalization and a linear model was fitted to identify a set of differentially expressed genes using the R limma package. The columns show the Affymetrix probe set (A), fold-change in expression level (B), p-value (C), FBgn identifier (D) and gene name (E). Genes are sorted by p-value.List of differentially expressed genes  in (0.37 MB XLS)Click here for additional data file.Table S3Lnk mutants. Genes listed in Drosophila metabolic pathways Metabolic pathways affected in (0.04 MB XLS)Click here for additional data file.Table S4Lnk mutants. Genes listed in et al. (2008). Nutritional control of protein biosynthetic capacity by insulin via Myc in Drosophila. Cell Metabolism 7: 21-32). Genes listed in grey were not reliably detected on the arrays.Genes in the IIS and Ras/MapK pathways that show differential expression in (0.05 MB XLS)Click here for additional data file.


Chronic fatiguing illness remains a poorly understood syndrome of unknown pathogenesis. We attempted to identify biomarkers for chronic fatiguing illness using microarrays to query the transcriptome in peripheral blood leukocytes.Cases were 44 individuals who were clinically evaluated and found to meet standard international criteria for chronic fatigue syndrome or idiopathic chronic fatigue, and controls were their monozygotic co-twins who were clinically evaluated and never had even one month of impairing fatigue. Biological sampling conditions were standardized and RNA stabilizing media were used. These methodological features provide rigorous control for bias resulting from case-control mismatched ancestry and experimental error. Individual gene expression profiles were assessed using Affymetrix Human Genome U133 Plus 2.0 arrays.There were no significant differences in gene expression for any transcript.Contrary to our expectations, we were unable to identify a biomarker for chronic fatiguing illness in the transcriptome of peripheral blood leukocytes suggesting that positive findings in prior studies may have resulted from experimental bias. The etiology of chronic fatigue syndrome (CFS) is unknown MSN, moesin). Gene expression studies of peripheral blood leukocytes (PBLs) are a potentially promising source of biomarkers for CFS Given the lack of clarity in existing studies of CFS, we undertook an “unbiased”, transcriptome-wide search for gene expression changes associated with CFS in PBLs. Our study had two notable design features. First, we used a control group optimal for detecting state-related gene expression changes and minimized false-positive findings due to genetic mismatching between cases and controls as we contrasted 44 individuals with clinically-evaluated chronic fatiguing illness with their 44 unaffected monozygotic co-twins. Use of rigorously discordant monozygotic twins provides the best control for genetic background currently possible in humans and allows use of paired statistics with greater statistical power. Second, we carefully standardized sampling conditions so that PBL samples were drawn into RNA-stabilizing media and taken from both members of a twin pair at the same time and place.The protocol was approved in advance by the ethical review board at UNC-CH and the Karolinska Institutet and all subjects provided written informed consent.We screened ∼61,000 individual twins from the Swedish Twin Registry for the symptoms of fatiguing illness Of 140 monozygotic and preliminarily discordant twin pairs, one or both twins declined participation in 23 pairs, 25 pairs were concordant for CFS-like illness, and inclusion criteria were not met in 35 pairs . After excluding these 83 pairs, 57 pairs of twins attended the clinical evaluation sessions, and 10 pairs were found not to meet inclusion criteria . Zygosity was confirmed by genotyping 46 single nucleotide polymorphisms using two Sequenom iPlex panels. In 3 pairs, microarray data of satisfactory quality could not be obtained.The analysis sample consisted of 44 pairs of rigorously discordant and genetically proven monozygotic twins. Discordance was defined as one twin meeting criteria for either idiopathic chronic fatigue  or CFS (32 pairs) Transient/situational factors can influence gene expression measurements. Biological sampling was standardized by having samples drawn from both members of a twin pair at the same place and time (∼0900) after an overnight fast. We required that all subjects be in their usual state of health on the day of sampling . It was neither practical nor ethical to study subjects medication-free, but we delayed assessment if there had been a recent significant dosage change.ex vivo gene expression). Total RNA was purified using the PAXgene blood RNA kit following the manufacturer's instructions (Qiagen). RNA quality was determined using the Agilent 2100 Bioanalyzer. Total RNA (5.0 µg) was labeled with the one-cycle cDNA synthesis kit (Invitrogen) and spiked with eukaryotic Poly-A RNA controls to check the target labeling process (Affymetrix). Synthesized cDNA was transcribed in vitro using the GeneChip IVT labeling kit (Affymetrix). The biotin labeled cRNA product (20 µg) was purified with a sample cleanup module (Qiagen) and samples were fragmented with the fragmentation buffer from Affymetrix at 94°C for 35 minutes. Fragmented and labeled targets (together with hybridization and oligo B2 controls) were hybridized to Affymetrix Human Genome U133 Plus 2.0 arrays at 45°C for 16 hours. Washing and staining of the arrays were performed on the Affymetrix fluidics station using the EukGE-WS2v5_450 protocol. Imaging of the arrays and signal quantification were performed with the Affymetrix GeneChip Scanner 3000 and GeneChip Operating Software. For verification, we used qRT-PCR. RNA was converted to cDNA with Superscript III (Invitrogen) and qRT-PCR was run with ABI's Taqman gene expression assays (with 18S rRNA as control). The ΔΔCt method was used for the calculations.Peripheral venous blood was drawn using sterile technique into PAXgene tubes manufactured in the same batch  under accession number GSE16059 and were prepared in accordance with MIAME 2.0 standards.Array images were manually checked for defects using DChip 43 = 0.32, p = 0.75). Body mass index was similar between the affected and unaffected twins. Two affected individuals (4.5%) reported sudden onset of fatigue. Affected twins had significantly worse physical and mental functioning on the SF-36 http://www.sf-36.org/nbscalc/index.shtml, accessed 12 December 2008).The analytic data set consisted of microarray data from 44 pairs of monozygotic twins discordant for clinically-evaluated chronic fatiguing illness Table 1.CXCR4 p = 0.03 and RAP2C p = 0.04), a degree of overlap that does not depart from chance expectations. At the single transcript level, there was no biological evidence of altered gene expression in PBLs that correlated with chronic, impairing, and medically unexplained fatiguing illness. For verification, we used qRT-PCR to assess the expression of seven genes selected from the CFS gene expression literature and our empirical findings . All p-values from paired t-tests contrasting affected and unaffected monozygotic twins were ≥0.26.The main analyses contrasted gene expression in PBLs in 44 pairs of monozygotic twins affected with CFS or ICF to that of their unaffected co-twins. As inclusion of males could increase noise, the second planned analysis compared 39 female pairs with CFS or ICF and the third planned analysis compared the 28 pairs of female twins with CFS. For each of these three sets of statistical comparisons, the observed results did not deviate from those expected by chance  plausibly involved in the pathophysiology of this idiopathic syndrome. We attempted to correct methodological issues in prior reports by careful control of sources of bias . We found no evidence of differential PBL gene expression that characterized the presence or absence of CFS or ICF. Therefore, unlike most prior published studies, we did not find evidence of a gene expression biomarker for chronic fatiguing illness.−66) with the expression level of glutathione S-transferase theta 1, GSTT1) These results may hold a lesson for case-control gene expression studies in humans. There are certainly examples where transcriptomic studies have yielded results that have the potential to improve disease prognosis and management  Use of discordant monozygotic twins represents the best control for genetic background currently possible in humans. Assuming identify at the DNA level and control for experimental bias, gene expression differences in discordant monozygotic twins can be cleanly attributed to disease state. It is reasonable to consider if use of discordant monozygotic twins represents “over-matching”. In comparisons of unrelated cases and controls, gene expression differences are an amalgam of disease state, the RNA-level impact of genetic loci causal to the trait, and the effects of case-control genetic mismatching . Use of discordant monozygotic twins yields more interpretable results particularly as there are large numbers of non-causal loci under genetic control. We would also argue that a gene expression study is a poor way to identify genetic loci causal to a disease when an alternative study design (the genome-wide association study) has been so successful We were unable to identify a biomarker for chronic fatiguing illness in the transcriptome of peripheral blood leukocytes suggesting that positive findings in prior studies may have resulted from experimental bias.Table S1Shown are results from pathway analyses using SAFE to investigate KEGG pathways, GO keywords , and PFAM protein family groupings. These all had an empirical p-value (from permutation) <0.005 and were composed of ≥10 transcripts.(0.11 MB DOC)Click here for additional data file.Figure S1Quantile-quantile (QQ) plots from the planned paired analyses contrasting monozygotic (MZ) twins affected with chronic fatiguing illness versus their unaffected co-twins. CFS = chronic fatigue syndrome, ICF = idiopathic chronic fatigue. The observed distribution of statistical results conform to chance expectations.(0.11 MB DOC)Click here for additional data file.


Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing.In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling.We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsize sequencing centers. Massively parallel sequencing is currently revolutionizing sequencing data generation in biology Sample preparation is one of the challenges associated with massive DNA sequencing In this study, an automated DNA library preparation method for the GS FLX Titanium sequencing system is described, which utilizes the precipitation of DNA on generic carboxylic acid-coated superparamagnetic beads as a general approach for PEG-mediated precipitation of DNA prior to sequencing. This approach can be used to readily remove shorter DNA fragments from a sample, thus cleaning it up prior to sequencing. By varying the composition of PEG in the precipitation buffer, the protocol can be adjusted to specifically suit the fragment length appropriate for the starting material, or adjusted to suit the read-length of the sequencing system. The automated protocol was evaluated by comparing it to the standard manual GS FLX Titanium library preparation protocol with respect to yield, sample throughput, robustness and sequence bias. For this task, the automated protocol was approximately two times faster, while increasing the sample throughput three-fold each run (with the possibility of running 3 preparations per day), and produced 3–5 times higher yields compared to a standard manual library preparationAutomated library preparations were set up using a Magnatrix™ 1200 Biomagnetic Workstation  capable of running custom made scripts. The robot, equipped with a 12-tip head and in-tip magnet processing, is highly suitable for magnetic bead based applications and was instructed to perform all reactions as specified by the GS FLX Titanium Library Preparation Protocol. The robot also features a Peltier unit (4–95°C) where all reactions were performed. For cooling inside the instrument, but outside the Peltier unit, a PCR-cooler  was used. The GS FLX Titanium Library Preparation Protocol begins with a standard fragmentation of DNA by nebulization, followed by purification and concentration with MinElute columns. The standard protocol uses AMPure® beads, calibrated to remove fragments below 400 bp, for the DNA fragment length separation after nebulization. The sample is then subjected to the following reactions and purifications in the following order: fragment end polishing, MinElute purification, adaptor ligation, MinElute purification, AMPure bead fragment length separation, library immobilization using streptavidin coated beads, fill-in reaction, and finally NaOH elution to isolate the ssDNA library containing emulsion PCR (emPCR) amplification primer sites. The automated protocol uses PEG/NaCl precipitation on MyOne™carboxilic acid-coated superparamagnetic beads (Invitrogen) as solid support for purification (CA-purification) instead of the MinElute™  purification steps and the AMPure beads for “double SPRI-method” used in the standard manual library preparation . Bovine PEG 6000  and NaCl (Merck) were dissolved in MilliQ water to a final concentration of 0.9 M NaCl and varying final PEG concentrations. The automated protocol takes 100 µl of PEG/NaCl precipitation solution to 10–100 µl MyOne™ CA-beads , and captures the DNA in 50 µl sample. The amount of CA-beads was adjusted to the amount of DNA to precipitate, where 10 µl was seen to be sufficient for 0.5 µg of DNA (data not shown). EB buffer (Qiagen) was used to elute the samples from the CA-beads.The fragment length necessary for precipitation was investigated by varying the final PEG concentration and precipitating DNA ladders 100–10,000 bp and 25–700 bp respectively . The results were analyzed using a 2100 Bioanalyzer , with the DNA 7500 kit and DNA 1000 kit respectively. The yield was investigated by precipitating a 3,000 bp PCR fragment, and analyzed using a NanoDrop™ ND-1000 .To estimate the variance of CA-purification nebulized lambda DNA was CA-purified in 11 parallel reactions during one instrument run, and analyzed using the Bioanalyzer with the DNA 7500 kit, and concentration measurements using the NanoDrop.Chironomus tentans (C. tentans) or bacteriophage lambda) was nebulized as instructed by the manufacturer .Prior to automatic and manual handling, DNA samples were nebulized using standard GS FLX library preparation nebulizers. For library preparation and sequencing, genomic DNA .All library reagents were taken from GS FLX Titanium kits (Roche) except the Titanium Multiplex Identifier (MID) adaptors, which were synthesized by Thermo Scientific according to the manufacturer's specifications (Roche). To evalTo compare the automated protocol to the manual handling one sample was prepared according to Roche protocol for double-SPRI purification. The manual library was prepared using MID6 (“sample SPRI”). The automated protocol was used to prepare four samples in parallel, three samples using MID1–3 respectively (“sample 1–3”), and one using MID4 (“sample 4”). The final PEG concentration for samples 1–3 and sample 4 in the precipitation reactions were 8.1% and 7.5% respectively. The two PEG concentrations were used to evaluate the impact on sequence read length.C. tentans DNA were prepared in parallel, each library with a different MID-adaptor. The automated protocol included a more stringent wash routine of the immobilization beads, compared to the first sequencing library preparations to make sure that all non-immobilized DNA were removed. All preparations were analyzed with the Bioanalyzer 7500 kit prior to library preparation, and the final ssDNA libraries were analyzed with the Bioanalyzer RNA 6000 Pico kit. Sample concentrations in the final ssDNA libraries were measured using Qubit-IT ssDNA kit (Invitrogen).To test MID-adaptors 1–12, 12 libraries of 7 and 106 molecules/µl according to Qubit and Bioanalyzer measurements; these dilutions were then amplified in triplicates using 200 nM emPCR primers . PCR started at 94°C for 4 min, followed by 50 cycles of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min 30 s. Fluorescence was measured after each cycle. The average primer efficiency (Peff) was calculated based on the 10-fold molecule difference of the dilutions of the libraries and the difference of cycle threshold (Ct) the samples started to amplify. . When Peff was determined, the relative difference between the libraries was calculated from the average cycle thresholds where the 107 diluted triplicates started to amplify. Nebulized C. tentans DNA was used as a negative control.Quantitative PCR (qPCR) can be used to estimate the number of amplifiable molecules of an ssDNA library The emPCR titration by quantification, amplification and sequencing were performed according to the manufacturer's instructions. The manual and the automated library preparations were sequenced using a GS FLX Titanium instrument.Different pools of libraries were set up and sequenced on individual lanes in duplicates to compensate for loading effects. Two SV  reactions were used per sample from the SV emPCR kit (Roche). Libraries were set up as follows: a pool of all libraries (P1), a pool of sample 1–3 (P2), sample 4 individually and sample SPRI individually. A 16 lane format was used to allow assessment of each library independently, where 11 lanes where used for this study. The SPRI sample was loaded in only one lane due to poor amplification in the emPCR, resulting in only 159,000 enriched beads  The firsTwo equimolar pools of all the 12 MID-libraries were set up. One pooled according to Bioanalyzer concentration measurements, and the other pooled according to the number of amplifiable molecules estimated by qPCR. The qPCR pool was sequenced on lane 1, and the Bioanalyzer pool was sequenced on lane 2. A total of two million beads were loaded on each lane resulting in 883,053 reads that passed all GS FLX quality control filters.The CA-purification was performed on DNA ladders from Fermentas and nebulized bacteriophage lambda DNA to determine its robustness and the DNA precipitation length cut-off at varying final PEG concentrations. The CA-purification showed great flexibility and could handle all reaction buffers tested without need for additional purifications and without visual sample loss in the instrument. The lower limit of DNA fragment precipitation was approximately 100 bp–1,000 bp depending on the final PEG concentration . The yieC. tentans DNA sample prepared using the automatic protocol showed very reproducible ssDNA size distributions when analyzed on the Bioanalyzer , capable of automating fragmentation in a 96 well format. These methods in combination would even further alleviate the increasing need for multiplexing, as sequencing platform capacity continues to increase (e.g. for gene expression studies with multiple patients). Specifically, recent efforts by Roche to improve the library preparation protocol for GS FLX Titanium sequencing by reducing the number of steps and reagents, in combination with expected future upgrades of the instrument, likely will amount to a need for further multiplexing. The simple, flexible and robust automated protocol described in this study could easily be adapted to address this need.In conclusion, we have demonstrated an automated DNA library preparation method for the GS FLX Titanium instrument that quickly and reproducibly generates higher yields of ssDNA compared to manual execution. Furthermore, the sample throughput of the automated protocol is up to 9 times higher than the manual protocol. The fragment size selection method we describe can be used as a general approach whenever DNA size separation needs to be performed, facilitating fast and automated handling of samples. Using the same strategy, similar automated protocols stand to alleviate workload and increase throughput for other massively parallel sequencing platforms.Table S1MID adaptor oligonucleotide sequences used for library preparations.(0.13 MB PDF)Click here for additional data file.Table S2Lane set-up from first sequencing run. Omitted lanes were used by other projects.(0.02 MB XLS)Click here for additional data file.Table S3Work schedule showing the preparation of 36 samples in one day. Green color indicates an automated procedure.(0.03 MB XLS)Click here for additional data file.Figure S1Read distribution between MID-libraries generated from standard equimolar pooling of all libraries in the first sequencing trial (gray) compared to library concentration difference factors of the individual library dilutions for those samples, determined by relative qPCR (blue) normalized to the least efficient library (MID3). The blue pie chart illustrates the predicted outcome when sequencing equal pooling of these library dilutions (percentage), based on the qPCR detected relative concentration difference (numbers).(1.02 MB TIF)Click here for additional data file.Figure S2Read length distribution of each prepared library from first sequencing run. MID1–4 was automatically prepared, sample SPRI (MID6) was manually prepared. Number of reads (y-axis) is plotted against read length (x-axis).(1.68 MB TIF)Click here for additional data file.


Regulated on activation, normal T cell expressed and secreted (RANTES) is a chemokine that is produced by fibroblasts, lymphoid and epithelial cells of the mucosa in response to various external stimuli. RANTES expression has been demonstrated in a variety of diseases characterized by inflammation, including asthma, transplantationassociated accelerated atherosclerosis, endometriosis and fibrosis. RANTES mRNA is quickly up-regulated by tumor necrosis factor (TNF)-α stimulation. Cyclosporine A (CsA) is widely used in organ transplant patients, often causing various side-effects including gingival overgrowth, which is fibrotic in nature. This study was carried out to assess the mRNA expression of TNF-α and RANTES in healthy individual, chronic periodontitis and CsAinduced gingival overgrowth tissues.Gingival tissue samples were collected from chronic periodontitis, CsAinduced gingival overgrowth patients and healthy individuals. Total RNA was isolated and reverse transcription polymerase chain reaction (RT-PCR) was performed for TNF-α and RANTES expression.The results suggest that CsAinduced gingival overgrowth tissues expressed significantly increased TNF-α and RANTES compared to control and chronic periodontitis.The findings of the present study suggest that CsA can modify the expression of TNF-α and RANTES in drug-induced human gingival overgrowth. Regulated on activation, normal T cell expressed and secreted (RANTES) is a member of the C-C chemokine subfamily and is produced by fibroblasts, lymphoid and epithelial cells of the mucosa in response to various external stimuli.–3 RANTES4Gingivitis is an inflammatory disease of bacterial origin initiated and perpetuated by pathogenic bacteria. The disease is characterized by infiltration of inflammatory cells like neutrophils, macrophages, etc.8 PeriodoGingival tissues were collected at the time of resective surgery from eight chronic periodontitis and eight CsAinduced gingival overgrowth patients aged 40-60 years. Eight control samples were also obtained from healthy individuals who had undergone orthodontic treatment. Drug-induced gingival overgrowth tissue samples were obtained from CsA-medicated organ transplant recipients during gingivectomies required to treat drug-induced gingival overgrowth. All the samples represented a moderate to severe degree of gingival overgrowth. All the diseased samples expressed clinical signs of inflammation and were taken from sites with a pocket depth >6 mm, with evidence of bleeding on probing. The control subjects were systemically and periodontally healthy without gingival inflammation and showed no evidence of bleeding on probing, with a pocket depth <4 mm. All the patients and healthy subjects were nonsmokers from enquiry on personal history. People who were medically compromised, who had antibiotic intake within 6 months and who had undergone any periodontal therapy were excluded from the study. Informed consent was obtained from all subjects and the study was approved by the Institutional Review Board of Sri Ramachandra Medical College and Research Institute.The total RNA was isolated from the gingival tissues by a single-step, acid guanidine thiocyanate-phenol-chloroform extraction method. The totaP <0.05 was considered statistically significant.Results are expressed as mean ± SD. Statistical analysis was performed by ANOVA. A value of The expression of TNF-α, RANTES and β-actin in human gingival tissues with chronic periodontitis and drug-induced gingival overgrowth was examined. The amount of β-actin PCR product was used as the standard for the analysis of TNF-α and RANTES mRNA expressions. TNF-α and RANTES mRNA were not only expressed in the drug-induced gingival overgrowth and periodontitis but also, weakly, in the uninflamed gingival tissues . The expStudies of chemokines are currently being undertaken to further understand their role in the pathogenesis of a number of diseases because of their potential use as targets for therapy. The present experiment is performed to study the role of RANTES and the cytokine TNF-α expression in human gingival tissues with chronic periodontitis and CsAinduced gingival overgrowth compared with normal healthy gingival tissues. All control tissues expressed a small amount of RANTES and proinflammatory cytokine TNF-α. Studies have shown that the numbers of healthy gingival tissue sections identified positive for RANTES expression.17 Severa182032


TLR4) non-synonymous single nucleotide polymorphisms Asp299Gly and Thr399Ile in this process remains highly controversial. Moreover, what differential impact these polymorphisms might have in at risk populations with respiratory dysfunction, such as current asthma or a history of infantile bronchiolitis, has never been examined. Here we determine the importance of these polymorphisms in modulating LPS and respiratory syncytial virus (RSV) - driven cytokine responses. We focus on both healthy children and those with clinically relevant respiratory dysfunction.A broad variety of natural environmental stimuli, genotypic influences and timing all contribute to expression of protective versus maladaptive immune responses and the resulting clinical outcomes in humans. The role of commonly co-segregating Toll-like receptor 4 (TLR4 Asp299Gly and Thr399Ile on cytokine production, we assessed multiple immune parameters in over 200 pediatric subjects aged 7–9. Genotyping was followed by quantification of pro- and anti-inflammatory cytokine responses by fresh peripheral blood mononuclear cells upon acute exposure to LPS or RSV.To elucidate the impact of In contrast to early reports, neither SNP influenced immune responses evoked by LPS exposure or RSV infection, as measured by the intermediate phenotype of pro- and anti-inflammatory cytokine responses to these ubiquitous agents. There is no evidence of altered sensitivity in populations with “at risk” clinical phenotypes.TLR4 Asp299Gly/Thr399Ile haplotype is of no clinical benefit in predicting the nature or intensity of cytokine production in children whether currently healthy or among specific at-risk groups characterized by prior infantile broncholitis or current asthma.Genomic medicine seeks to inform clinical practice. Determination of the  Genomic medicine seeks to inform clinical practice. It is widely anticipated that identifying specific components of an individual's genome will help determine the optimal approach to health care; for example, by facilitating the prediction and prevention of diseases such as asthma The role of Toll-like receptor 4 (TLR4) polymorphisms in influencing clinical and immunologic responses to environmental stimuli is an area of much continuing and conflicting attention. TLR4 is specifically involved in generating immune responses against a diverse panel of agonists TLR4 gene harbours two commonly co-segregating single nucleotide polymorphisms (SNP)—Asp299Gly and Thr399Ile—that alter the amino acid sequence of the TLR4 protein. Experimental systems indicate that the 299Gly SNP is associated with hyporesponsiveness to TLR4 ligands such as LPS in vivo and in vitro, and that it acts in a dominant fashion with respect to the more common Asp299 allele TLR4 299Gly allele at a higher frequency than children with mild disease not requiring hospitalization The human TLR4 Asp299Gly and Thr399Ile polymorphisms on the human immune response to both LPS exposure and, independently, to RSV infection.LPS and RSV are ubiquitous in the environment. Pediatric populations may experience substantially higher exposure to both agents and exhibit a higher burden of disease than adults. Given the morbidity associated with RSV bronchiolitis and the observation that RSV infection increases the risk of subsequent wheezing and asthma, we undertook a rigorous examination of the functional impact of the TLR4 variants, or use of inbred animal models, the results of which are often directly extrapolated to highly diverse human populations, (ii) use of human cells ex vivo as previously frozen samples or following pre-conditioning regimens with addition of cytokines (ie IFNγ) to culture, both of which have demonstrated potential to skew the resulting immune responses, (iii) small sample sizes (frequently less than 100), and (iv) use of different, or undefined, variants of RSV for re-stimulation. Importantly, the majority of studies attempt to link SNPs or haplotypes directly to clinical outcomes, without analysis of the intermediate phenotype of immune function. Thus, notwithstanding extensive evidence of the impact of gene by environment by time interactions on a wide variety of immune and clinical outcomes The most significant challenges associated with prior studies of this question include (i) use of cell lines, reliance on artificial experimental systems in which cell lines are transfected with TLR4 Asp299Gly and Thr399Ile polymorphisms on putatively distinct cytokine production in human populations that interact with a broad diversity of natural environmental stimuli throughout childhood, we measured multiple immune parameters in over 200 pediatric subjects aged 7–9 years following acute in vitro re-exposure to LPS or RSV. The results demonstrate that while some reports link TLR4 Asp299Gly polymorphism with clinical outcomes of RSV infection, the haplotype comprised of Asp299Gly and Thr399Ile does not detectably impact the immune response to RSV, as measured by the intermediate phenotype of RSV-stimulated cytokine responses nor LPS-driven cytokine production. This conclusion applies equally to healthy children and those with “at risk clinical phenotype” characterized by current asthma or a history of severe bronchiolitis in infancy.As an alternative approach to better elucidate the immunological impact of the Study approval was obtained from the University of Manitoba Faculty Committee on Use of Human Subjects in Research. This 1995 birth cohort  was created from the Manitoba Canada universal provincial health care registry In prior studies, 100% of this age group was seropositive for prior RSV infection, reinforcing the ubiquity of these infections in the general population. These 7–9 year old children had no evidence of current upper or lower respiratory tract infection within one month of recruitment based on histories from child and parent (no “colds”) and no current evidence of a URI on physical examination.20), according to the Canadian Asthma Consensus Guidelines Pediatric allergist assessment was used as the gold standard for diagnosis of current asthma (ie. within the last 12 months). Diagnosis was based on history and physical assessment  diagnosis of “acute bronchitis and bronchiolitis” (466 code) between birth and age 2, which was identified using the Manitoba Center for Health Policy databases.Blood was collected in tubes containing EDTA, peripheral blood mononuclear cells (PBMC) were isolated by density centrifugation, counted (>95% viability by trypan blue exclusion), and used immediately for short-term primary culture in the absence of exogenously added cytokines.50 performed in 96-well microtiter plates using tenfold dilutions.RSV strain Long was cultured on Hep-2 cells at 37°C in Eagle's MEM supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml), 30 µg/ml L-glutamine (Life Technologies) and 1% fetal calf sera (Sigma). RSV was titrated by the quantal assay TCID6 cells/ml in 200 µl in 96 well U bottom plates. Duplicate cultures were stimulated with LPS  from Escherichia coli serotype 00011:B4 at 0.05, 0.5 and 5.0 ng/ml from Sigma  or RSV strain Long (104.9 TCID50/ml). LPS purity was validated by i) its inability to activate other human TLRs expressed in transfected HEK 293 cells, and ii) the finding that pretreatment with TLR4 blocking antibodies , but not IgG2a isotype control antibodies (Ebioscience), blocks the capacity of LPS to stimulate cytokine production demonstrate its specificity for TLR4 . Based on data obtained in preliminary time course experiments (data not shown), culture supernatants were harvested following 1 or 6 days culture, the respective time of peak LPS and RSV driven responses for the cytokines evaluated. The optimal concentration of LPS stimulation used to evaluate IL-10 and CCL2 responses was 0.05 ng/ml, with 0.5 ng/ml for IL-1β and TNFα responses. While the intensity of responses evoked was weaker in most individuals when sub-optimal concentrations were used, the conclusions from the data were identical (data not shown).Freshly isolated PBMC were suspended in complete medium  with 2.5×10As a safety precaution against infection, culture supernatants were UV irradiated for one hour to inactivate residual virus prior to ELISA analysis. Experiments (data not shown) demonstrated that this had no impact on the sensitivity or precision of the assays used to evaluate cytokine concentrations. Anti-cytokine capture and biotinylated detection antibodies were purchased from BD-Pharmingen , Endogen , Biolegend  or R&D Systems  and recombinant cytokine standards from BD Pharmingen, Endogen or Peprotech . They were used as previously described TLR4 allelic variants Asp299Gly (refSNP ID: rs4986790) and Thr399Ile (refSNP ID: rs4986791) were genotyped by quantitative PCR assay as previously described The Associations between Ag-driven responses by subjects were compared using 2-tailed Mann-Whitney tests (nonparametric data). All statistics were performed using GraphPad Prism version 3.02 for Windows .TLR4 Asp299Gly and Thr399Ile SNPs were in Hardy-Weinberg equilibrium, with the common homozygote genotypes co-segregating in 202 of 207 individuals (97.5%). This cosegregation is consistent with data demonstrating that these SNPs are located on the same allele in European populations TLR4 haplotypes formed by the Asp299Gly and Thr399Ile polymorphisms, because different haplotype structures are observed in different human populations TLR4 haplotypes.We genotyped 207 7–9 year old children from the SAGE cohort . TLR4 AsTLR4 Asp299Gly and Thr399Ile polymorphisms on LPS responsiveness remains highly controversial ex vivo are required. Using acute assays of primary PBMC cell cultures from 202 healthy children, we found that LPS stimulated secretion of four characteristic TLR4-driven pro- and anti-inflammatory cytokines, IL-10, IL-1β, TNFα and CCL2, was indistinguishable between subjects with the common homozygous haplotype (Asp/Asp+Thr/Thr) and those with the heterozygous haplotype (Asp/Gly+Thr/Ile) (TLR4 Asp299Gly polymorphism across this pediatric population (data not shown).The impact of the Thr/Ile) Figure 1n of 20–100, demonstrated an association between TLR4 polymorphisms and LPS hyporesponsiveness or clinical outcomes of airway dysfunction such as asthma and bronchiolitis Many functionally important genetic associations are better revealed in at-risk groups than in general population studies. Given that some prior studies, often with an TLR4 haplotypes.Among children carrying the homozygous haplotype (Asp/Asp+Thr/Thr), asthmatics produce higher levels of IL-10 (p<0.03) in response to stimulation with LPS relative to non-asthmatics Figure 2TLR4 haplotype  or the heterozygous haplotype (Asp/Gly+Thr/Ile)   and 6. ThesTLR4 Asp299Gly and Thr399Ile polymorphisms in shaping immune activation and subsequent clinical outcomes in humans remains highly controversial. Much of this controversy may stem from the diversity of experimental systems employed. Here, using freshly derived in vitro stimulated PBMC in short term culture, we determined whether children with the TLR4 heterozygous haplotype differ from the majority of the population, which carries the homozygous haplotype, in their capacity to mount LPS or RSV-driven immunoregulatory cytokine responses. This cohort of >200 individuals demonstrates that both LPS and RSV-driven responses are indistinguishable in both groups, regardless of haplotype. Further stratification of the cohort based on clinical phenotype  also revealed similar LPS- and RSV-driven responses among individuals of the two genotypes. A third independent approach, with cohort stratification based on a history of moderate to severe bronchiolitis/bronchitis during early infancy, also revealed that indistinguishable cytokine responses are elicited in these populations. Thus, the TLR4 Asp299Gly + Thr399Ile heterozygous haplotype has no detectable effect on the capacity of randomly selected 7–9 year old children to mount either RSV-stimulated or LPS-driven responses regardless of whether the children exhibited a clinical phenotype of current asthma or those with a history of infantile bronchiolitis.The role of the haplotype comprised of TLR4 Asp299Gly polymorphism on LPS responsiveness is likely affected by the disparity in the cosegregation frequency of the Asp299Gly and Thr399Ile alleles in various study populations. In the only large study completed to date other than our own, (n = 245 African subjects) LPS-induced cytokine production by PBMC was not influenced by TLR4 Asp299Gly genotype The diversity of findings evident in the literature from examination of the putative effects of the TLR4 haplotype (Asp/Asp+Thr/Thr) produced marginally higher levels of IL-10 than did carriers of the heterozygous haplotype (Asp/Gly+Thr/Ile) if their cells were stimulated at the higher concentration (0.5 ng/ml) of LPS (p<0.04) . None of the other cytokines tested in this population differed. While this borderline statistical significance may be a consequence of multiple comparisons, it does raise the possibility that under conditions of increased LPS exposure, TLR4 haplotype may influence LPS-driven IL-10 production in select children who exhibited severe pathological symptoms during RSV infection in infancy. Further experiments specifically addressing this hypothesis, in a larger cohort specifically constructed for this purpose, would be required to discriminate between these possibilities.Interestingly, among the 64 statistical comparisons made between the two haplotypes in this study which showed no statistically significant differences in production of a variety of cytokines, a single exception was observed — that amongst children with prior bronchiolitis. Children with prior bronchiolitis in infancy who were carriers of the common homozygous TLR4 Asp299Gly polymorphism on RSV specific immune responses from individuals with a prior history of bronchiolitis or airway dysfunction. Here, we used the intermediate phenotype of RSV-specific cytokine responses to attempt to bridge the gap between genotype and clinical phenotype. In contrast to the recent study by Tulic et al., which found associations between RSV-driven cytokine production and the Asp299Gly (or Thr399Ile) genotype Despite extensive evidence of the impact of gene by environment by time interactions on a wide variety of immune and clinical outcomes, studies to date, due to their experimental design, were unable to evaluate the impact of the TLR4 Asp299Gly polymorphism with clinical outcomes of RSV infection, our data show that the haplotype comprised of Asp299Gly and Thr399Ile does not detectably impact the human immune response to RSV, as measured by the intermediate phenotype of RSV-stimulated cytokine responses or that of a well validated TLR4 ligand, LPS. In this study we did not try to directly link TLR4 SNPs to clinical outcome, but rather stratified by clinical phenotype (asthma or bronchiolitis) to see if differences might be enriched in these groups. Cytokine production from PBMC in a ∼200 member cohort, or in subcohorts characterized by current asthma or a history of severe bronchiolitis in infancy consistently yield indistinguishable pro- and anti-inflammatory cytokine production. As both LPS and RSV can also interact with alternate pattern recognition receptors In summary, multiple pattern recognition receptors including various TLR and RLR continue to be linked to resistance to and resolution of RSV infection in humans and animal models Figure S1Anti-TLR4 blocking antibodies abrogate LPS-driven cytokine production. IL-6, IFNγ and IL-10 responses in response to acute activation with 0.5 ng/ml LPS plus/minus blocking antibodies are shown. Bars represent mean population responses +/− SEM.(0.29 MB TIF)Click here for additional data file.Figure S2LPS-driven IL-10 production is distinct between children with the homozygous (Asp/Asp+Thr/Thr) and heterozygous (Asp/Gly+Thr/Ile) TLR4 haplotypes in a sub-population that experienced infantile bronchiolitis. IL-10 responses in response to acute activation with 0.5 ng/ml LPS are shown with black bars represent median population responses, derived from individual children. * P = 0.03.(0.33 MB TIF)Click here for additional data file.


Reelin or double null for Vldlr and Apoer2, Purkinje cell clusters fail to disperse. Here we report that animals null for either Vldlr or Apoer2 individually, exhibit specific and parasagittally-restricted Purkinje cell ectopias. For example, in mice lacking Apoer2 function immunostaining reveals ectopic Purkinje cells that are largely restricted to the zebrin II-immunonegative population of the anterior vermis. In contrast, mice null for Vldlr have a much larger population of ectopic Purkinje cells that includes members from both the zebrin II-immunonegative and -immunopositive phenotypes. HSP25 immunoreactivity reveals that in Vldlr null animals a large portion of zebrin II-immunopositive ectopic cells are probably destined to become stripes in the central zone (lobules VI–VII). A small population of ectopic zebrin II-immunonegative Purkinje cells is also observed in animals heterozygous for both receptors (+/−: Vldlr+/−)Apoer2, but no ectopia is present in mice heterozygous for either receptor alone. These results indicate that Apoer2 and Vldlr coordinate the dispersal of distinct, but overlapping subsets of Purkinje cells in the developing cerebellum.The adult cerebellar cortex is comprised of reproducible arrays of transverse zones and parasagittal stripes of Purkinje cells. Adult stripes are created through the perinatal rostrocaudal dispersion of embryonic Purkinje cell clusters, triggered by signaling through the Reelin pathway. Reelin is secreted by neurons in the external granular layer and deep cerebellar nuclei and binds to two high affinity extracellular receptors on Purkinje cells-the Very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 (Apoer2). In mice null for either  Heterogeneous protein expression has been observed as early as E14.5 Purkinje cells in the adult cerebellum are grouped into discrete modules based on two arrays of orthogonal boundaries–transverse zones and parasagittal stripes Reelin show no Purkinje cell dispersal and are profoundly ataxic . Reelin binds to two extracellular receptors–the very low density lipoprotein receptor (Vldlr) and apolipoprotein E receptor 2 Apoer2 and Vldlr show Purkinje cell dispersal deficits nearly identical to reeler, indicating that both receptors are necessary for full Reelin signaling dab1 also have reeler-like Purkinje dispersal deficits Signaling by Reelin is crucial to the maturation of this complex topography. After their terminal division, Purkinje cells migrate out of the ventricular zone and accumulate in clusters in the cerebellar anlage. Reelin signaling subsequently triggers the perinatal dispersal of the Purkinje cells into the adult monolayer Apoer2 or Vldlr. Our goal in this study is to develop a map of Purkinje cell ectopia in each mutant animal by using markers that identify Purkinje cells or spatially restricted subsets of these neurons in the mature cerebellum. We have identified both unique and overlapping populations of ectopic Purkinje cells in different mutant combinations. Taken together these data suggest that each Reelin receptor directs the dispersal of distinct Purkinje cell subsets during development.To understand better the role of Reelin signaling in the regulation of Purkinje cell migration during development, cerebella were examined from heterozygous and homozygous null animals for either The Guide to the Care and Use of Experimental Animals as outlined by the Canadian Council for Animal Care. Vldlr and Apoer2 null mice were raised from stocks originally created through targeted deletion of each individual gene All procedures using animals conformed to UT Southwestern IACUC approved protocols as well as Mice were anesthetized using isoflurane and perfused transcardially with 5–10 ml of 0.9% ice-cold saline, followed by 40 ml of freshly prepared 4% paraformaldehyde in PBS . Following perfusion, the brains were removed from the skull and stored in 4% paraformaldehyde for at least 48h. For cryosectioned tissue, brains were first cryoprotected through an ascending series of sucrose solutions until they sank . The tissue was embedded by freezing in OCT  and 40 µm sections cut in either the sagittal or transverse plane. Tissue sections were stored in Millonig's solution (PBS+0.05% sodium azide) at 4°C until required.Section immunohistochemistry was performed free floating as previously described 45Ca-binding spot of calbindin D-28k  in a two-dimensional gel of mouse brain homogenate (manufacturer's information)) and used here diluted 1:5000 in blocking serum. Rabbit anti-calbindin was raised against full-length recombinant CaBP . Both anti-calbindin antibodies yielded Purkinje cell specific staining identical to that previously reported ApteronotusA mouse monoclonal antibody against CaBP was obtained from Swant ; raised against chicken CaBP and specifically binds to the 2O2 in PBS. Tissue immunoreacted with anti-PLCß4 antibodies was post-fixed in methanol (MeOH) at this stage by sequential 15 min incubations in 50% MeOH/PBS, 100% MeOH, and 50% MeOH/PBS . Next, the tissue was rinsed in PBS and blocked for 1h at room temperature in PBS containing 10% normal goat serum  and 0.1% Triton X-100 . Sections were incubated overnight at 4°C with gentle agitation in primary antibodies diluted in blocking solution, and washed the following morning. Following the primary incubation, sections were washed and incubated with an appropriate biotinylated secondary antibody . Antibody binding sites were revealed by using the Vectastain ABC kit  with diaminobenzidine as the chromagen. Tissue sections were mounted on slides and allowed to dry for at least two hours, dehydrated through an ascending alcohol series, cleared in Histoclear , and coverslipped with Entellan mounting medium .For peroxidase immunocytochemistry, cerebellar sections were washed 2 × 5 minutes in PBS followed by a 30 minute incubation in 0.1% HFor immunofluorescence, the tissue was blocked as above and incubated overnight with primary antibodies . Following incubation, the sections were washed 3×10 min in blocking solution and incubated with a fluorescently labeled secondary antibody diluted in blocking solution for 2 h at room temperature. All fluorescent secondary antibodies were obtained from Molecular Probes  as Alexa fluorophore conjugates . Sections were washed 3×10 minutes in PBS, mounted on slides and allowed to dry for 2 h at room temperature. The tissue was rehydrated for 5 minutes in PBS and mounted in Aqua Polymount .Whole mount immunocytochemistry was performed as previously described For conventional histological examination cryosections were mounted on slides and allowed to adhere for at least two hours at room temperature. Tissue was first fixed in 10% neutral buffered formalin  for five minutes, dehydrated in an ascending series of alcohol and xylene, and rehydrated back to distilled water. Sections were stained for 30 s in 1% w/v cresyl violet acetate in distilled water, then rinsed in several changes of distilled water until clear. Sections were re-dehydrated through an ascending alcohol series. At 100% ethanol, they were incubated 2×5 min in 20 ml ethanol containing 5 drops of 100% acetic acid, before completing the dehydration in xylene. Finally, sections were coverslipped in Entellan mounting medium .Lobule lengths were measured from photographs of three randomly selected vermal mid-sagittal sections from animals of each genotype (n = 3) by using Zeiss Axiovision 3.1 . The midline vermis was defined as lying between the two medial cerebellar nuclei. Boundaries between lobules were defined as the deepest part of the sulcus separating each lobule and the length of each lobule was defined as the distance between the deepest point of each sulcus. Lobule lengths were measured at the level of the Purkinje cell layer for both wild type and mutants. Lobule lengths, as well as overall cerebellar length, were compared to wild type by ANOVA and a difference of p<0.05 was considered significant.Apoer2 and Vldlr is necessary to block all perinatal Purkinje cell dispersal and recapitulate the reeler phenotype Apoer2 or Vldlr null cerebella were stained with cresyl violet to assess lobular and cytoarchitectural abnormalities . In general, anti-zebrin II immunostaining of the Apoer2 null cerebellum appears normal, suggesting that few zebrin II-immunopositive Purkinje cells are ectopic  of the ectopic Purkinje cells in the ebrin II , phosphoebrin II  and heatebrin II . Zebrin ebrin II ; Fig. 2B ectopic . Occasio ectopic . The larApoer2 null cerebellum in order to better understand the organization of transverse zones in these mutants. HSP25 immunoreactivity in the vermis marks a subpopulation of zebrin II-immunopositive Purkinje cells in both the NZ and CZ Apoer2 null cerebellum resembles wild type and therefore appears unaffected . However, while the rostral limit of HSP25 expression in the wild type cerebellum normally ends in the anterior face of lobule VI within the primary fissure Apoer2 null CZ is the caudal face of lobule Via (data not shown). This caudal displacement of the limit of HSP25 expression in the −/−Apoer2 CZ suggests that cerebellar lobulation has shifted with respect to the rostral boundary of the CZ.Finally we conducted HSP25 immunolabeling using sagittal sections from the Apoer2 null cerebellum are zebrin II-immunonegative. To confirm this hypothesis, sections serial to those immunostained with calbindin above were immunostained by using antibodies against PLCß4, a positive antigenic marker of the zebrin II-immunonegative Purkinje cells  contains few HSP25-immunopositive Purkinje cells  express zebrin II in a pattern that is reminiscent of wild type, with broad zebrin II-immunopositive stripes separated by narrow stripes of zebrin II-immunonegative Purkinje cells .More caudally, Purkinje cells in the je cells . FinallyVldlr null transverse cryosections and cerebellar whole mounts were immunolabeled with anti-PLCß4 antibodies  and anti-KLC3, a cerebellar marker specific for cerebellar nuclear neurons Apoer2 and Vldlr null cerebella, Purkinje cells and cerebellar nuclear neurons form distinct, non-overlapping clusters.Purkinje cells and cerebellar nuclear neurons arise during development from the neuroepithelium of the fourth ventricle and rhombic lip, respectively Apoer2+/− and Vldlr+/−) have any Purkinje cell dispersal defects Apoer2+/−:: Vldlr+/−) reproducibly exhibited a subtle Purkinje cell ectopia , and mice lacking functional copies of both the Apoer2 and Vldlr genes meander tail and rostral cerebellar malformation mice each have ectopic Purkinje cells that are derived from the AZ population weaver cerebellum also has a small population of ectopic Purkinje cells, in this case derived from discrete parasagittal stripes in the CZ cerebellar deficient folia (cdf) mouse whereas the zebrin II-immunopositive Purkinje cells disperse normally Mutations in the Reelin signaling pathway cause a phenotype in which most Purkinje cells fail to disperse to the cerebellar cortex. Mutant mice with this phenotype include In the present report we show that a significant regulation of Purkinje neuron dispersal occurs at the level of the Reelin receptor.Recent work examining neuron dispersal in the cerebral cortex similarly indicates that Apoer2 and Vldlr also play diverging roles in regulating neuronal migration during cortical development Apoer2 and Vldlr null cerebella. The analysis depends on the assumption that Purkinje cells reliably express their normal phenotypic antigenic markers when in ectopic locations. Considerable evidence supports this hypothesis. First, studies of cerebellar development have shown that Purkinje cells are already committed to their adult phenotype at around their time of birth in the 4th ventricle  and that subsequent experimental manipulations cannot alter this weaver–40; zebrin II-immunonegative/PLCß4-immunopositive Purkinje cells from the AZ in cdf–41). Thirdly, in complete dispersal mutants, ectopic Purkinje cells show a mediolateral striped patterning consistent with their normal adult phenotypes despite the fact that they fail to disperse to the cerebellar cortex . It has been previously reported that Reelin signaling may affect glial morphology Apoer2 or Vldlr null cerebella.Our evidence demonstrates that Purkinje cell ectopia is restricted to parasagittal and/or transverse subsets of Purkinje cells in both Reelin receptor mutants takes two forms-in some cases, the failure to disperse is complete and the Purkinje cells form reproducible, tightly packed clusters in the cerebellar core . While the expression pattern for each receptor in the adult cerebellum is consistent with the ectopia that we observed in the mutants, it will be interesting to explore if this restricted expression pattern is present in Purkinje cells during development while dispersal is occurring. Interestingly, humans homozygous for a Vldlr deletion show profound Purkinje cell migration defects Apoer2 null .Support for a model where Apoer2 and Vldlr are selectively expressed in subsets of Purkinje cells comes in part from recent reports indicating that these receptors are differentially expressed in various neural populations including cortical neurons and Purkinje cells Apoer2 null is the most straightforward: the tight Purkinje cell ectopic clusters are all zebrin II-immunonegative/PLCß4-immunopositive  and zebrin II–immunonegative  Purkinje cells. Moreover, there should be no overlap between the Purkinje cells that fail to disperse from clusters in the two nulls, but this is not always the case. For example, zebrin II-immunonegative/PLCß4-immunopositive ectopic Purkinje cells form a tight cluster in the anterior cerebellum of the Vldlr null cerebellum (Apoer2 null (Apoer2::Vldlr heterozygote cerebella (Vldlr>Apoer2>Apoer2::Vldlr double heterozygote). The simplest explanation for this observation is that zebrin II-immunonegative cells comprise two or three subgroups, each of which expresses a different receptor combination . However, if indeed the Purkinje cells ectopic in Apoer2 nulls are a subset of those ectopic in Vldlr then this observation is not easy explained simply by receptor distributions. One possibility is that co-ectopia arises non cell-autonomously. For example, wild type <-> scrambler chimeras reveal a community effect wherein Purkinje cells with defective Reelin signaling negatively influence the dispersal of wild type cells No simple correlation between adult Purkinje cell antigenic phenotype and the ectopia observed in the three mutants is apparent. The ive e.g. . The samrozygote . Howeverve e.g.,  and zebrve e.g.,  Purkinjerebellum . Ectopicer2 null  and doubIt will be interesting to determine the interplay between Apoer2 and Vldlr receptors and its role in neuronal migration. It is unclear if each receptor regulates the dispersal of unique populations of cells, if they have a synergistic relationship, or some combination of the two, especially as Hack and colleagues tottering mouse express a mutant alpha 1a calcium channel but only the zebrin II-immunonegative population dies lurcher (Lc/+), an ataxic mouse strain with gross cerebellar deficits due to a gain-of-function point mutation in the orphan delta 2 glutamate receptor gene (GluRδ2/Grid2: 63–65). Possible roles for Apoer2 in apoptotic and excitotoxic neuronal death are discussed in weaver mouse, in which all Purkinje cells express a mutated version of the inwardly rectifying K+ channel girk2Catna2 gene, encoding for alpha-N-catenin, is truncated in the cdf null mutant Catna2 is expressed in all Purkinje cells only those that are zebrin II-immunonegative are ectopic Selective ectopia can also be explained by postulating that the distribution of Apoer2 and Vldlr in Purkinje cells is homogeneous and selective ectopia is due to the differential sensitivity to the mutation of Purkinje cell subsets. There are many examples in which an entire population of Purkinje cells expresses a mutant protein but only a subpopulation is adversely affected. For example, in mouse models of Niemann-Pick type C disease, all Purkinje cells express the mutant NPC1 protein but zebrin II-immunonegative Purkinje cells are far more susceptible to its effects Apoer2 and Vldlr null cerebella (Finally, we have observed a significant reduction compared to wild type in the length of several individual cerebellar lobules as well as in the total lengths of both erebella . Such re