Cigarette smoke disrupts the protective barrier established by the airway epithelium through direct damage to the epithelial cells, leading to cell death. Since the morphology of the airway epithelium of smokers does not typically demonstrate necrosis, the most likely mechanism for epithelial cell death in response to cigarette smoke is apoptosis. We hypothesized that cigarette smoke directly up-regulates expression of apoptotic genes, which could play a role in airway epithelial apoptosis.In vitro studies using human bronchial cells exposed to cigarette smoke extract (CSE) and an adenovirus vector encoding the pirin cDNA (AdPirin) were performed to test the direct effect of cigarette smoke on pirin expression and the effect of pirin expression on apoptosis.Microarray analysis of airway epithelium obtained by bronchoscopy on matched cohorts of 13 phenotypically normal smokers and 9 non-smokers was used to identify specific genes modulated by smoking that were associated with apoptosis. Among the up-regulated apoptotic genes was pirin , an iron-binding nuclear protein and transcription cofactor. Quantitative TaqMan RT-PCR confirmed a 2-fold increase in pirin expression in the airway epithelium of smokers compared to non-smokers (p < 0.02). CSE applied to primary human bronchial epithelial cell cultures demonstrated that pirin mRNA levels increase in a time-and concentration-dependent manner .Overexpression of pirin, using the vector AdPirin, in human bronchial epithelial cells was associated with an increase in the number of apoptotic cells assessed by both TUNEL assay  and ELISA for cytoplasmic nucleosomes  compared to control adenovirus vector.These observations suggest that up-regulation of pirin may represent one mechanism by which cigarette smoke induces apoptosis in the airway epithelium, an observation that has implications for the pathogenesis of cigarette smoke-induced diseases. Although the airway epithelium has several defense mechanisms that respond to the stress imposed by cigarette smoke, in some individuals these defenses are exaggerated, resulting in chronic inflammation, and eventually, chronic bronchitis -5. One oper se is more important than dysfunction of the junctions between epithelial cells .AdPirin-induced apoptosis was assessed in the human airway epithelial BEAS-2B cell line . BEAS-2BTwo assays were used to assess apoptosis: TdT-mediated dUTP nick end labeling (TUNEL) assay and cytoplasmic nucleosome ELISA. For the TUNEL assay, cells were fixed to the coverslips using 4% paraformaldehyde and then permeabilized with 0.2% Triton X-100 in PBS. Cells were equilibrated with equilibration buffer, nucleotide mix, and rTdT enzyme  for 60 min and then washed. DAPI nuclear counterstain was applied before cells were mounted onto slides and evaluated under fluorescent microscope. The percentage of apoptotic cells per 10× field were manually counted in 10 fields per slide. For the cytoplasmic nucleosome ELISA assay , BEAS-2B cells were lysed with lysis buffer, centrifuged for 10 min at 200 × g to pellet nuclei. The supernatant (20 μl) was added to the immunoreagent containing anti-histone biotin and anti-DNA horseradish peroxidase (HRP). Sample wells were placed on shaker at 300 rpm for 2 hr, 23°C. 2, 2azino-di[3-ethylbenzthiazolin-sulfonate] (ABTS) solution was added and photometric analysis was measured at 405 nm, subtracted from background 490 nm. For each sample, the fluorescent value was normalized to the internal negative control of the experiment to generate an apoptotic index, which reflects the fold change in the number of apoptotic cells for experimental condition compared to control.32P-labeled pirin specific DNA probe was synthesized using strip EZ labeling kit  from a pirin cDNA template amplified from human genomic DNA. RNA electrophoresis was performed in 1% agarose gel followed by transfer to nitrocellulose membrane and UV crosslinking. The membrane was then hybridized with DNA probe and exposed to X ray film for 1 hr.A in vitro studies, comparisons between RNA expression levels for pirin and percentage of apoptotic cells were made using the Student's two-tailed t-test.The microarray data was analyzed using the GeneSpring software . Normalization was carried out sequentially: per microarray sample  and then per gene . Data from probe sets representing genes that failed the Affymetrix detection criteria  in all 44 microarrays were eliminated from further analysis. The p value for each gene was calculated comparing the non-smokers with smokers using the Welch t-test with a Benjamini-Hochberg correction for false discovery rate. For the vs non-smokers. Of the 4,512 genes that were expressed, there were a total of 85 probesets that were significantly (p < 0.05) up-regulated and 13 probesets down-regulated in smokers compared to non-smokers. The 98 probesets were functionally annotated by manual review of public databases  into categories that described their cellular processes. Of these, 7 of the up-regulated genes were identified to be associated with apoptosis, including pirin, retinoic acid receptor responder 2, prostrate differentiation factor, insulin-like growth factor binding protein 5, bone morphogenic protein 7, carcinoembryonic antigen-related cell adhesion molecule 6, and S100 calcium-binding protein A10  a significant up-regulation of pirin in the airway epithelium in the smokers , 2.1-fold increase for cells exposed to 103 AdPirin (p < 0.01) and 7.9 fold increase for cells exposed to 50% CSE  and 133.7-fold (p < 0.01) increase in pirin RNA expression level, respectively  and evaluated for apoptosis and pirin RNA level. This assay, which uses an increase in fluorescent signal compared to the naive negative control to generate an apoptotic index, demonstrated a 19.3-fold increase for cells exposed to 10y Figure .Cigarette smoking is the major environmental exposure that leads to the pathogenesis of COPD -5. The ovs non-smokers for the up-regulation of candidate proteins using microarray technology. Affymetrix gene chip assessment of airway epithelial cells from phenotypically normal ~20 pack-yr smokers compared to normal non-smokers demonstrated the up-regulation of several proteins that have been linked to apoptosis in the airway epithelium of smokers compared to non-smokers. Among the up-regulated proteins that were found to be associated with apoptosis was pirin, a nuclear transcription cofactor that is part of the cupin superfamily [Apoptosis of airway epithelial cells is relevant to the pathogenesis of chronic bronchitis because disruption of epithelial integrity in the central airways appears to be an early event in response to cigarette smoke -8,32. Baerfamily ,33,34.in vivo observation, an in vitro model was designed to test the effect of cigarette smoke on pirin expression in epithelial cell culture. Using primary cultures of human bronchial epithelial cells, pirin expression was shown to respond to increasing concentrations of cigarette smoke extract relatively acutely, within 24 to 48 hr. A similar increase in pirin RNA expression level was observed in the BEAS-2B cell line when exposed to cigarette smoke extract. The potential for pirin to induce apoptosis in bronchial epithelial cells was assessed by using AdPirin, an adenovirus gene transfer vector expressing the human pirin cDNA, to over-express pirin in cultures of transformed airway epithelial cells. The data demonstrate that pirin over-expression results in significant epithelial cell apoptosis, a possible mechanism that leads to a breakdown of the epithelium in response to cigarette smoke. Although the magnitude of pirin over-expression is greater in the in vitro AdPirin experiments than that observed in vivo, it is important to recognize that the in vivo upregulation of pirin expression occurs after chronic smoking (~20 pack-yr), and this chronic, low level up-regulation over an extended period of time is impossible to emulate in vitro. It is therefore not surprising, that in order to demonstrate an in vitro effect on apoptosis over a much shorter time course (2–3 days), a higher expression of the gene was required. Interestingly, in the experiments where bronchial epithelial cells (BEAS-2B) were exposed to cigarette smoke extract, there was a similar 2 fold increase in pirin expression to 10% CSE and a significant increase in apoptosis, thus paralleling the in vivo observation.TaqMan real-time RT-PCR independently confirmed the microarray observed changes in pirin mRNA expression. To validate this th to 14th generation bronchi), inflammatory exudates can accumulate in the lumen of the bronchi resulting in airflow obstruction [One of the main functions of the airway epithelium is to provide a physical barrier to protect the underlying tissue from the harmful effects of cigarette smoke ,2,4,5. Ttruction .99mTC-DTPA lung clearance in human subjects, has been shown to be increased in chronic smokers compared to non-smokers [in vitro cellular studies have shown that alveolar epithelial cell viability decreases after exposure to cigarette smoke in a time and concentration dependent manner [Epithelial permeability, as measured by -smokers ,32. Stud-smokers ,11. This-smokers ,11. Const manner .in vivo studies of the airway in humans.The mechanism of cell death for the epithelium in response to cigarette smoke may involve some necrosis, but apoptosis is likely the dominant mechanism . Mouse min vitro studies have shown that apoptosis can occur in response to cigarette smoke extract (CSE) in a variety of cell types, including airway epithelial cells, alveolar macrophages, endothelial cells, alveolar epithelial cells, and fibroblasts [in vitro studies into consideration, one potential explanation is that at low concentrations, CSE causes DNA damage that is reparable until a threshhold concentration is reached, after which the cell initiates apoptosis. Very high levels of CSE likely cause overt necrosis.Several roblasts -15. In croblasts  found throblasts . Liu et roblasts  reportedroblasts  et al usPirin was first identified by Wendler et al , where i2+ ion in the highly conserved N-terminal region [Pirin was later found to bind to the oncoprotein B-cell lymphoma 3-encoded (Bcl-3), a member of the IκB family. Bcl-3 il region . This mePirin stabilizes the formation of quaternary complexes between Bcl-3, NF-κB, and its DNA target. While it is not clear exactly how pirin influences the interaction between Bcl-3 and NF-κB, based on our results and others, it seems possible that pirin directs NF-κB DNA binding towards a pro-apoptotic response. Elucidating this mechanism will require further investigation to understanding the signaling that results from pirin expression. This may not be possible through gene expression studies alone, since several apoptotic pathways are induced by mechanisms other than up-regulation of mRNA levels of pro-apoptotic genes. This might explain why there was no observed upregulation of traditional apoptotic or inflammatory pathway genes, e.g., caspases and Bcl-2, in our microarray data obtained from human airway epithelium. The lack of up-regulation of pro-inflammatory genes in the airway epithelium of smokers is also supported by the transcriptome analysis of Spira et al . One posle-pirin, an ortholog to human pirin, mRNA levels are dramatically increased. In the cyanobacterium Synechocystis, pirin orthologs are highly induced under conditions of severe salt stress [Evidence for pirin involvement in apoptosis comes from experiments in tomatoes . When tot stress .We originally noted pirin to be up-regulated in microarrays of phenotypically normal individuals that smoke , a findiOur current study suggests that pirin expression in the airway epithelium is in response to the acute oxidative stress imposed by cigarette smoke. Our study also suggests that pirin over-expression in response to cigarette smoke may play a role in epithelial cell apoptosis, and one possible mechanism is by interacting with NF-κB to induce the expression of pro-apoptotic genes, in cells injured by cigarette smoke . Theoret


Plasmodium falciparum parasites carrying mutations associated with resistance to each of these drugs were present in 2001 but did not cause a significant loss of therapeutic efficacy among children receiving the combination CQ/SP. We measured their effect on parasite transmission to Anopheles gambiae mosquitoes.In the Gambia, chloroquine (CQ) plus sulphadoxine-pyrimethamine (SP) is the first-line antimalarial treatment. We conducted a single-blind, randomised, controlled trial with follow-up over 28 d. Mosquito feeding experiments were carried out 7, 10, or 14 d after treatment.The study took place in the town of Farafenni and surrounding villages in the Gambia.P. falciparum malaria.Participants were 500 children aged 6 mo to 10 y with uncomplicated Children were randomised to receive CQ, SP, or CQ/SP.A. gambiae mosquitoes with blood from 70 gametocyte-positive patients. Mutations at seven loci in four genes associated with drug resistance were measured pre- and posttreatment and in the midguts of infected mosquitoes.Outcomes related to transmission were determined, including posttreatment gametocyte prevalence and density. Infectiousness was assessed by membrane-feeding pfcrt-76T, pfmdr1-86Y, pfdhfr-59R, and pfdhps-437G) was associated with significantly higher oocyst burdens after treatment with the combination CQ/SP.After SP treatment, the infectiousness of gametocytes was delayed, compared to the other two treatment groups, despite comparable gametocyte densities. Among bloodmeal gametocytes and the midguts of infected mosquitoes, the presence of the four-locus multidrug-resistant haplotype TYRG (consisting of mutations Parasites with a multidrug-resistant genotype had a substantial transmission advantage after CQ/SP treatment but did not have a significant impact on in vivo efficacy of this drug combination. Protocols that include measuring transmission endpoints as well as therapeutic outcomes may be a useful strategy when monitoring the evolution of drug resistance in malaria parasites in vivo. Background: In an accompanying paper published in PLoS Clinical Trials [l Trials , the invWhat the trial shows: In this trial it was found that children treated with chloroquine and sulfadoxine-pyrimethamine were no more or less likely to carry sexual forms of the malaria parasite compared to children treated with chloroquine, over 28 days of follow-up. Transmission of the malaria parasites back to mosquitoes also did not differ significantly between children treated with chloroquine and sulfadoxine-pyrimethamine versus chloroquine alone. Parasites bearing the TYRG genotype, associated with drug resistance, were more likely to be transmitted to mosquitoes from children treated with chloroquine/sulfadoxine-pyrimethamine than from chloroquine treated children.Strengths and limitations: The addition of data about parasite transmission within the setting of a randomized trial is valuable and can provide information about the evolution of drug resistance. However, the results may not completely represent the current situation, as the trial was originally conducted in 2001 and levels of resistance may now be higher in the Gambia.Contribution to the evidence: This follow-up study to a randomized trial provides additional evidence that treatment with chloroquine and sulfadoxine-pyrimethamine, as compared to chloroquine, does not reduce the extent to which malaria parasites can produce sexual forms and transmit them back to mosquitoes. The finding that parasites bearing the TYRG genotype are more likely than other parasites to be transmitted back to mosquitoes following chloroquine and sulfadoxine-pyrimethamine treatment is also novel. Plasmodium falciparum in Africa, it is likely that parasites harbouring mutations conferring resistance to both CQ and SP circulate in many areas. Policy changes towards the implementation of artemisinin-containing combination therapies (ACTs) are now occurring in many countries , 3.68 [p = 0.001], and 31.2 [p < 0.001], respectively). Gametocyte density in the CQ/SP-treated group was higher than in the CQ group at day 3 , but there was no significant difference between these two groups at day 7  or day 14 . At day 28, gametocyte density in the CQ/SP-treated group was significantly lower than in the CQ group .A pairwise comparison of mean gametocyte density between treatment groups at each time point was carried out, including all evaluable children who were not gametocyte carriers at presentation. Gametocyte density in the SP-treated group was significantly higher than in the CQ/SP group at days 7, 14, and 28 , despite high gametocyte densities in the latter group  were contributing significantly to parasitological failure after treatment with SP. Parasites with the TYRG genotype were also present in some cases after treatment with CQ/SP and thus likely to be selected with widespread combination drug pressure. We therefore tested for associations between either posttreatment gametocyte emergence or transmission success and resistance-associated alleles at seven loci alone and in combination.We report elsewhere on the prevalence of resistance-associated parasite genotypes in the study population, and the contribution of such parasites to treatment failure . It was pfcrt-76T and pfmdr1-86Y, but not for pfmdr1-184F. Similarly, the difference was significant when analysing the pyrimethamine-resistant mutations in the pfdhfr gene  both individually and together as presence of the “triple mutant.” Pfdhps-437G was the only sulphadoxine-resistant mutation detected in this population, and was found at a higher prevalence in gametocyte donors at day 0, but this difference was not significant. When grouping mutations into the multidrug resistance-associated genotype TYRG as defined in Dunyo et al. [–17.0; two-sided Fisher exact, p = 0.11), although this was also not statistically significant.Carriage of genetically resistant parasites at presentation was associated with the presence of gametocytes during the follow-up period, as shown in o et al.  we foundpfcrt-76K to T after CQ treatment [We have previously presented evidence of within-host directional selection from reatment . Howeverpfdhfr and pfdhps-437 sites examined. In one further feed (MF59), six of seven infected midguts carried pfmdr1-184F and one had pfmdr1-184Y. In all other cases where multiple oocyst-positive midguts arose from a single feed, they contained identical parasite genotypes (In two feeds (MF27 and MF40), mixed infections were detected in some midguts, but not others, at the enotypes .pfcrt-76T, pfmdr1-86Y, and pfmdr1-184F were detected pre- and posttreatment and in all 11 midguts. The pfdhfr genotype was mixed with both wild-type and resistance-associated alleles detected at positions 51, 59, and 108 both pre- and posttreatment. However, none of these wild-type alleles were detected in the resulting 11 midguts, all of which harboured pure pfdhfr triple mutants .The most infectious gametocyte sample (MF68) originated from a CQ/SP-treated patient, producing 13 infected midguts with a mean of 42 oocysts on each . Eleven of the 13 midguts were PCR-positive for all four resistance-associated genes. CQR alleles N = 644 dissected mosquitoes: mean ratio, 4.14; 95% CI, 0.722–23.8; p = 0.111; for CQ/SP, N = 415: mean ratio, 5.53; 95% CI, 0.742–41.2; p = 0.095). In contrast, in the CQ group, only one donor harboured TYRG parasites at day 0, and there was no transmission from this child's gametocytes. Parasites carrying CQ resistance markers at pfcrt and pfmdr1 without accompanying antifolate resistance mutations in pfdhfr and pfdhps were highly transmissible in the CQ group . This is consistent with previous findings [Very few changes in parasite genotype at resistance-associated loci were observed from pretreatment sampling to identification of markers in infected mosquito midguts. Therefore, we used day 0 data to look for associations between multidrug-resistant genotypes and mean oocyst count in mosquitoes membrane-fed on posttreatment emergent gametocytes . The presence of parasites with the four-locus multidrug-resistant genotype TYRG at day 0 was associated with higher oocyst numbers in SP and CQ/SP groups, but this was not statistically significant in either group . However, after CQ/SP treatment, parasites carrying TYRG resulted in significantly greater transmission success compared to other genotypes in this treatment group.The presence of TYRG in gametocyte samples and in oocyst DNA was associated with a significantly lower oocyst burden from CQ-treated individuals. In the SP-treated group, TYRG gametocytes also led to lower oocyst burdens, with borderline significance  was also more common among gametocyte carriers, and among children donating gametocytes for feeding experiments, than among noncarriers, although this was not statistically significant. In the accompanying paper, the TYRG genotype was found to be weakly associated with parasitological failure after SP treatment, but not after CQ/SP treatment [Resistance-associated alleles at five of the seven loci examined were more common among gametocyte carriers than among noncarriers in a randomly selected group of pretreatment isolates, as was the nd 108-N . The mulreatment .P. falciparum oocysts present in infected mosquito midguts was associated with significantly higher oocyst burdens in the CQ/SP combination group. This was independent of feed type or the presence of gametocytes prior to treatment, and was much stronger than the association between this genotype and treatment failure [The presence of the TYRG multilocus genotype among either gametocytes membrane-fed to mosquitoes, or among  failure . TYRG ca failure .pfcrt-76T and pfdhfr-108N, were at a high starting prevalence and so wild-type alleles were rare.Our data did not provide clear evidence of within-host selection for the TYRG genotype, as we have previously observed in CQ-treated patients ,13. Thispfdhfr and pfdhps mutation prevalences remained stable  . However, CQ monotherapy remained first line treatment at this time and CQ/SP was not being commonly used. The apparent anomaly between the widespread prevalence of the pfdhfr triple mutant in Africa and its weak association with risk of treatment failure [Taken together, our analyses of therapeutic and transmission endpoints of this clinical trial demonstrate that multidrug-resistant parasites enjoyed a survival advantage in terms of their likelihood of transmission to mosquitoes, but that the particular genotypes encountered in the Gambia did not threaten the therapeutic efficacy of CQ/SP ,15. Pret failure  may simpP. falciparum are prevalent in West Africa and that transmission of these genotypes is favoured when CQ and SP are combined for treatment of uncomplicated malaria in children. Thus, evolution of resistance to this combination will continue despite any reduction in its therapeutic efficacy, so far. These findings demonstrate the importance of measuring transmission endpoints in studies of the evolution of resistance to antimalarial chemotherapy.The work presented in this paper extends our previous studies of CQ-treated, SP-treated, and CQ/SP-treated patients in this study area –11,13. TCONSORT ChecklistClick here for additional data file.(50 KB DOC)Trial ProtocolClick here for additional data file.(88 KB DOC)Table S1(343 KB DOC)Click here for additional data file.


We compared the accuracy of haplotype inferences at a 6 Mb region on chromosome 7 where significant linkage between a brain oscillation phenotype and a cholinergic muscarinic receptor gene was previously reported. Individual haplotype assignments and haplotype frequencies were estimated using 5, 10, and 14 consecutive Illumina single-nucleotide polymorphisms (SNPs) within the 1-LOD unit support interval of the chromosome 7 linkage peak. Initially, haplotypes were constructed incorporating phase information provided by relatives using the pedigree analysis package MERLIN. Population-based haplotypes were inferred using the haplotype estimation software HAPLO.STATS and PHASE, using unrelated individuals.The 14 SNPs within this region exhibited markedly low linkage disequilibrium, and the average D' estimate between SNPs was 0.18 (range: 0.01–0.97). In comparison to the family-based haplotypes calculated in MERLIN, the computational inferences of individual haplotype assignments were most accurate when considering 5 consecutive SNPs, but decayed dramatically when considering 10 or 14 SNPs in both PHASE and HAPLO.STATS. When comparing the two haplotype inference methods, both PHASE and HAPLO.STATS performed poorly. These analyses underscore the difficulties of haplotype estimation in the presence of low linkage disequilibrium and stress the importance of careful consideration of confidence measures when using estimated haplotype frequencies and individual assignments in biomedical research. The advent of inexpensive high-throughput single-nucleotide polymorphism (SNP) genotyping ,2 and veThe International HapMap Project  was initIn this study we assessed the accuracy of computational inferences of individual haplotypes and haplotype frequencies at a region on chromosome 7 where Jones et al. [COGA began in 1989 to elucidate genetic mechanisms that influence susceptibility to alcohol abuse, alcohol dependence, and related phenotypes [We selected the 14 SNPS from the cleaned Illumina SNP dataset  that were within the 1-LOD unit support interval (6 Mb) for the quantitative trait locus (QTL) on chromosome 7 initially reported by Jones et al. [n = 21 individuals) also had members removed to comply with MERLIN pedigree size restrictions.Individual haplotypes were first determined using all related individuals with the pedigree analysis package MERLIN and the -best option, which provides haplotypes corresponding to the most likely pattern of gene flow within a pedigree . Four faTo examine population level statistics, we selected 1 individual with complete SNP data for the 14 SNPs from each of the 91 families. We determined both the amount of linkage disequilibrium (LD) between SNPs and Hardy-Weinberg equilibrium using the computer program HAPLOVIEW . We usedIF and IH, where IF is a metric of agreement between family-based and population-based haplotypes and is given by . pek and ptk represent the population-based and family-based frequencies for the kth haplotype and h is the number of possible haplotypes. IH compares the number of population-based haplotypes to the number of actual haplotypes and ranges from 0 to 1  and is defined as IH = 2(mtrue - mmissed) / mtrue + mest, where mtrue represents the number of actual haplotypes, mest is the number of population-based haplotypes, and mmissed corresponds to the number of family-based haplotypes that were not inferred. To assess individual haplotype inference we also calculated an overall error rate, defined as the proportion of individuals whose population-based haplotype differed from the true haplotype.Excoffier and Slatkin  proposed2 estimates between the SNPs were 0.18 (range: 0.01–0.97) and 0.04 (range: 0–0.64), respectively. Eleven individuals (12%) had haplotypes with at least 1 SNP for which phase could not be determined using MERLIN and were excluded from all subsequent analyses.All 14 SNPs were in Hardy-Weinberg equilibrium, with minor allele frequencies ranging from 0.269–0.473. The SNPs exhibited markedly low LD, as the average D' . Thus, more investigators will face the challenge of creating haplotypes from large SNP sets. Our results suggest that haplotypes estimated from population-based data should be interpreted with caution. Even though many features of haplotype inference are found to be consistent from one dataset to the next, it is not yet clear how general these tendencies will prove to be in the context of very low LD , and future research is warranted.While both programs had high levels of inaccuracy, statistical measures of confidence, such as a posterior probability estimate for each individual haplotype, are provided. For example, in the 5-SNP haplotypes estimated in PHASE, the incorrectly specified haplotypes had a mean posterior probability estimate of 0.52 (range: 0.34–0.66). Clearly, such uncertainty in haplotype assignment should be incorporated into subsequent statistical analyses incorporating these haplotypes. Unfortunately, such practices do not routinely appear in the literature.Both haplotype estimation packages performed similarly and poorly when 5, 10, and 14 SNP sets were considered, although PHASE slightly outperformed HAPLO.STATS. Thus, our findings underscore the difficulties of computational haplotype inference under less-than-ideal conditions (linkage region with low LD) and stress the importance of careful consideration of confidence measures when employing estimated haplotypes in biomedical research. Further, the definition of haplotype blocks should be considered carefully on a case-by-case basis, with careful attention to the number of underlying sites and the pattern of LD.COGA: Collaborative Study of the Genetics of AlcoholismGAW14: Genetic Analysis Workshop 14LD: Linkage disequilibriumQTL: Quantitative trait locusSNP: Single-nucleotide polymorphismCLA and LJM performed statistical analyses, interpreted results, and collaborated to write the manuscript; JTW and KEN aided in interpretation of results and collaborated to write the manuscript. All authors read and approved the final manuscript.


Percentages of neutrophils and eosinophils increased with greater intensity of WTC exposure . MMP-9 levels positively correlated to neutrophil counts . Particles were larger and more irregularly shaped in FDNY-FFs  than in TA-FFs . PSD was similar to that of WTC dust samples. In conclusion, IS from highly exposed FDNY-FFs demonstrated inflammation, PSD, and particle composition that was different from nonexposed controls and consistent with WTC dust exposure.New York City Firefighters (FDNY-FFs) were exposed to particulate matter and combustion/pyrolysis products during and after the World Trade Center (WTC) collapse. Ten months after the collapse, induced sputum (IS) samples were obtained from 39 highly exposed FDNY-FFs (caught in the dust cloud during the collapse on 11 September 2001) and compared to controls to determine whether a unique pattern of inflammation and particulate matter deposition, compatible with WTC dust, was present. Control subjects were 12 Tel-Aviv, Israel, firefighters (TA-FFs) and 8 Israeli healthcare workers who were not exposed to WTC dust. All controls volunteered for this study, had never smoked, and did not have respiratory illness. IS was processed by conventional methods. Retrieved cells were differentially counted, and metalloproteinase-9 (MMP-9), particle size distribution (PSD), and mineral composition were measured. Differential cell counts of FDNY-FF IS differed from those of health care worker controls ( In the aftermath of September 11th, the clouds of dust and smoke that stood for days in place of the World Trade Center’s (WTC) twin towers raised serious health concerns among exposed workers and residents. The Fire Department of New York City (FDNY) operated a continuous rescue/recovery effort from 11 September 2001 through May 2002. Nearly every FDNY firefighter (FDNY-FF) worked at the site during the first weeks, reporting numerous exposures to airborne particulates and products of combustion/pyrolysis  that hav3 and 1 mg/m3, respectively  recovered significant quantities of fly ash, degraded fibrous glass, and asbestos fibers along with evidence for a significant inflammatory response (70% eosinophils and increased levels of interleukin-5) in one FDNY-FF hospitalized with acute eosinophilic pneumonitis several weeks after WTC exposure . AlthougInduced sputum (IS) provides a non-invasive alternative method to study respired particulate matter and the lung’s inflammatory response . QualitaThis study is the first to use IS methodology to assess respired particulate matter and the inflammatory response of the lung after exposure to WTC dust. Our objective was to determine if IS collected from highly exposed FDNY-FFs 10 months after the collapse demonstrates a unique pattern of inflammation and particulate matter deposition compatible with WTC dust. Inflammation was assessed by differential cell counts and by measuring metalloproteinase-9 (MMP-9), a cytokine involved in airways inflammation  and remoTen months after the WTC collapse, we studied 39 male FDNY-FFs who worked at the WTC, 12 male firefighters who lived in Tel-Aviv, Israel (TA-FF), and 8 male Israeli hospital workers, all apparently free of respiratory disease. Firefighters were recruited from those undergoing medical monitoring during June 2002. The only inclusion criterion was that firefighters must have worked in the WTC dust cloud the morning of 11 September 2001. Current or past tobacco smokers were excluded. IS induction was voluntary, and all were informed about the ongoing research. For FDNY-FFs, cumulative WTC exposure was measured in workdays reported on a self-administered questionnaire, followed by a confirmatory interview before IS induction. The Institutional Review Boards at Tel-Aviv Medical Center and Montefiore Medical Center approved this study.IS was obtained at the FDNY Bureau of Health Services as previously described . After pSamples from the cellular fraction were resuspended and processed to cytospin slides, centrifuged, air-dried, and stained with Giemsa. Differential cell counts were measured by scanning cytospin slides with high power (×500) magnification. We counted 200 nonsquamous cells and expressed cell differentials as percentages of total nonsquamous cell counts.We determined the absolute value of MMP-9 in IS supernatants using an ELISA commercial kit . MMP-9 values expressed total levels of active and pro-MMP-9 (0.1–25 ng/mL). We performed spike experiments using 10 and 20 ng/mL pure MMP-9 protein to assess the efficacy of recovery. We found that only 10–12% of protein was equally denatured in the pure protein and in all IS samples by DTT. No other metabolites were measured in the study.6 cells/mL) introduced into a quartz cuvette containing stirred water for IS samples and water with glycerol (1:1) for the WTC settled dust sample collected 7 days postcollapse on Cortlandt Street, one block east of the WTC. Each result was an average of three consecutive measurements.After separation of the plugs and viscous materials, all fractions of IS were preserved in 10% formalin and stored at 4°C until analysis of mineral particles. We used the samples containing both extracellular and intracellular particles for the SEM analysis; samples were treated with 14% formamide solution and filtered onto a 0.8-μm carbon-coated Nuclepore filter . Particles with a diameter > 0.4 μm were analyzed by a JEOL 840 SEM  equipped with a Link 10,000 energy-dispersive system . The spectrometer of the EDS system separated the elements according to energy rather than wavelength. In addition, we used a petrographic microscope to identify minerals , 1999b. t-test, Mann-Whitney test, and chi-square test. In addition, group means of all percent cells and particle size, adjusted for MMP9, were compared by a one-way analysis of covariance with the natural logarithm of MMP-9 as covariate. Natural logarithm transformation was applied to MMP-9 because of its skewed distribution (Ln MMP-9). The association between percent parameters was evaluated by Pearson correlation coefficients. For all tests, p-values < 0.05 were considered statistically significant. The data were analyzed using Statistical Package for the Social Sciences (SPSS) for Windows software, Version 11.0 .Demographic comparisons between groups were performed by parametric or nonparametric  analysis of variance (ANOVA). FDNY-FFs were analyzed according to cumulative exposure days using a continuous scale and dichotomized based on the median (< 10 days vs. 10 days). Groups were compared by independent p = 0.109, respectively) or their respective fire-fighting work tenure . The average age of the nonfirefighter controls was 41.6 years. All 39 FDNY-FFs were caught in the WTC dust cloud during the morning of 11 September 2001, representing the highest acute exposure group as defined by arrival time (n = 23) or 10 days (n = 16).We found no significant differences in age between the FDNY-FFs and the TA-FFs , and the combined levels for both groups were significantly higher than for the controls . Independent of exposure group, the levels of MMP-9 were positively correlated to the percentage of neutrophils  and > 5 μm (p = 0.0001) compared with the TA-FF samples. We found no significant differences in PSD measurements when they were correlated to cumulative WTC exposure . Most of the particles showed an irregular shape (We determined PSD in 35 of 39 FDNY-FF samples (4 samples were eliminated because of contamination before measurement) and 12 of 12 TA-FF samples. ar shape .n = 39; We also demonstrated compatibility between the PSD of the settled raw dust samples from Cortlandt Street (located one block east of the WTC) collected 7 days after the collapse with measurements from the IS of FDNY-FFs n = 39; . Both PSChemical and mineralogic analyses were performed on IS samples from 4 FDNY-FFs and 2 TA-FFs randomly chosen from high quality specimens. Chemical analysis of the FDNY-FF samples revealed many elements, for example, titanium, zinc, mercury, gold, tin, and nickel , which wIn FDNY-FFs—all highly exposed to WTC dust and combustion/pyrolysis products during the morning of the collapse and nearly all with additional cumulative workday exposures to WTC dust—IS analysis were significantly different from controls, in inflammation  and particulate matter deposition .It is not surprising to find abnormalities in lung particulate matter and inflammation months after exposure. After chronic occupational exposures, IS has shown increased eosinophil counts in sensitized asthmatics  and in aFirefighters are exposed to numerous irritants , and such exposures have the potential to alter lung permeability . Inflamma) plays an important role in neutrophil recruitment to the lung (b) is detectable in IS with reliable and reproducible results (c) is increased in IS from workers exposed to hazardous dust  are a family of zinc- and calcium-dependent endopeptidases with a central role in inflammation and combined ability to degrade components of connective tissue matrices . MMPs arthe lung ; b) is d results ; and c) ous dust . We founous dust  and otheous dust  after exPSD measurements demonstrated significant differences between FDNY-FF and TA-FF IS samples. We found a high load of relatively large particles (1–50 μm in diameter) with irregularly shaped structures in FDNY-FF IS samples that were completely different from the smaller, regular shaped particles found in TA-FF IS samples. Chemical and mineral analyses also demonstrated differences between FDNY-FF and TA-FF IS samples. TA-FF IS showed findings typical of soil contaminants from the Tel-Aviv area. In contrast, a heterogeneous mixture was found in FDNY-FF IS, consistent with exposures to aerosolized building debris and dust and smoke generated by the collapse and fires . Minerala) all FDNY-FFs had significant WTC dust exposures because they were caught in the dust cloud during the collapse and then continued to work at the WTC site for days thereafter; b) IS provided an assessment of persistent inflammation and cumulative particle deposition because the enormity of the disaster prevented us from collecting IS until 10 months after the collapse; c) IS assessments were not biased toward acute, transient inflammation and particle deposition because at least 1 month had elapsed between collecting IS and the last workday at the WTC site; and d) controls were truly unexposed because they were recruited from outside this region—a necessity because by this time nearly every FDNY-FF and most other rescue workers and many Manhattan residents had reported some level of exposure.Despite our finding significant correlations between inflammation and cumulative WTC exposure and significant differences in PSD between FDNY-FFs and TA-FFs, we could not detect a significant effect of cumulative WTC exposure on PSD. This may be related to other physical factors at the WTC site, such as differences in work location, minute ventilation , specific work task–related exposures, and the use of respiratory protection  . It may We believe the differences between FDNY-FFs, TA-FFs, and controls demonstrate a unique exposure following the WTC collapse  and 4. TAlthough helpful, measurements obtained from settled dust samples, which are often enriched with larger particles, are not entirely representative of the particle types or size distribution of aerosolized, potentially respirable dust during the height of exposure. In fact, the majority of particles in FDNY-FF IS samples were < 2.0 μm in diameter: 74% of FDNY-FFs had > 60% of PSD < 2.0 μm in diameter . CompareOur findings support both the practical utility and scientific usefulness of IS as a non-invasive method for screening and follow-up monitoring of populations exposed to high concentrations of aerosolized particulates following a disaster—natural or man-made. IS is superior to BAL because it is noninvasive and collection can occur at nearly any field location. In contrast to traditional blood and urine biomonitoring, IS directly samples the lung, the specific target organ of interest following an inhalation exposure. For example, blood and urine samples were collected 1 month after the collapse in a different group of FDNY-FFs, and only a few of the 110 chemicals measured showed significant, yet small, differences when WTC-exposed FDNY-FFs were compared with nonexposed FDNY-FFs . In contIn conclusion, IS from FDNY-FFs caught in the WTC dust cloud during the morning of the collapse showed an influx of inflammatory cells, percentages of neutrophils and eosinophils that increased with exposure intensity, increased MMP-9 levels, a shift in PSD toward larger-size particles, and chemical/mineral analyses consistent with exposure to building debris, smoke, and dust generated by the attack on the WTC. If future population studies demonstrate that IS measures of inflammation or PSD correlate with health outcomes, then IS evaluations would be a valuable addition to medical screening/monitoring programs following inhalation exposures.


Increased oxidative stress has been associated with the pathogenesis of chronic diabetic complications, including cardiomyopathy. Recent studies indicate that curcumin, a potent antioxidant, may be beneficial in preventing diabetes-induced oxidative stress and subsequent secondary complications. We have investigated the effects of curcumin on the nitric oxide (NO) pathway in cardiac tissues and cultured cells.Streptozotocin-induced diabetic rats were treated with curcumin for a period of one month. Heart tissues were then analyzed for endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) mRNA expression. Oxidative protein and DNA damage were assessed by immunohistochemical analysis of nitrotyrosine and 8-hydroxy-2'-deoxyguanosine (8-OHdG). Heart tissues were further subjected to endothelin-1 (ET-1) mRNA expression. In order to further characterize the effects of curcumin, we assayed microvascular endothelial cells (MVECs). Cultured MVECs, exposed either to glucose or glucose and varying concentrations of curcumin, were assessed for alterations of NOS expression and activation of nuclear factor-κB (NF-κB) and activating protein-1 (AP-1). Oxidative stress and ET-1 expression levels were also assayed.Our results indicate that one month of diabetes causes an upregulation of both eNOS and iNOS mRNA levels, and nitrotyrosine and 8-OHdG immunoreactivity in the heart. Treatment of diabetic rats with curcumin reduced eNOS and iNOS levels in association with reduced oxidative DNA and protein damage. Interestingly, curcumin further increased vasoconstrictor ET-1 in the heart. Exposure of MVECs to high glucose increased both eNOS and iNOS levels and oxidative stress. Curcumin prevented NOS alteration and oxidative stress in a dose-dependent manner which was mediated by nuclear factor-κB and activating protein-1. Exposure to curcumin also increased ET-1 levels in the MVECs.Our studies indicate the differential effects of curcumin in vasoactive factor expression in the heart and indicate the importance of tissue microenvironment in the treatment of diabetic complications. Nearly 40% of diabetic patients develop secondary complications -3. TheseAn interesting pathway suggested to be involved in augmenting oxidative stress is the nitric oxide (NO) pathway ,11. NO iCurcuma Long Linn), was originally used in tradition Indian medicine over 3000 years ago [Curcumin, the active component in Tumeric Rhizomes , weighing approximately 200–250 g, were randomly divided into three groups (n = 6): controls (CO), diabetic animals (DM), and diabetic animals treated with curcumin (DM-C). Diabetes was induced by a single intravenous injection of streptozotocin  , while t2 in endothelial growth medium (Clonetics) as previously described [Human microvascular endothelial cells  were cultured at 2500 cells/cmescribed . Cells wescribed ,31. Afte2, 1 μL each of 10 mM forward and reverse primers , an oxidative DNA damage marker . ParaffiElectrophoretic mobility shift assay (EMSA) was performed to assess activation of transcription factors, NF-κB and activating protein-1 (AP-1), as described previously ,34.The data are expressed at mean ± S.E.M. and were analyzed by ANOVA followed by student's t-test. Statistical differences were considered when p < 0.05.Animals were monitored for body weight gain and blood glucose levels to assess diabetic dysmetabolism. Diabetic animals exhibited decreased body weight gain and increased blood glucose levels as compared to non-diabetic controls Table . CurcumiHeart tissues from diabetic animals exhibited increased eNOS and iNOS mRNA levels as compared to control rats Figure . ET-1 mRTreatment of diabetic animals with curcumin prevented eNOS and iNOS mRNA upregulation Figure . InteresIn order to investigate the mechanisms of curcumin-mediated downregulation of NOS and oxidative stress, we assayed microvascular ECs. Exposure of ECs to high glucose levels (25 mmol/L) increased both eNOS and iNOS mRNA levels Figure . TreatmeIn order to assess whether curcumin prevents high glucose-induced oxidative stress, we employed the early molecular marker of oxidative damage, HO-1 and HO-2. Our results show that glucose-induced HO-1 and HO-2 expression is normalized by pre-treatment with curcumin Figure .In the present study, we showed that diabetes causes an increase in eNOS and iNOS mRNA levels in the cardiac tissues, in association with increased oxidative protein and DNA damage. Treatment of diabetic animals with curcumin prevents diabetes-induced eNOS/iNOS upregulation and oxidative stress. We also report, for the first time, that curcumin potentiates diabetes-induced ET-1 mRNA levels in the heart. Furthermore, the mechanism of NOS alteration by curcumin involves activation of NF-κB and AP-1.Over the past several decades, researchers have provided substantial evidence that oxidative stress is augmented in diabetic complications, including cardiomyopathy ,36. SeveCurcumin, a potent antioxidant and anti-inflammatory compound, has been shown to mediate many biological affects such as downregulation of NOS -40. A reOne other interesting finding of the study is the effect of curcumin on ET-1 expression in the heart as well as cultured ECs. ET-1 was found to be further increased in curcumin-treated diabetic animals and ECs exposed to high glucose levels. It is possible that curcumin may have a differential effect on the vasoactive factors. Our results show that curcumin decreases eNOS/iNOS levels in the heart and cultured ECs but increases ET-1 levels. The mechanism of such action and potential significance remains to be determined. The differential effects of curcumin on eNOS/iNOS and ET-1 may be due to the activation of different transcriptional regulators. Another possible explanation may be that the downregulation of eNOS/iNOS could have a secondary effect on ET-1. It is also plausible that such an alteration may represent the importance of tissue microenvironment and bystander cells. In support of such a notion, our studies indicate that curcumin decreases ET-1 levels in the kidneys and the retina (data not shown). Further studies are necessary to elucidate whether such differences represent the different molecular aberrations in the target organs of chronic diabetic complications.Our results support the findings that curcumin prevents diabetes-induced oxidative protein and DNA damage, in association with decreasing NOS levels. Our findings also suggest that the use of curcumin in novel therapeutic modalities should be approached with caution, as curcumin may augment vasoactive peptide ET-1 and lead to vascular dysfunction in diabetes.The author(s) declare that they have no competing interests.in vitro and in vivo experiments, including helping maintain the diabetic animals. HF also assisted in drafting the manuscript and preparing it for publication.HF conducted the majority of the in vivo and in vitro data. ZAK also helped draft the manuscript and revised it critically for intellectual content.ZAK maintained the diabetic animals and helped to interpret the SC helped with conducting the molecular techniques and assisted in interpreting the results.SC* assisted in the experimental design, revised the manuscript critically for intellectual content, and gave the final approval for the manuscript to be submitted for publication. In addition, all the experiments were conducted under his grant approval.


However, the precise activity of ERK during axonal regeneration in vivo remains unclear. Peripheral axotomy has been shown to activate ERK in the cell bodies of primary afferent neurons and associated satellite cells. Nevertheless, whether ERK is also activated in the axons and surrounded Schwann cells which also play a key role in the regeneration process has not been clarified.Activation of extracellular signal-regulated protein kinase (ERK), a member of mitogen-activated protein kinase (MAPK) family, has been proposed to mediate neurite outgrowth-promoting effects of several neurotrophic factors Phosphorylation of ERK in the sciatic nerve in several time-points after crush injury has been examined. Higher phosphorylation of ERK was observed in the proximal and distal nerve stumps compared to the contralateral intact nerve from one day to one month after crush. The activation of ERK was mainly localized in the axons of the proximal segments. In the distal segments, however, active ERK was predominantly found in Schwann cells forming Bungner's bands.in vivo remains to be elucidated.The findings indicate that ERK is activated in both the proximal and distal nerve stumps following nerve injury. The role of activated ERK in Wallerian degeneration and subsequent regeneration  Peripheral axotomy can activate several signaling pathways in neurons and associated glial cells leading to two opposing consequences: cell death or adaptation to regenerate neurites. The principal signaling pathways that have been demonstrated to be involved in axonal regeneration are PI3K-Akt -3 and JA2-terminal kinase (JNK) and p38 kinase (p38). JNK along with its main transcription factor, c-Jun, are activated in the dorsal root ganglia (DRG) after sciatic nerve transection . Sample buffer  was added to the samples before boiling for 5 min. These homogenized samples were stored at -20°C until use.Concentration of protein in each sample was determined using Bramhall protein assay . SDS-PAGThe data were imported to SPSS for Windows version 10 and checked for normal distribution and homogeneity of variances. If these assumptions were met, one-way analysis of variance (ANOVA) was employed to compare means from different time-points. However, if the measurements were extremely skewed from normal distribution and/or had a markedly significant difference in variances, a non-parametric test  was used instead. Where statistically significant differences were observed in ANOVA or Kruskal-Wallis test, pair-wise or post-hoc comparisons were achieved by using Tukey's HSD or Mann-Whitney U test, respectively. Statistically significant differences were considered when p < 0.05 unless otherwise stated.Sciatic nerves kept in 20% sucrose in PBS were embedded in OCT medium and 9 μm-thick slices were cut by cryostat section. Proximal and distal segments of sciatic nerves were sectioned either transversely or longitudinally. The slides with sections were blocked in 10% normal serum (Sigma) and incubated in the primary antibody to phosphorylated ERK  for 48 hours at 4°C. After washing, the slides were incubated in the secondary antibody conjugated with fluorescein isothiocyanate (FITC)  for 2 hours at room temperature. The slides were then mounted with anti-fading mounting medium , cover-slipped and examined under fluorescence microscope. In some sections, double-staining with either anti-pan-neurofilament  or anti-S-100  antibodies overnight at 4°C followed by secondary antibody conjugated with rhodamine  was done to locate the axons or Schwann cells, respectively.SA carried out the Western blot and immunohistochemical experiments, analysis of the data and drafting the manuscript.AK and NT carried out the animal experiment and collection of tissues.VC participated in the design of the study, gave advise during the experiments and correcting the manuscript.


Three transcription factors that are expressed at high levels in embryonic stem cells (ESCs) are Nanog, Oct-4 and Sox-2. These transcription factors regulate the expression of other genes during development and are found at high levels in the pluripotent cells of the inner cell mass. The downregulation of these three transcription factors correlates with the loss of pluripotency and self-renewal, and the beginning of subsequent differentiation steps. The roles of Nanog, Oct-4 and Sox-2 have not been fully elucidated. They are important in embryonic development and maintenance of pluripotency in ESCs. We studied the expression of these transcription factors in porcine umbilical cord (PUC) matrix cells.Cells were isolated from Wharton's jelly of porcine umbilical cords (PUC) and histochemically assayed for the presence of alkaline phosphatase and the presence of Nanog, Oct-4 and Sox-2 mRNA and protein. PCR amplicons were sequenced and compared with known sequences. The synthesis of Oct-4 and Nanog protein was analyzed using immunocytochemistry. FACS analysis was utilized to evaluate Hoechst 33342 dye-stained cells.PUC isolates were maintained in culture and formed colonies that express alkaline phosphatase. FACS analysis revealed a side population of Hoechst dye-excluding cells, the Hoechst exclusion was verapamil sensitive. Quantitative and non-quantitative RT-PCR reactions revealed expression of Nanog, Oct-4 and Sox-2 in day 15 embryonic discs, PUC cell isolates and porcine fibroblasts. Immunocytochemical analysis detected Nanog immunoreactivity in PUC cell nuclei, and faint labeling in fibroblasts. Oct-4 immunoreactivity was detected in the nuclei of some PUC cells, but not in fibroblasts.Cells isolated from PUC express three transcription factors found in pluripotent stem cell markers both at the mRNA and protein level. The presence of these transcription factors, along with the other characteristics of PUC cells such as their colony-forming ability, Hoechst dye-excluding side population and alkaline phosphatase expression, suggests that PUC cells have properties of primitive pluripotent stem cells. Furthermore, PUC cells are an easily and inexpensively obtained source of stem cells that are not hampered by the ethical or legal issues associated with ESCs. In addition, these cells can be cryogenically stored and expanded. Stem cells constitute a population of cells that are present in all stages of development from preimplantation embryos through adulthood. Pluripotent stem cells have the ability to self-renew for indefinite periods in culture and to differentiate, e.g., give rise to all of the specialized cells in an animal. Currently, pluripotent stem cells can be isolated consistently only from the inner cell mass of embryos or from the gonadal ridge . In contThree transcription factors found in mouse and human ESCs play a central role in the regulation of pluripotency and self-renewal. These factors include the POU (Pit/Oct/Unc) domain-containing protein Oct-4 -5, Sox-2in vitro are important considerations. Therapeutically, stem cells from non-embryonic sources have been used clinically and have successfully treated a number of diseases. To advance animal biotechnology, it will be necessary to produce ESCs and EGCs that display germ line transmission in species other than the mouse; so far this has not met with success  and 97% (392/401 bp) homology to the ovine sequence [GenBank: X96997] [see Nanog primers produced a single amplified product of expected size from PUC isolations with 86% (326/379 bp) homology to a human Nanog sequence [Genbank: AB093576] and a 268 bp segment with 99% homology to the published Sus scrofa Nanog partial sequence [GenBank: AY596464] [see Oct-4 PCR amplified products from PUC isolations displayed >99% (298/300) homology to the Sus scrofa Oct-4 sequence [TIGR: TC205936] [see The sequence of 997] see . Analysi464] see . Oct-4 P936] see .Immunocytochemical analysis was performed to test for expression of Nanog and Oct-4 protein. Positive labeling for Nanog in PUC cell nuclei Fig.  as well Nuclear immunoreactivity for Oct-4 Fig.  was alsoNanog, Oct-4 and Sox-2 in embryonic discs, PUC cells and porcine fibroblasts. Results indicate that differences in the expression levels were apparent between cell types . For Oct-4 and Sox-2, the embryonic disc sample expressed the highest copy number of target RNA  followed by PUC cells , when normalized to the expression levels of fibroblasts. The copy number for Nanog RNA ranged from 1.5 to 40-fold and 1.4 to 8.6-fold higher than fibroblasts for embryonic disc and PUC cells, respectively. Importantly, the results show that these stem cell markers are expressed in greater copy number in PUC cells than in porcine fibroblasts, although less than in porcine embryonic disc.Quantitative RT-PCR was conducted to determine the relative expression levels of pes Fig. . ResultsNanog RNA at higher levels than porcine fibroblasts. Both Oct-4 and Sox-2 message was present in discs in substantially greater amounts than in PUC cells, and PUC cells contained more message than fibroblasts. Nanog, Oct-4, and Sox-2 have been reported to occupy the promoters of at least 2,260 human genes and these transcription factors co-occupy 353 gene promoters [Here we report expression of Nanog, Oct-4 and Sox-2 in cultured PUC matrix cells. These transcription factors are considered key regulators of gene transcription in primitive stem cells. Quantitative RT-PCR revealed that PUC cells and porcine embryonic discs express romoters . Thus, tNanog in both PUC cells and embryonic discs suggests that the regulatory pathways influenced by Nanog in the pig may be similarly activated or inactivated in the embryonic disc and PUC cells. However, the reduced expression of Oct-4 and Sox-2 in PUC cells relative to the embryonic disc could indicate that genes regulated by these transcription factors may have different expression patterns in these two tissues. Because genetic studies of the functions of these transcription factors are not available in the pig, the meaning of the lower expression of Oct-4 and Sox-2 mRNA in PUC cells is unknown. The significance of expression, albeit at low levels, of all three transcription factors in fibroblasts is also unclear but may indicate that quantitative regulation is determinative of the differentiation state in this species.We observed gene expression of RNA for all three transcription factors in porcine embryonic discs, PUC cells, and fibroblasts. Therefore, quantitative RT-PCR data on the expression of these transcription factors was undertaken. In particular, the high level of mRNA expression of Nanog RNA is present in both the embryonic and extraembryonic porcine conceptus tissues at day 15 of pregnancy [A recent paper described colony formation and Oct-4 expression in fetal murine and porcine fibroblasts, but failed to replicate these findings in adult-derived porcine fibroblasts . Expressregnancy .Nanog, Oct-4 and Sox-2 (as compared to fibroblasts) appears to vary in embryonic discs and PUC cells with embryonic discs clearly expressing more Oct-4 and Sox-2. Numerous factors may explain these results. First, PUC cells and fibroblasts may contain stem cells with similar gene expression levels to those present in embryonic discs, but have a smaller stem cell to non-stem cell ratio than embryonic discs. However, immunocytochemical analysis suggests that while protein levels may differ in PUC cells and fibroblasts, both cell types display nuclear labeling in a uniform manner suggesting that all cells express (or do not express) Nanog and Oct-4 protein at similar levels. Alternatively, posttranslational modifications may account for the reduced expression of Nanog protein and lack of Oct-4 protein in porcine fibroblasts despite the detection of mRNA for both genes. It is unknown whether no protein or an alternative form of the protein is being expressed in fibroblasts. Nonetheless, PUC cells express Nanog, Sox-2 and Oct-4 at the mRNA level with nuclear immunoreactivity for Nanog and Oct-4 being clearly and uniformly present.The relative levels of mRNA expression for Nanog, Oct-4 and Sox-2 was detected by RT-PCR in PUC cells. The amplification of these transcription factors using RT-PCR techniques produced bands of expected sizes for each PCR product. The subsequent sequencing of the product and high homology to published human and pig sequences is a strong indication of the presence of these genes. Three separate quantitative analysis experiments revealed higher mRNA expression levels of Nanog and Sox-2 in PUC cells compared to fibroblasts, while two out of three experiments revealed higher levels of Oct-4 mRNA in PUC cells compared to fibroblasts. Nuclear immunocytochemical labeling for Oct-4 and Nanog support this result. Recent studies have identified several processed pseudogenes for Nanog and Oct-4 in the human and mouse genome, including two that have been identified in expressed sequence tags [The expression of nce tags ,34. It iOct-4 upregulation results in differentiation of ESCs into extraembryonic mesoderm or endoderm while Oct-4 knockout ESCs differentiate into trophectoderm [Nanog allows mouse ESCs to remain pluripotent without the need for leukemia inhibitory factor (LIF) supplementation in the growth medium [Oct-4, Nanog and Sox-2 are central regulators for maintaining a pluripotent state in mouse and human ESCs. In the mouse, artificial ectoderm ,36. Upreh medium ,8. Sox-2en route to their final destination in the gonadal ridge. It is possible that some of these migrating germ cells or their descendents remain in the umbilical cord matrix.Alkaline phosphatase activity was observed in colony-forming PUC isolations. Alkaline phosphatase is an enzyme long-known to be expressed in ESCs as well as primordial germ cells . The realow side population that was detected by flow cytometry. Hematopoietic stem cells contain a small population of Hoechst dye-excluding cells (termed side population or SP) when examined by flow cytometry, and this property is inhibited in the presence of verapamil [low cells in bone marrow samples is less than 1% of the total population. Our observation of approximately 7%  suggests that a higher concentration of stem-like cells are present in PUC cell isolations.PUC cells displayed a Hoeschterapamil ,39. Altherapamil . Typicalin vivo environments, but the characteristics identified here suggest that PUC cells will have a wide potential.The present results are consistent with the interpretation that PUC cells represent a stem cell population that expresses the central regulators required for mouse embryonic stem cells. Transplantation studies will be required to define the potential of PUC cells when placed in various We conclude that PUC matrix cells contain unique and primitive cells whose potential is as yet undefined. Ease of collection and propagation, and abundant numbers make PUC matrix cells an attractive candidate as a resource for stem cell biotechnology and biomedical research.Sequence alignment of Nanog PCR products. Alignment of Nanog PCR product sequence from embryonic disc (Disc), porcine ear fibroblasts (Ear), PUC cells (PUC) and Sus scrofa Nanog gene exon 2 and partial coding sequence from GenBank (AY596464). Disc, Ear and PUC sequences share a 268 bp segment with 99% homology to the published Sus scrofa sequence.Click here for fileSequence alignment of Sox-2 PCR products. Direct sequencing of embryonic disc (Day15_Disc) and PUC cells (PUC1) using Sox-2 primers were aligned to the human and ovine Sox-2 sequence. PUC sequence alignment revealed 96% homology to the human Sox-2 sequence and 97% homology to the ovine sequence.Click here for fileSequence alignment of Oct-4 PCR products. The sequence of two separate PUC isolations using Oct-4 primers were aligned to the TIGR Sus scrofa Oct-4 sequence . Alignment revealed 98% (PUC1) and 99% (PUC2) homology.Click here for file


Chlamydia pneumoniae, chlamydial heat shock protein (Ch-hsp) 60 and human heat shock protein (h-hsp) 60 infection sero-status and development of secondary cardiovascular events. Patients with diabetes might be at higher risk since they are prone to infections. The objective of this study was to investigate prospectively the role of Chlamydia pneumoniae (CP), chlamydial heat shock protein (Ch-hsp) 60 and a possible intermediate role of human heat shock protein (h-hsp) 60 sero-status in the development of secondary cardiovascular disease (CVD) events in patients with coronary heart disease (CHD) under special consideration of diabetes mellitus.There have been suggestions of an association between th Rev. pos. 410–414) between January 1999 and May 2000 in one of two participating rehabilitation clinics in Germany were included in this analysis. Chlamydia pneumoniae (CP), chlamydial heat shock protein (Ch-hsp) 60 and human heat shock protein (h-hsp) 60 status at baseline were measured by serum immunoglobulin G and A antibodies. Secondary CVD events  were recorded during a mean follow-up period of 33.5 months (response = 87%).Patients aged 30–70 undergoing an in-patient rehabilitation program after acute manifestation of coronary heart disease  participants. Occurrence of a secondary CVD event was more common among CP (IgA) and CP (IgG) sero-positive than among sero-negative patients . The risk of secondary CVD events was increased among patients with both a positive CP sero-status and diabetes compared to infection negative, non-diabetic patients and in general, sero-positivity added a hazard to diabetes. The interaction term between infection sero-status and diabetes was not statistically significant. We were not able to show an intermediate role of human heat shock protein (h-hsp) 60 sero-status in the development of secondary CVD events in patients with CHD.Results from this cohort of 1052 patients with pre-existing CHD cannot exclude a possible moderate increase in risk of secondary CVD events among patients with a positive infection sero-status. However, our study showed no intermediate role of human heat shock protein (h-hsp) 60 sero-status in the development of secondary CVD events in patients with CHD. Larger studies or meta-analysis of multiple studies are needed to address the interaction between infection sero-status and diabetes with adequate power. Chlamydia pneumoniae (CP) is an obligate intracellular organism. Sero-positivity is low in early childhood (< 5 years), rising rapidly during school age such that at age 20 years approximately 50% of individuals will have detectable antibodies. Seroprevalence continues to rise, at a slower pace, in adulthood and reaches 75% in the elderly population [C. pneumoniae infection appeared in 1985 [pulation . The firChlamydia pneumoniae is one infectious agent that has received particular attention as a potent atherogenic stimulus. Correlative studies suggest a possible relationship between atherosclerosis and CP infection [nfection ,4. Pathonfection . Severalnfection ,7, howevInflammation, infection and autoimmunity to human heat shock protein 60 (h-hsp60) have emerged as novel risk factors for coronary heart disease during the past decade. Heat shock proteins are a class of highly conserved proteins produced by all organisms as a result of stress and damage caused by, for instance, exposure to chemical and environmental factors, inflammation and infections . BecauseChlamydia pneumoniae infection [Chlamydia pneumoniae infection and atherosclerosis [Two independent groups have shown that an elevated anti-human hsp60 antibody level is a risk factor for primary coronary atherosclerosis, especially when it is present with nfection ,12. Therclerosis . Elevateclerosis . In a reclerosis .The role of heat-shock proteins in patients with prevalent coronary heart disease on prognosis has been rarely investigated. Furthermore, despite the fact that patients with diabetes are immuno-compromised, remain persistently at risk for infectious complications and are prone to develop accelerated atherosclerosis , there aChlamydia pneumoniae, chlamydial heat shock protein 60 and a possible intermediate role of human heat shock protein 60 sero-status in the development of secondary CVD events in patients with coronary heart disease (CHD) under special consideration of diabetes, also comparing the relationship between chlamydial sero-markers of infection, heat shock proteins and secondary cardiovascular disease (CVD) events in patients with diabetes with those without.In this study we are investigating prospectively the role of In addition, the association of CP sero-status and serum concentration of C-reactive protein (CRP) was explored, as CRP is considered a main determinant of secondary CVD events.th Rev. pos. 410–414) or after coronary artery revascularisation between January 1999 and May 2000 in one of two participating rehabilitation clinics in Germany  were enrolled in the study. Germany has a very comprehensive in-patient rehabilitation program and patients who were hospitalized due to acute coronary syndrome  or coronary artery revascularisation  have the possibility to undergo an in-patient rehabilitation program (rehab) after discharge from the acute care hospital. The aims of this 3-weeks program are the reduction of cardiovascular disease risk factors, improvement of health related quality of life, and the preservation of the ability to work (latter only if a subject was working at onset of disease otherwise prevention of nursing care). This in-patient rehabilitation program begins approximately three weeks after the acute coronary syndrome or the revascularisation procedure but this time interval may be longer in some cases. In the current study only patients who were admitted within 3 months after the acute coronary syndrome or revascularisation procedure were included.All patients aged 30–70 years participating in an in-patient rehabilitation program after acute coronary syndrome .Active follow-up was conducted one and three years after discharge from the rehab clinic. At both times information was obtained from patients via a mailed standardized questionnaire. A standardized questionnaire regarding cardiovascular disease events and treatment since discharge from the in-patient rehabilitation clinic was also obtained from the patient's primary care physician. If a subject died during follow-up, the death certificate was obtained from local Public Health authorities and the main cause of death was coded according to the International Classification of Diseases (9Chlamydia pneumoniae (CP), chlamydial heat shock protein (Ch-hsp) 60 and human heat shock protein (h-hsp) 60 and physician's questionnaire at follow up were included. Among 1206 patients with coronary heart disease recruited at baseline, follow-up information by physician's questionnaire or by death certificate could be obtained for 1052 patients (87%), of whom infection sero-status could be determined for 1030 (98%) patients for Chlamydia pneumoniae. H-hsp 60 was available in 983 (93%) patients.Secondary cardiovascular disease (CVD) events were defined as CVD being main cause of death, or a physician's diagnosis of a non-fatal myocardial infarction or ischemic cerebrovascular event. For the analyses in this study, only subjects with sero-status of Chlamydia pneumoniae by using specific enzyme immunoassay for the qualitative and semiquantitative detection of specific IgG or IgA antibodies .Blood samples drawn before hospital discharge from the rehab clinic were prepared and serum for serology was stored at -80°C. Serum samples were analysed for The sero-positivity for the semiquantitative analysis was defined as cut-off index  and evaluated as titer. Cut-off index ≤ 1.1 was defined as negative with titer < 1:50, cut-off index >1.1 and ≤ 1.8 was defined as positive with titer 1:50, cut-off index >1.8 and ≤ 3.6 was defined as positive with titer 1:100, cut-off index >3.6 and ≤ 7.2 was defined as positive with titer 1:200. Serum samples were also analysed by using recombinant enzyme immunoassays for chlamydial heat shock protein 60  and human heat shock protein 60 immunoglobulin G antibodies . Serum samples were also analysed by using recombinant enzyme immunoassays for chlamydial heat shock protein 60  and human heat shock protein 60 immunoglobulin G antibodies . Blood lipids were measured by routine methods and CRP was measured using a high sensitivity CRP (hsCRP) method . All laboratory measures were done in blinded fashion.We first described the study population according to basic socioeconomic and medical characteristics. We then calculated the prevalence of sero-positivity to CP, Ch-hsp60 and h-hsp60 at baseline in the whole study population and in various age groups stratified for gender. We quantified possible associations with age by a chi-square statistic.Furthermore, we described the incidence of secondary cardiovascular disease (CVD) events  and their combination during follow up. The occurrence of fatal and non-fatal CVD events during follow up according to sero-prevalence of CP, Ch-hsp60 and h-hsp60 at baseline was analysed by the life table method.Finally, we used the Cox proportional hazards model to assess a possible association of sero-positivity to CP, Ch-hsp60 and h-hsp60 and diabetes  at baseline with fatal and non-fatal CVD events during follow up while adjusting for the following covariates: age (years), gender , HDL-cholesterol , history of smoking , alcohol consumption within last 12 months , school education , marital status , history of myocardial infarction , study centre (Bad Nauheim/Isny). Associations were quantified by hazard ratios (HR) and their 95% confidence interval (CI). In addition, a possible interaction between CP, Ch-hsp60 and h-hsp60 sero-status and diabetes was assessed by introducing a product term of each of the infection markers and history of diabetes in the adjusted model. In addition, age and gender adjusted geometric mean values of CRP (which is considered as a feature of atherosclerosis) were calculated and compared according to CP, Ch-hsp60 and h-hsp60 sero-status and history of diabetes by means of General Linear Regression methods. All analyses were carried out using the SAS software package, version 8.2.2 while 27.6% of patients with an additional history of diabetes had a body mass index > 30 kg/m2.Table Chlamydia pneumoniae (CP), chlamydial heat shock protein (ch-hsp) 60 and human heat shock protein (h-hsp) 60 sero-status at baseline in various age groups stratified by gender. Sero-positivity for CP IgA was 38.1% and 33.3% for males and females respectively and for CP IgG, it was 39.2% and 39.7% for males and females respectively. Sero-positivity for chlamydial heat shock protein (Ch-hsp) 60 (IgG) was 21.9% in males and 28.1% in females . Sero-positivity for human heat shock protein (h-hsp) 60 (IgG) was (8.7%) in males and (7.3%) in females .Table Table Table Table Chlamydia pneumoniae (CP), chlamydial heat shock protein (ch-hsp) 60 with history of diabetes and secondary CVD event. The risk of secondary CVD events was highest among patients with both a positive sero-status for CP IgA, CP IgG, Ch-hsp60 and diabetes and in general, sero-positivity tentatively increased the HRs of diabetes.Table Furthermore, we found associations between CP IgA, CP IgG sero-positivity and CRP levels at baseline after adjusting for age and gender ), ). Our analyses also showed no association between Ch-hsp60 IgG and CRP levels at baseline after adjusting for age and gender.Chlamydia pneumoniae (CP), Chlamydia heat shock protein (Ch-hsp) 60 and a possible intermediate role of human heat shock protein (h-hsp) 60 antibodies in the development of secondary cardiovascular events in patients with coronary heart disease (CHD) under special consideration of diabetes mellitus. The results from this study showed that the occurrence of a secondary CVD event was more common among CP (IgA) and tentatively CP (IgG) sero-positive than among sero-negative patients in crude analysis. However, the associations and a possible interaction between the sero-status of these infections and history of diabetes were not statistically significant after multivariate adjustment for covariates. Although the study clearly demonstrated an increased risk in developing a secondary CVD event among sero-positive patients with a history of diabetes compared to sero-negative without and sero-positivity adding a hazard to diabetes, it is suggested that mainly diabetes was responsible for the increased risk. Furthermore, this study showed that human heat shock protein 60 (h-hsp60) is unlikely an intermediate factor for developing secondary CVD events in the population studied.In this prospective study, we investigated the role of and Chlamydia pneumoniae is one infectious agent that has received particular attention as a potent atherogenic stimulus. However, the question whether serological evidence of prior infection with CP and the presence of Ch-hsp60 and h-hsp60 is associated with MI and CHD death remains a source of controversy, as several prospective studies have failed to demonstrate consistent associations between the presence of IgG antibodies to CP and incident MI [ident MI -19. Examident MI . Resultsident MI .Further examples of the limited number of studies that looked at CP sero-positivity and secondary CVD events include a prospective study in the context of the Heart Outcomes Prevention Evaluation (HOPE) Study. Their study showed that among patients with pre-existing CVD, neither CP IgG  nor CP IgA  predicted cardiovascular events during the 4.5 years of follow-up. . AnotherChlamydia pneumoniae (CP) infection, persistently elevated human heat-shock protein 60 (h-hsp60) antibodies and C-reactive protein (CRP) on coronary risk. Results showed that, compared with persistently low levels, the risk of coronary events was 2-fold for persistently elevated IgA antibodies to CP  and also for IgA antibodies to h-hsp60 . The risks associated with elevated antibodies were much higher when CRP was also elevated [C. pneumoniae antibodies in our study, elevated hsCRP levels of patients did not have any type of joint effect in predicting secondary cardiac event (data not shown). This is in accordance with observations of others [In addition, another study investigated the role of infections, autoimmunity and inflammation in atherosclerosis. They studied the joint effect of chronic elevated . In a prf others ,26. CRP Furthermore, all patients included in this study had an acute cardiovascular event within last 3 months before baseline. However, such an event may also reduce antibody titers, as has been shown for (h-hsp) 60  and thisC. pneumoniae, although the majority of antibodies against chlamdial LPS are due to C. pneumoniae.The following limitations of our study should be considered. Although we had a large sample of patients with CHD (over 50% with a MI), fatal-CVD events were rare in this study population. This might be explained by the fact that mortality of MI is highest within the pre- and early hospital phase. As the acute events leading to diagnosis of CHD or MI had occurred at least 3 weeks before inclusion in this study, we were dealing with a selection of patients with a better prognosis compared to a patient population within the early phase of a newly diagnosed CHD. Furthermore, not all patients of course are willing or able to participate in a rehab program. This is another reason why severely ill-patients are probably underrepresented in our study sample. In addition, proof of chlamydial heat shock protein 60 immunoglobulin G is genus specific and not specific to Although inflammation represents an important feature in all stages of atherogenesis and its complications ,29, we cDespite the above mentioned limitations, results of our study cannot exclude a possible moderate increase in risk of secondary CVD events among patients with a positive infection sero-status, however, they clearly indicate an increased risk among diabetic patients. This study further showed that h-hsp60 is unlikely an intermediate factor for developing secondary CVD events considering mechanisms involved in autoimmunity. Larger studies or meta-analysis of multiple studies are needed to address the interaction between infection sero-status and diabetes with adequate power. Notwithstanding the need of further evidence from such studies, patients with diabetes are at special risk for developing secondary CVD events and should be a target group for preventive measures.Results from this cohort of 1052 patients with pre-existing CHD cannot exclude a possible moderate increase in risk of secondary CVD events among patients with a positive infection sero-status. However, our study showed no intermediate role of human heat shock protein (h-hsp) 60 sero-status in the development of secondary CVD events in patients with CHD. Larger studies or meta-analysis of multiple studies are needed to address the interaction between infection sero-status and diabetes with adequate power.CI = confidence intervalHbA1c = hemoglobin A1cOR = Odds ratioThe author(s) declare that they have no competing interests.MGO did the statistical analysis and wrote the initial draft.HB and DR had the idea of the study and HB, TD and DR did study design and conduct. All authors critically revised the MS and read and approved the final manuscript.The pre-publication history for this paper can be accessed here: