In about 50–80% of ST-segment elevation myocardial infarction (STEMI) patients there is significant atherosclerotic disease in other coronary arteries in addition to the culprit vessel. There is substantial controversy as to the optimal revascularization approach in these patients. We sought to compare the outcomes of STEMI patients with multi-vessel disease (MVD) treated with culprit-only primary percutaneous coronary intervention (PPCI) without significant ischemia on subsequent non-invasive testing, to those of STEMI patients with single-vessel disease (SVD).Between 2001–2010, 1,540 consecutive patients treated with primary PCI for STEMI were prospectively observed and entered into a comprehensive clinical database. The primary end point was a composite of major adverse cardiac events (MACE), consisting of mortality, re-infarction and revascularization within 1 and 3 years following PPCI (excluding events occurring during the first 30 days). Patients with cardiogenic shock were excluded. The study included 720 patients with SVD and 185 patients with MVD who underwent culprit-only PPCI and had no residual ischemia on subsequent non-invasive stress testing. Patients with MVD were older, more likely to have hypertension or previous MI and less likely to be smokers and present with anterior MI than patients with SVD. One and 3-year MACE rates were similar between the groups. On cox proportional-hazards regression MVD without residual ischemia was not independently associated with MACE and its components.STEMI patients with MVD treated with culprit only-PCI without significant residual ischemia on non-invasive stress testing appear to have similar prognosis to STEMI patients with SVD. The main goal of primary percutaneous coronary intervention (PCI) in the setting of ST elevation myocardial infraction (STEMI) is to re-perfuse the myocardium by opening the culprit (infarct-related) coronary artery. However, in as many as 50–80% of patients with STEMI there is significant atherosclerotic disease in other coronary arteries in addition to the culprit vessel; a state which is associated with adverse outcomes –7. Many A central question that may guide decision-making in this controversial issue is what the prognosis is of patients with STEMI with multi-vessel disease undergoing culprit vessel PCI without significant ischemia on subsequent non-invasive testing (and thus they did not undergo further PCI).We hypothesized that the prognosis of these patients is similar to that of patients with single vessel disease undergoing successful primary PCI. Hence, the aim of this study was to compare the prognosis of these two groups of patients.Between January 2001 and December 2010, 1,540 consecutive patients with STEMI undergoing primary PCI at the Rabin Medical Center, Israel, were prospectively observed and entered into a comprehensive clinical database. Acute STEMI was defined as the presence of typical chest pain and accompanying symptoms for a duration of at least 30 minutes but < 12 hours in the presence of ST-segment elevation ≥1 mm in at least 2 contiguous leads, or new or undetermined duration of left bundle branch block in association with elevated cardiac enzymes . The registry included demographic, clinical, angiographic, procedural and echocardiographic data. This registry was approved by the ethics committee of the Rabin Medical Centre, in compliance with the Declaration of Helsinki.Patient records/information was anonymized and de-identified prior to analysis.All patients were treated with aspirin 300 mg before the PCI and clopidogrel 600 mg. Unfractionated heparin (70 U/Kg loading) was given before PCI and adjusted to achieve an activated clotting time of 200 to 275 seconds during the intervention. Glycoprotein IIb/IIIa receptor inhibition by eptifibatide was used at the discretion of the operator only after crossing the culprit lesion with a guidewire. Coronary angiography was performed through the femoral or radial rout according to the operator’s discretion. All patients had PCI performed only to the culprit vessel according to the institutional policy at the time of data collection (2001–2010). Selection of stent type, pre-dilatation with undersized balloons, and post-dilatation with larger balloons also were left to the operator’s discretion. Procedural success was defined as an angiographic residual stenosis of 20% or less by visual estimation or quantitative coronary angiography with optimized angiographic flow  flow 3). All patients were prescribed lifelong aspirin and clopidogrel (75 mg daily) for 12 months. Baseline clinical characteristics, angiographic details, quantitative coronary angiography, TIMI flow and clinical outcomes were collected.Patients were stratified according to the presence or absence of multi-vessel disease, which was defined as ≥ 70% stenosis of ≥ 2 epicardial coronary arteries or their major branches. Patients with multi-vessel disease were further classified according to whether or not they had undergone staged PCI to the non-culprit vessels based on the results of non-invasive stress testing for residual ischemia performed 4–6 weeks after the index PCI. The test was considered diagnostic when heart rate reached at least 85% of target heart rate.Exclusion criteria for the current analysis were:Cardiogenic shock at presentationPrevious coronary artery bypass grafting (CABG)Death, revascularization or re-infarction within 1 month of the index eventMultivessel disease without non-invasive testing for residual ischemia or with findings of significant ischemia following the index procedureThe study primary endpoint was the 1-year and 3-year cumulative rates of major adverse cardiovascular events (MACE) defined as a composite of all-cause mortality, revascularization (i.e. coronary artery bypass grafting and/or catheter-based target vessel revascularization) and re-infarction (excluding events occurring during the first 30 days after the index PCI). Secondary endpoints included the rates of individual components of MACE at 1 and 3-years.All events were further adjudicated by a research coordinator and reviewed by an experienced cardiologist from our research team. For each patient, a standardized questionnaire was completed either by telephone or in the outpatient clinic at 1, 6, 12, 24 and 36-month follow-ups. Mortality was confirmed by the records of the Interior Ministry of Israel. Repeat revascularization procedures and episodes of reinfarction were confirmed using the hospital as well as affiliated hospitals databases. These databases were searched for all patients in the study to gather information regarding repeat events. The diagnosis of reinfarction during follow-up was based on recurrent chest pain, suggestive of acute MI, accompanied by re-elevation of the cardiac enzyme with at least one value above the 99th percentile upper reference limit at least 48 hours after PCI and/or new ST elevation, new left bundle branch block or development of pathological Q waves in the electrocardiogram. Target vessel revascularization was defined as any revascularization that involved the target vessel. Stent thrombosis was defined according to the Academic Research Consortium definitions as “definite” in the context of acute coronary syndrome and/or reinfarction in the culprit coronary territory with angiographically proven thrombosis (thrombus or occlusion) of the previously implanted stent.t testing or Mann Whitney testing, as appropriate. Categorical variables were compared using chi-square statistics or Fischer’s exact testing, as appropriate.Data are presented as mean ± standard deviation for normally distributed variables and as median (interquartile range (IQR)) for non-normally distributed variables. Continuous variables were compared using Student’s Time-to-event curves using the Kaplan-Meier method were calculated and compared using with the Log-Rank testing. Cox proportional hazards regression model using the enter method to control for confounders that are expected to be related to long-term outcomes  was performed.All tests were two-tailed, and a P-value <0.05 was considered significant. Analyses were performed using SPSS 21.0 statistical software package (IBM SPSS Inc).A total of 905 patients were included in the present analysis, of whom 720 had single-vessel disease and 185 had multi-vessel disease (73 with 3-vessel disease) without evidence of significant residual ischemia on subsequent non-invasive stress testing .Patients with multi-vessel disease were older than patients with single vessel disease . They were more likely to have hypertension and less likely to be current or ex-smokers than patients with single-vessel disease. Previous MI was more prevalent amongst patients with multi-vessel disease than in patients with single-vessel disease . A greater proportion of patients with single-vessel disease presented with anterior MI as compared with patients with multi-vessel disease . Neither the Killip class nor the left ventricular systolic function differed significantly between the 2 groups.Mean follow up was 83.6 ± 32 months. Clinical outcomes are presented in MACE rates calculated by time to first event up to 1 and 3-years were similar between the 2 groups , Fig 2A.On cox proportional hazards regression model, multi-vessel disease without residual ischemia was not independently associated with MACE and its components at 1 and 3-years, whereas diabetes was independently associated with both MACE at 1 and 3-years and female sex for MACE at 1-year Tables .Patients with adverse outcomes occurring during the first 30 days following the procedure were not included in the final analysis . A sub-analysis of patients who experienced an adverse outcome during the first 30 days after the index event revealed that the MACE rates at 30 days did not differ significantly between patients with single-vessel disease and patients with multi-vessel disease treated with culprit-PCI only . Mortality rates at 30-days were higher in the multi-vessel group, as compared with the single vessel group  but after adjustment for confounders multi-vessel disease per se did not remain independently associated with mortality at 30-days.Multi-vessel coronary artery disease is prevalent amongst patients undergoing primary PCI for STEMI ,2. WhileWe have found that patients with STEMI and multi-vessel coronary disease treated with primary PCI of the culprit vessel-only, who had not demonstrated significant residual ischemia on subsequent non-invasive stress testing, had similar outcomes at 1 and 3-years after the index event to those patients with single vessel disease. Interestingly, 1-year mortality rates were lower in the multi-vessel disease group than in the single-vessel disease group. A possible explanation is the exclusion of patients who experienced an adverse event within the first 30 days following the index event from the analysis.We have also shown that patients with multi-vessel disease treated with culprit-only PCI had similar 30-day outcomes to patients with single vessel disease. These data may imply that it is safe to postpone stress testing following STEMI until a few weeks have elapsed.Notably in the vast majority of patients with multi-vessel disease who had undergone non-invasive stress testing in our cohort (83.7%) the non-culprit arteries were not associated with significant residual ischemia on noninvasive stress testing performed 4–6 weeks after the index event. Indeed, Hanratty et al have previously shown that a significant exaggeration of non-culprit stenosis severity occurred at the acute MI angiography in 21% of patients . MoreoveIt is noteworthy, that in contrast to the aforementioned studies, the recent PRAMI study, has found that preventive PCI at time of the index procedure (in culprit and non-culprit coronary arteries with significant stenosis) was superior to PCI limited to the culprit artery in reduction of MACE . HoweverThe current study has several limitations. First, it was a retrospective and nonrandomized single center study that was subjected to our local mode of practice. Second, only 40% of patients with multi-vessel disease underwent non-invasive stress testing. Third, we excluded patients who had an adverse event during the first 30 days following the index procedure, as patients underwent non-invasive testing usually within 4 weeks following the index procedure. Hence, the outcome analysis did not include events occurring up to 30 days after the index procedure. Information about the cause of death was not available; therefore we could only assess the rate of all cause mortality. Pervious studies have shown that approximately 30% of patients have residual stress defects following PCI and are known to be at higher risk of adverse events. However, since performing non-invasive stress testing following PPCI in patients with SVD is not common at our medical center we cannot rule out residual ischemia in patients with SVD.We have shown that patients with STEMI and multi-vessel disease treated with culprit-only PCI without significant residual myocardial ischemia on non-invasive stress testing have similar short- and long-term prognosis to STEMI patients with single vessel disease. These findings support a strategy of employing non-invasive stress testing for the assessment of ischemia in patients with STEMI and multi-vessel disease following the culprit artery PCI. Such strategy can guide the management and prevent unnecessary interventions in this STEMI patient population.S1 File(XLS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Autoimmune diseases (ADs) are associated with a number of B-cell neoplasms but the associations are selective in regard to the type of neoplasm and the conferred risks are variable. So far no mechanistic bases for these differential associations have been demonstrated. We speculate that developmental origin of B-cells might propose a mechanistic rationale for their carcinogenic response to autoimmune stimuli and tested the hypothesis on our previous studies on the risks of B-cell neoplasms after any of 33 ADs. We found that predominantly germinal center (GC)-derived B-cells showed multiple associations with ADs: diffuse large B cell lymphoma associated with 15 ADs, follicular lymphoma with 7 ADs and Hodgkin lymphoma with 11 ADs. Notably, these neoplasms shared significant associations with 5 ADs . By contrast, primarily non-GC neoplasms, acute lymphocytic leukemia, chronic lymphocytic leukemia and myeloma associated with 2 ADs only and mantle cell lymphoma with 1 AD. None of the neoplasms shared associated ADs. These data may suggest that autoimmune stimulation critically interferes with the rapid cell division, somatic hypermutation, class switch recombination and immunological selection of maturing B-cell in the GC and delivers damage contributing to transformation. Autoimmune diseases (ADs) are characterized by the reaction of T or B lymphocytes towards own (self or auto) antigens and destruction of body’s own constituents , 2. ADs We speculated that developmental origin of B-cells might propose a mechanistic rationale for their carcinogenic response to autoimmune stimuli. It has been suggested that most B-cell lymphomas derive from germinal center (GC) cells which are undergoing somatic hypermutation and class switch recombination . We hypoWe adopted the cell of origin classification of Seifert and coworkers . As GC-dAD patients were identified from 4 published studies based on the Swedish Hospital Discharge Register operating regionally since 1964 and nation-wide since 1986 . The stuPerson-years of follow-up were calculated from date of discharge with the first main diagnosis of AD until death, emigration, or closing date, December 31, 2008 or 2010. Standardized incidence ratios (SIRs) were calculated as the ratio of observed (O) to expected number of cases. Expected numbers were calculated for anyone not hospitalized for AD. The expected numbers were calculated as age, sex, period, region and socioeconomic status-specific standardized incidence rates. Additional adjustments were made for hospitalization for obesity, chronic obstructive pulmonary disease (surrogate of smoking) and alcohol use disorders. We show data for ADs with at least 3 neoplasms in the most common AD category. Differences were called ‘significant’ when the 95% confidence intervals (95%CIs) did not overlap with 1.00.The study was approved by the regional ethical review board at Lund. The methods were carried out in accordance with the approved guidelines; only anonymized data and no human tissues were used.In Our studies on AD and cancer have been very large as they have covered all hospitalized cases of AD in Sweden over an extended period. However, an essential question is whether they find support in other studies and whether they can be generalized. The international InterLymp study reported associations of NHL subtypes with 16 ADs (924 cases) . The stuThe InterLymph data as well as ours on B- and T-cell NHL in AD patients showed that both types of cells were vulnerable to transformation by autoimmune stimulation and that there was not much selectivity whether the initial immune activation was through B-cells  or T cells  , 11. T-cIn conclusion, we propose here a model, based on the developmental origin of B-cells, explaining why autoimmune stimulation differentially affects B-cell neoplasms and why certain ADs appear to exert selectivity among the target neoplasms and the magnitude of risk. A further mechanistic understanding of the cellular events in B-cell neoplasia will help to validate the model.


Gasterosteus aculeatus, Linnaeus 1758) and preferred by nine-spined stickleback . After 1979, the separation between both sticklebacks became weaker, corresponding to a decreased influence of local groundwater on stream water quality. The way both species changed their distribution in the field provides a strong indication that they differ in their susceptibility to iron-rich conditions. These observed differences correspond with differences in their respiration physiology, tolerance of poor oxygen conditions and overall life-history strategy documented in the literature. Our results exemplify how species can partition niche along a non-structural niche axis, such as sublethal iron-rich conditions. Other fish species may similarly segregate along concentration gradients in iron, while sublethal concentrations of other metals such as copper may similarly impact fish via respiratory impairment and reduced aerobic scope.Acute exposure to iron can be lethal to fish, but long-term sublethal impacts of iron require further study. Here we investigated whether the spatial and temporal distribution (1967–2004) of two closely related species of stickleback matched the spatial distribution of iron concentrations in the groundwater. We used the ‘Northern Peel region’, a historically iron-rich peat landscape in The Netherlands as a case study. This allowed us to test the hypothesis that niche segregation in two closely related species of stickleback occurred along a physiological axis. Patterns in stickleback occurrence were strongly associated with spatial patterns in iron concentrations before 1979: iron-rich grid cells were avoided by three-spined stickleback (The online version of this article (doi:10.1007/s10452-012-9395-y) contains supplementary material, which is available to authorized users. Metal toxicity can cause a decline or loss of fishery resources  on the three-spined stickleback  is due to their acidity and that the ferric ion (Fe3+) has little or no lethal effect. Although not fatal, iron-induced respiratory impairment may decrease aerobic scope (the capacity to perform aerobic activity), which likely impacts growth, reproduction and population dynamics in the long term  are currently not available, ecological studies unequivocally show that the nine-spined stickleback has a superior tolerance to poor oxygen conditions  was redirected and used to feed these streams to ensure continuous water supply for agricultural reasons  . Grid cells located on the boundary of the iron-rich subregion were assigned to the same group as the upstream grid cell. This map was reported in van den Munckhof  and base2 grid cells in a certain year. Historical literature  and from the total number of sampled grid cells (60 in the first period versus 240 in the second period). From these numbers, it follows that sampling intensity (number of sampling occasions averaged per grid cell) roughly halved .While this does not affect the spatial comparison (iron-rich versus iron-poor), it does put restrictions on the temporal comparison (period 1 vs. period 2), as an expanded distribution could in principle result from the greater spatial coverage of sampling. However, we can document temporal shifts in the distribution of the two species relative to each other and investigate how spatial patterns of co-occurrence have changed over time, because both species are easy to catch and identify and any sampling bias towards any one species is unlikely. Finally, calculating changes in the number of records can be done by applying a straightforward correction for sampling intensity .Gobio gobio [L 1758]) increased strongly in the second period; Verberk et al. Our analysis focused on the Northern Peel region and analysed stickleback distribution patterns using all stickleback records before 1979 (560 records) and after 1979 (949 records). Grid cells were categorised into three groups, based on whether only three-spined stickleback was recorded (group 1), only nine-spined stickleback was recorded (group 2), or both species co-occurred (group 3). Since the focus of this study was on changes in co-occurrence of the two species, grid cells that were not sampled and those that were sampled but did not yield any sticklebacks (‘empty’ grid cells) were excluded from the analysis. Given that sampling effort was not documented in the database (see above), a change in the number of these ‘empty’ grid cells could be also be caused by increases in non-focal species leading to spurious results if we had included them (e.g. gudgeon (To test for differences in species (co-)occurrence across space and time, we performed a log-linear analysis on counts of grid cells in each of the three groups, with period (before/after 1979) and region (iron rich/iron poor) as explanatory variables. The log-linear analyses employed here are a specialised case of generalised linear models for Poisson-distributed data. This version of chi-square analysis, suited for 3-way contingency tables, is best suited to analyse the type of data reported here, which deals with counts and discrete blocks Fienberg . For eacOur study revealed marked differences in the distribution patterns of three- and nine-spined stickleback. In the first period (1967–1978), the nine-spined stickleback was the most wide ranging species occupying 58 grid cells, compared to only 34 grid cells for the three-spined stickleback (Table S1). The distribution of the three-spined stickleback was balanced towards the iron-poor region, while the nine-spined stickleback was equally distributed across both regions Fig. a. In theG2 = 32.98; df = 7; P < 0.0001; Table S1; Fig. S1). In the first period, the majority (~76 %) of iron-rich grid cells were occupied exclusively by nine-spined stickleback, while the majority of iron-poor grid cells were shared by both species. Virtually none of the grid cells (~3 %) were exclusively occupied by three-spined stickleback. In the second period, more grid cells (~17 %) were exclusively occupied by three-spine stickleback, and the difference between iron-rich and iron-poor grid cells was much reduced. The fraction of iron-rich grid cells occupied exclusively by nine-spined stickleback was markedly lower (reduced from ~76 to ~42 %).The spatial pattern of co-occurrence of both species differed between regions, and this pattern changed over time  and the nine-spined stickleback . The patterns in record counts were largely congruent with those for grid cells described above. The number of records for three-spined stickleback was much higher in the iron-poor region in the first period compared to the iron-rich region. Given that an almost equal number of grid cells was sampled in both regions, this indicates that three-spined stickleback avoided the iron-rich region . This avoidance disappeared in the second period . Nevertheless, the sublethal effects can affect fish distributions on the long term, possibly by reducing aerobic scope and altering competitive strength. Niche partitioning is considered an important mechanism to reduce competition between species and promote their coexistence Gause . The difThe preference of nine-spined stickleback to reside and nest in dense vegetation, away from the bottom substratum where oxygen deficiency is likely more pronounced Below is the link to the electronic supplementary material.


We analysed 8929 regions harboring genes for potential long non-coding RNA that were retrieved from data from the ENCODE project. Of these regions, 60% were annotated as intergenic, which implies that they do not overlap with protein-coding genes. Focusing on the intergenic regions, and using stringent analysis of the allele-specific expression data, we detected robust cis-regulatory SNPs in 258 out of 489 informative intergenic regions included in the analysis. The cis-regulatory SNPs that were significantly associated with allele-specific expression of long non-coding RNAs were enriched to enhancer regions marked for active or bivalent, poised chromatin by histone modifications. Out of the lncRNA regions regulated by cis-acting regulatory SNPs, 20% (n = 52) were co-regulated with the closest protein coding gene. We compared the identified cis-regulatory SNPs with those in the catalog of SNPs identified by genome-wide association studies of human diseases and traits. This comparison identified 32 SNPs in loci from genome-wide association studies that displayed a strong association signal with allele-specific expression of non-coding RNAs in monocytes, with p-values ranging from 6.7×10−7 to 9.5×10−89. The identified cis-regulatory SNPs are associated with diseases of the immune system, like multiple sclerosis and rheumatoid arthritis.We applied genome-wide allele-specific expression analysis of monocytes from 188 samples. Monocytes were purified from white blood cells of healthy blood donors to detect  ASE analysis is more powerful for detecting cis-regulated gene expression than the total expression levels of genes determined by regular eQTL analysis cis-regulatory SNPs (cis-rSNPs) stems from the fact that the differential expression of the two alleles of a SNP are measured in the same environment and have been exposed to the same environmental conditions in the cells from which the RNA was extracted trans-acting and environmental factors that may affect gene expression are controlled for in ASE analysis. To adjust for possible methodological differences in the efficiency of the genotyping assay to measure the two alleles of a SNP, such as the sequence context of a SNP or copy number variations, the relative amounts of the alleles measured in DNA, is used as a reference for quantification of the allelic expression. Genome-wide ASE analysis by SNP genotyping has been applied to map cis-regulation of protein coding genes associated with human diseases and traits in lymphoblastoid cell lines Determination of allele-specific gene expression (ASE) levels by quantitative genotyping of heterozygous SNPs on the RNA level cis, and especially with genes that overlap with the antisense strand of a lncRNA. By investigating the effect of regulatory SNPs on the expression of lncRNAs using traditional microarray-based expressed quantiative trait locus (eQTL) analysis of peripheral blood cells a recent study identified 112 cis-regulated lncRNAs Long non-coding RNAs (lncRNAs) are involved in gene regulation and other cellular processes. LncRNAs such as XIST and TSIX, which are involved in X chromosome inactivation cis-acting SNPs that regulate the expression of lncRNAs and to highlight previously unknown roles for lncRNAs in human complex diseases. We reasoned that ASE-analysis that is highly sensitive for detecting cis-rSNPs would be a powerful tool for studying cis-regulation of lncRNAs that are expressed at lower levels than protein-coding genes. To our knowledge ASE-analysis has not been previously used for studies of lncRNAs.The aim of our study was to apply ASE-analysis to identify cis-acting regulatory SNPs out of which 3910 are located in intergenic regions and 571 of these are located in enhancer regions marked as active or poised by histone modifications in monocytes. We also compared the indentified SNPs that regulate expression of lncRNAs with risk SNPs for complex diseases and traits previously identified in genome-wide association studies (GWAS). In this way we were able to obtain new functional clues for these disease associated GWAS loci. Additionally, we analysed co-expression between lncRNA genes and nearby protein coding genes, and found that 20% of the intergenic lncRNAs with a cis-rSNP were co-regulated with a protein-coding gene.We analysed RNA extracted from human monocytes purified from white blood cells of 188 healthy blood donors using a genome-wide panel of SNPs. Monocytes were selected for analysis because they are a relevant cell type for multiple diseases. Our analysis included 8929 genomic regions that harbor genes for potential long non-coding RNA that were retrieved from the GENCODE database Circulating monocytes were collected from healthy adult blood donors of European origin (n = 188) recruited from the United Kingdom National Blood Service Centre in Cambridge, UK as part of the Cardiogenics Transcriptomic Study DNA was extracted from peripheral blood leukocytes using the guanidine hydrochloride - chloroform method. CD14+ magnetic microbeads  were used to isolate monocytes from whole blood. RNA was extracted from cell pellets of freshly isolated monocytes by homogenization with Trizol-reagent , chloroform-ethanol extraction and purification using Qiagen RNAeasy columns and reagents, followed by on-column DNase treatment. cDNA was synthesized using reagents from the Illumina TotalPrep RNA Amplification Kit, except that the poly-dT primers were substituted by random decamers .All participants in the study provided their written consent after a personal meeting with a study nurse. Written consent from each participant was recorded by name, date and signature on a standardized consent form. Both the consent form and the study has been reviewed and approved by the Cambridgeshire 1 Research Ethics Committee. The Research Ethics Committee is independent from the research institute and part of the UK Health Research Authority.A total of 8929 genomic regions containing lncRNAs were retrieved from the GENCODE version 7 database cis-rSNPs and the ASE-levels of lncRNAS in a genomic region. Genotypes were called in gDNA using Genome Studio version 2009.2 (Illumina) with a call rate of 99% as the threshold for genotype calls for SNPs and 98% for samples. SNPs were further filtered on deviations from Hardy-Weinberg equilibrium with a p-value cutoff of 10−6 (Chi-squared test). One sample with higher than 40% call rate in cDNA were removed due to possible DNA contamination. The raw two-colour fluorescence signals from the Infinium assay were normalized to remove dependency of the allele fractions on the signal intensities of the fluorophores using a quadratic function with medians of bins for the two fluorescence colors. From the regression, the intensities were predicted based on the log10 values of the raw signal, which were used to adjust the allele fraction, see cDNA/(Allele1cDNA+Allele2cDNA)] – [Allele1gDNA/(Allele1gDNA+Allele2gDNA)]. Here, gDNA, where the two SNP alleles are present in 1∶1 ratio serves as a quantification standard for the relative expression levels of the two alleles in RNA.RNA (cDNA) and genomic DNA (gDNA) from the monocyte samples were genotyped using the Infinium assay and Human 1.2 M Duo custom BeadChips  as described previously cis-rSNPs  was 42797. To retain only protein coding genes, all genes annotated as RNA genes in the GeneCards database Protein coding regions extracted from the Refseq database were analysed to detect SNPs with association signals that overlap with those from lncRNAs. The total number of transcripts in Refseq  data was retrieved from the Blueprint project cis-rSNP were correlated with gene expression of the closest protein coding gene. For this purpose we divided the samples into three groups based on three genotype combinations of the most significantly associated cis-rSNP . For each expressed gene region we calculated the average signal intensity for both alleles and used this value as a measure of gene expression levels. Next, we performed linear regression analysis with the three genotype groups as x-values and the average total expression levels as y-values in order to obtain a p-value for the co-expression.We investigated to what extent expression of lncRNA genes with a cis-regulation of 8929 genomic regions harboring lncRNA from the ENCODE project using allele-specific expression (ASE) analysis of 188 RNA samples from human primary monocytes. To determine ASE, we genotyped RNA (cDNA) and genomic DNA from the monocytes using a genome-wide panel of 1.2 million SNP markers We explored The definitions of exon and intron boundaries were taken from Refseq. As protein coding genes are on average expressed at higher levels than lncRNAs, the ASE signal for lncRNA that overlap with protein coding genes is hard to distinguish from that of a protein-coding gene. Because of this, we focus on the intergenic lncRNA regions where the ASE data is most reliable.−6, based on the number of tests performed and adjusted for SNPs in high LD (>0.9), was used to correct the association signals for multiple testing. In addition a threshold of 0.05 for the slope of the regression line was applied to exclude SNPs with low effects on ASE. This value corresponds to approximately a 20% expression difference between the two alleles and was set as a reasonable threshold for biological relevance. To avoid false positives with inflated p-values, we only retained associations that were based on at least four data points for each allele combination for the calculation of the p-value cis-rSNP (−6) in the 188 analysed monocyte samples, and for the intergenic lncRNA regions 3910 out of 79628 tested SNPs (4.9%) were significantly associated cis-rSNPs.For the ASE analysis, the genotypes of 194530 SNPs located within 250 kb upstream and downstream of the lncRNA regions were tested for their association with the ASE levels of the corresponding expressed lncRNAs, using linear regression, see Manhattan plot in cis-rSNP . The asscis-rSNP out of 108881 tested SNPs (7.9%) in 471 out of 10345 regions (4.6%) that were associated with ASE of a Refseq gene.To allow comparison between ASE of lncRNA genes and protein coding Refseq genes, we applied the same procedure for ASE analysis to the Refseq gene regions as for the lncRNAs. This analysis identified 8604 cis-rSNPs located in enhancer regions marked as active or poised by histone modifications in monocytes. To determine if the cis-rSNPs for lncRNAs are enriched in enhancer regions, we calculated the fold enrichment of significantly associated cis-rSNPs defined by ASE analysis of intergenic lncRNA regions to histone marks for active enhancers (H3K27ac) and bivalent, poised enhancers (H3K4me1) in monocytes. The fold enrichment was determined by comparing the fraction of significantly associated SNPs in enhancer regions with the fraction of non-significant  SNPs in enhancer regions. Of the significantly associated SNPs, 2.1% were located in H3K27ac peaks compared to 1.2% for non-significantly associated SNPs, which corresponds to a 1.72-fold enrichment . For H3K4me1 we observed a 1.54-fold enrichment  of significantly associated SNPs, with 12.5% of the significant SNPs located in the enhancer peaks, while this number was 8.1% for non-significant SNPs. For protein coding genes the percentages and fold enrichment are similar to those for lncRNA for H3K27ac with 2.2% and 1.5% for significant and non-significant SNPs, respectively, and 1.43 fold enrichment  and less pronounced for H3K4me1 with 9.8% and 8.5% for significant and non-significant SNPs, respectively, and 1.15 fold enrichment . This result shows that enhancers have a regulatory cis-effect on expression of not only protein coding genes, but also lncRNAs.We identified 571 cis-rSNP. In our data 52 (20%) of the identified intergenic lncRNA regions to which a cis-acting regulatory SNP was associated were also correlated with the expression level of a protein-coding gene at a false discovery rate (FDR) of 10%, suggesting co-regulation of the genes, see cis-rSNPs are significantly associated both with ASE of a lncRNA and ASE of the closest protein coding gene, which implies that the expression of the lncRNA and the protein coding genes are regulated by the same cis-rSNP. For 19 cis-rSNPs the ASE signal is unique to a lncRNA, which could be an indication of involvement of the lncRNA in the regulation of the expression of an adjacent protein coding gene as has been suggested in previous studies cis-rSNPs had a distance >250 kb to the nearest protein coding gene and the ASE signal could therefore not be evaluated.Next we investigated whether the lncRNA genes and nearby protein coding genes are co-expressed and co-regulated by a cis-rSNP associated with ASE of intergenic lncRNAs in monocytes with SNPs identified in GWAS of human diseases and traits, listed in the National Human Genome Research Institute (NHGRI) GWAS Catalog th of January 2013) includes 9617 entries, 641 traits and 7797 unique SNPs. We identified SNPs associated with 32 loci high-lighted by GWAS that are also cis-rSNP for lncRNAs. The cis-rSNP are the same as the GWAS associated SNP for 25 of the loci, while seven cis-SNPs are linkage disequilibrium (LD) proxies to the lead GWAS SNPs, with five SNPs having LD = 1.0 and two having LD> = 0.88. cis-rSNP associated with ASE of lncRNA regions as well as the phenotypes to which these SNPs were associated in GWAS. cis-rSNP that were significantly associated with ASE of a lncRNA are located in an intron of a protein coding gene, illustrating a direct or indirect regulatory function of an intronic SNP. One locus is reported by GWAS to be associated with a non-coding gene (genomic region: 2:19067188–19369296), which we confirm here by a cis-rSNP for the same lncRNA gene. Furthermore, when searching for overlaps between cis-rSNP associated with lncRNA and GWAS SNPs that are located in active enhancer regions in monocytes, we detected three GWAS SNPs that are located in a ChIP-seq peak for H3K4me1, and seven additional SNPs that are in the 2.5 kb enhancer regions flanking the histone mark. For H3K27ac no overlap between the cis-rSNP and the actual ChIP-seq peak was found, but five SNPs were located in the 2.5 kb flanking region. The number of overlapping GWAS SNPs – cis-rSNPs that map to an enhancer region are enriched at the same level as cis-rSNPs in general. For these GWAS SNPs the effect on the enhancer could be the functional mechanism.Next we superimposed the cis-rSNP or a proxy cis-rSNP in high LD . An advantage of RNA-seq compared to ASE analysis using SNP genotyping is that alternatively spliced transcripts and strand specific gene expression can be detected, provided that the sequencing coverage is sufficient. However, ASE analysis using RNA-seq suffers from the drawback of being highly affected by the reference bias The combination of a large number of samples from primary monocytes and the sensitive genotyping method for detecting cis-rSNP. We observed correlated expression of 20% of the lncRNAs with their neighbouring protein-coding genes, and co-regulation of a lncRNA and a neighbouring protein coding gene by the same cis-rSNP for approximately half of these lncRNA genes. This finding is consistent with a recent study of peripheral blood cells, where 75% of 112 lncRNAs that were mapped using traditional eQTL analysis were found to be regulated independently of nearby protein-coding genes cis-regulation, ASE analysis allows the dissection of cis-effects by lncRNAs that influence the transcription of nearby genes. If the expression level of a lncRNA and a nearby protein coding gene are correlated, and they both show significant ASE association with the same cis-rSNP, it is likely that the cis-rSNP directly regulates the protein coding gene. If the expression levels between a protein-coding and lncRNA gene are correlated, but there is no shared association with a cis-SNP, the lncRNA may be a regulatory factor that affects the expression of the adjacent protein coding gene. In our ASE data set we found that these two putative mechanisms occur at similar frequencies. However, using the ASE approach, trans-acting regulatory mechanisms for co-expressed genes would remain undetected. A recent study used traditional eQTL analysis of monocytes found several trans-regulated modules of co-expressed protein coding genes. However, lncRNA expression was not addressed in this study In this study we focused our ASE analysis of monocytes on 5346 lncRNAs that were annotated by the ENCODE project as intergenic without overlap with protein coding genes. Thus we ensured that our analysis targeted truly lncRNA genes and was not confounded by overlapping protein-coding genes. Using stringent criteria for calling ASE, we identified 258 lncRNAs that were regulated by at least one A drawback of our study is that this specific dataset was not replicated by an independent method. However, in a previous study we have shown that a similar method for genome-wide ASE-analysis using NS-12 BeadChips (Illumina) was highly reproducible between replicate samples, with correlation coefficients of 0.9969 for gDNA and 0.9956 for cDNA. ASE-levels determined using NS-12 BeadChips and those determined by quantitative Sanger sequencing were also strongly correlated (0.86) in nine representative genes cis-rSNPs that regulate the expression of lncRNAs have been identified in GWAS as risk variants for human diseases or traits. The diseases or phenotypic traits associated with these cis-regulatory SNPs according to the lncRNA ASE analysis, include diseases of the immune system; multiple sclerosis, Graves’ disease, eosinophilic esophagitis, celiac disease, rheumatoid arthritis, immune response to small pox vaccine, soluble E-selectin levels, soluble ICAM-1, and ankylosing spondylitis, for which monocytes are a relevant cell type . Figure adapted from Almlöf et al(PDF)Click here for additional data file.Figure S2Signal intensity threshold. The figure shows the distribution of signal intensities for the two alleles. The x-channel represents the A-allele and the y-channel represents the B-allele. In the xraw panel the fraction of SNPs with the BB genotype having intensity levels above the cutoff level of 1000 is very low and similarly for the yraw panel.(PDF)Click here for additional data file.Figure S3Regression lines. Regression lines for the 32 risk SNPs from GWAS that are significantly associated with a lncRNA region.(PDF)Click here for additional data file.Figure S4lncRNA in the GWAS catalog. Illustration of all lncRNA regions that have a significant association to a SNP that is also significantly associated in the GWAS catalog. The tracks are from top to bottom in each panel: Horizontal red bars represent transcript windows (with genomic coordinates) used for determination of ASE levels; grey lines show p-values for the association of GWAS SNPs with ASE levels in the transcript window; a grey line overlayed with a red dotted line indicates that this is the SNP that overlaps with the reported SNP in the GWAS catalog; red vertical lines are median ASE-levels for each SNP; annotated transcripts are shown in black below the tracks.(PDF)Click here for additional data file.Table S1Significantly associated lncRNA regions.(DOCX)Click here for additional data file.Table S2SNPs associated with allele-specific expression of Refseq protein coding genes with published trait- or disease-associations from genome-wide association studies.(DOCX)Click here for additional data file.Methods S1Quadratic normalization. Detailed explanation of the quadratic normalization performed on the intensity levels obtained from the genotyping.(DOCX)Click here for additional data file.


Bell pepper endornavirus (BPEV) is a double-stranded RNA virus infecting economically important crops, such as peppers. Next-generation sequencing of small RNAs extracted from the leaves of a pepper plant showing mild viral symptoms, along with subsequent analysis, identified BPEV. The complete genome of this isolate was cloned and sequenced. Endornavirus, a young genus in the family Endornaviridae  showing mild crinkling and chlorosis symptoms on young leaves with the mirPremier microRNA isolation kit . Small RNA libraries were constructed with the TruSeq small RNA sample prep kit , according to the supplier’s instructions, and sequenced using the MiSeq desktop sequencer (Illumina) with the MiSeq version 2 reagent 50-cycle paired-end (PE) kit (Illumina). After the removal of adapter sequences, the clean reads were assembled into large contigs. Searches against the NCBI database using the assembled contigs hit the genome sequence of BPEV with high (>98%) sequence similarity. Based on the contig sequences, primers were designed to clone the nearly full-length cDNA of this Canadian isolate. The 5′ and 3′ end sequences were obtained by rapid amplification of cDNA ends (RACE). The genome sequence of the Canadian isolate was determined to be 14,726 nucleotides (nt) in length. Like other endornaviruses, the genome of this isolate has a single open reading frame encoding a polyprotein containing viral methyltransferase (MTR), RNA helicase 1 (Hel-1), UDP-glucose-glycosyltransferase (UGT), and RNA-dependent RNA polymerase (RdRp). A comparison of the genome sequences revealed that the Canadian isolate shares sequence identity to the other five isolates in a range of 88 to 99% at the nucleotide level and 92 to 99% at the amino acid level. Phylogenetic analysis using the RdRp sequences was conducted. The results showed that this Canadian isolate is distinct from the YW isolate from the United States and the remaining four isolates from four different countries, which form a phylogenetic group. Taken together, we identified and determined the genomic sequence of a BPEV isolate that apparently has ability to induce typical viral symptoms in peppers.KT149366.The genome sequence of the isolate described in this work has been deposited in GenBank under the accession no.


P = .046). Use of this assay as an additional tool for managing allo-HCT recipients at risk for CMV reactivation needs further validation in future studies. Application of this new approach may reduce the duration and intensity of CMV monitoring and the duration of prophylaxis or treatment with antiviral agents in those who have achieved CMV-specific immune reconstitution.The ability to distinguish allogeneic hematopoietic cell transplant  recipients at risk for cytomegalovirus (CMV) reactivation from those who are not is central for optimal CMV management strategies. Interferon γ (IFN-γ) produced by CMV-challenged T cells may serve as an immune marker differentiating these 2 populations. We prospectively monitored 63 CMV-seropositive allo-HCT recipients with a CMV-specific enzyme-linked immunospot (ELISPOT) assay and for CMV infection from the period before transplantation to day 100 after transplantation. Assay results above certain thresholds (50 spots per 250 000 cells for immediate early 1 or 100 spots per 250 000 cells for phosphoprotein 65) identified patients who were protected against CMV infection as long as they had no graft-versus-host disease and/or were not receiving systemic corticosteroids. Based on the multivariable Cox proportional hazards regression model, the only significant factor for preventing CMV reactivation was a CMV-specific ELISPOT response above the determined thresholds (adjusted hazard ratio, 0.21; 95% confidence interval, .05–.97;  Cytomegalovirus (CMV) is a common cause of significant morbidity and mortality in patients with impaired cellular immunity, including patients with hematologic malignancies and recipients of solid organ and allogeneic hematopoietic cell transplants  –3. Like In immunocompromised hosts, the production and replication of CMV may lead to infection and a variety of end-organ diseases, such as retinitis in patients with AIDS and pneumonitis in HCT recipients. CMV has also been associated with rejection after solid organ transplantation , 6 and wIn allo-HCT recipients, prophylaxis and/or preemptive therapy have been used to manage CMV infection. Owing to the toxic effects of the antiviral agents presently available, the prophylactic approach has fallen out of favor for a preemptive strategy. Despite the lack of a well-established, validated CMV load or antigenemia threshold for the initiation of therapy, the preemptive strategy is currently the most widely accepted practice for preventing CMV-associated end-organ disease after allo-HCT , 3, 11.+ and CD8+ T cells are implicated in the protection against CMV infection; complex interactions involving a CD8+ T-cell response produce interferon γ (IFN-γ) and a host of other cytokines in response to the presence of CMV [+ T cells develop CMV infection more frequently than those who have these cells, and reconstitution of CD8+ T-cell responses correlates with protection against CMV [+ and CD8+ T cells. Patients infected with CMV have T cells that recognize these antigens and secrete IFN-γ in response; the detection of individual IFN-γ–producing cells is the basis of the assay and allows the assessment of the CMV-specific activity of the immune system.Cell-mediated immunity is considered the primary defense mechanism for controlling CMV replication . Both CDe of CMV . HCT recinst CMV , 15. IFNinst CMV . The relOne of the major challenges in treating allo-HCT recipients is the ability to distinguish patients at high risk for CMV reactivation who require close monitoring from those who have achieved a level of immune reconstitution sufficiently adequate to control latent CMV from reactivating. We hypothesize that the IFN-γ produced by CMV-challenged T cells can serve as an adaptive immune marker differentiating these 2 populations. This study is a proof-of-concept study to assess the ability of the CMV-specific ELISPOT assay to identify CMV-seropositive allo-HCT recipients who have achieved immune reconstitution and so are not at risk for CMV reactivation.For this prospective cohort study, conducted between October 2013 and June 2014, we enrolled CMV immunoglobulin–positive adult patients with leukemia who were in remission before they underwent allo-HCT. Patients were serially monitored until day 100 after transplantation via the CMV-specific ELISPOT assay and for CMV reactivation. Written informed consent was obtained from each participant, and all data collection and laboratory procedures were approved by the University of Texas MD Anderson Cancer Center Institution Review Board.CMV infection was managed in patients according to the standard of care at our institution, which consists of the preemptive strategy previously described . BrieflyWe collected data on various clinical characteristics, such as age, sex, race, CMV serostatus, type of transplant and conditioning regimen, results of routine laboratory tests , and all positive microbiological data, including CMV antigenemia and blood culture findings. In addition, we also collected data on GvHD; immunosuppressive therapy; anti-CMV antiviral therapy, including prophylaxis and/or treatment; and work-ups related to possible CMV disease, such as computed tomography findings and bronchoscopic, colonoscopic, and ophthalmic examinations.The primary end point of the study was the first episode of significant CMV reactivation, defined as the detection of CMV via the antigenemia assay in the patient's blood, after which anti-CMV therapy was initiated by the treating physician in accordance with our institutional guidelines. The secondary end point was CMV-associated end-organ disease as defined by Ljungman et al .Blood specimens were collected at 4 consecutive time points: before the initiation of the transplant conditioning regimen (pre-HCT) and day 30 (±7 days), day 60 (±7 days), and day 100 (±14 days) after transplantation. The samples were shipped on the same day as collection to Oxford Diagnostic Laboratories , where the CMV-specific ELISPOT assay was performed within 32 hours of blood specimen collection, in accordance with the validated test procedures. When the cell count ranged from 75 000 to <250 000 cells per well, spot counts for both IE-1 and pp65 antigens were standardized by multiplying the spot counts by a correction factor to adjust to the optimal cell count of 250 000 cells per well. If the cell counts were <75 000 cells per well, the results were excluded from the analyses because of an insufficient cell count. In addition, nil control spot counts were subtracted from the spots count obtained for IE-1 and pp65 if the nil control was ≤10, and the sample results were excluded from analyses if the nil control was >10. The laboratory personnel were blinded to the patients' clinical information to minimize potential bias. The results of the assay were reported in batches to the research team; however, they were not available to the treating physicians and thus did not affect treatment decisions.2 or Fisher exact tests, for categorical variables, and Student t tests, for continuous variables. Bivariable Cox proportional hazards regression analysis was used to discretize the 2 continuous variables into binary variables to identify their thresholds for predicting subsequent significant CMV reactivation . Further, patients were classified into 2 categories: those with a high response to the CMV-specific ELISPOT assay (if the level of any antigen was above the aforementioned threshold) and those with a low response to the CMV-specific ELISPOT assay (if levels of both antigens were below the aforementioned threshold). Kaplan–Meier failure curves were generated to demonstrate the difference in the probability of significant CMV reactivation between the patients with low and those with high responses, using values from both antigens in the CMV-specific ELISPOT assay performed before such reactivation. Finally, a multivariable Cox proportional hazards regression model was built to identify the effect of CMV-specific ELISPOT assay results on subsequent significant CMV reactivation, when adjusted for known risk factors. A 2-sided P value of .05 was considered statistically significant. All statistical analyses were performed using Stata, version 13.0 .The first episode of significant CMV reactivation was the primary outcome of interest for these analyses. Clinical characteristics of allo-HCT recipients who did and those who did not develop significant CMV reactivation during the study period were compared using χSupplementary Figure 1). No statistically significant differences in baseline characteristics were observed between those who experienced significant CMV reactivation and those who did not . The majority of patients were white (78%), male (59%), and undergoing allo-HCT while in remission from acute leukemia (60%). More than half of the patients underwent HCT with a transplant from a matched-unrelated donor (56%), and most received a myeloablative conditioning regimen (94%). A total of 22 patients (35%) received hematopoietic cells from a CMV-seronegative donor, and 19 patients had developed GvHD and/or received corticosteroids for suspected or confirmed GvHD before CMV reactivation. Interestingly, the first episode of significant CMV reactivation  occurred in 23 patient (37%) within the first 60 days after transplantation . Patients with a high response at day 30 after HCT  had a lower chance of developing CMV reactivation than those with a low response . Patients who received matched-unrelated donor or cord blood transplants were more likely to have reactivation than those who received a matched-related donor transplant; however, this factor was not significant after adjustment for other variables in the multivariable model. None of the other variables were significant predictors of CMV reactivation  and those who did not  with a persistent low response at day 60 had a second episode of CMV reactivation.None of the patients developed CMV-associated end-organ disease, but 6 had a relapse of leukemia during the study period. A total of 8 patients (13%) died within 100 days after transplantation; however, none of the deaths were related to CMV.For the IE-1 antigen, the sensitivity for identifying allo-HCT patients at high risk for CMV reactivation in case of a low response was 96%, and the negative predictive value (NPV) denoting protection in case of a high response was 86%. For the pp65 antigen, the sensitivity for identifying allo-HCT patients at high risk for CMV reactivation in case of a low response was 91%, and the NPV denoting protection in case of high response was 88%. The overall accuracy for both antigens combined was similar .In this study, we evaluated the feasibility of the CMV-specific ELISPOT assay for measuring IFN-γ release as a marker of protection against CMV reactivation in patients with leukemia following allo-HCT. We observed that patients with a high CMV-specific ELISPOT assay response had no risk of developing significant CMV reactivation, compared with those with a low response, unless they had GvHD and/or were treated with systemic corticosteroids. These results support use of the CMV-specific ELISPOT assay to determine whether patients are protected against CMV reactivation following transplantation.The major drawback of the preemptive approach is the uncertainty as to which patients remain at risk and require continuous monitoring and which patients have an immune system sufficiently reconstituted to control latent CMV infection, thereby obviating the need for further monitoring.A more personalized laboratory-guided preventive strategy can be developed by precisely identifying the HCT recipient's immune protection against CMV reactivation. CMV-specific ELISPOT assay results above certain thresholds (50 spots for IE-1 and 100 spots for pp65) identified patients who, as long as they were not treated with corticosteroids, were protected against CMV infection, as seen in 2 patients who had a high CMV-specific ELISPOT assay response at day 30 but a week later developed GvHD, which required treatment with corticosteroids. Four days after the initiation of steroid therapy, these 2 patients developed CMV reactivation, indicating in our opinion that GvHD and/or steroids may negate the protective effect that was measured by the CMV-specific ELISPOT assay. We recommend repeating the CMV-specific ELISPOT assay for patient for whom a change a circumstances might reasonably be expected to have yielded diminished T-cell function since a prior test result. It is also conceivable that a lack of protection against CMV reactivation in the presence of CMV-specific T-cell response may be explained by innate immune deficiencies such as deficiency of natural killer cells  or failuHLA-restricted T-lymphocyte cytotoxic responses have also been associated with recovery from CMV infection in allo-HCT recipients . Lower mWe demonstrated that allo-HCT recipients with higher numbers of CMV-specific IFN-γ–producing memory T cells were protected against CMV reactivation. This finding is consistent with other studies reporting similar clinical protection against CMV infection in both CMV-seropositive and CMV-seronegative kidney transplant recipients , 24. In CMV-seropositive patients receiving allo-HCT from seronegative donors are at higher risk of developing CMV reactivation . When adOn the basis of our findings, we propose the examination of a new approach that includes immune monitoring for CMV in allo-HCT recipients during the posttransplantation period Figure . This apThe overall benefit of the proposed immune-monitoring approach over the current preemptive strategy will be manifold. Application of this new approach may help reduce the duration and intensity of CMV monitoring and the duration of antiviral therapy or of prophylaxis, for those who have achieved CMV-specific immune reconstitution. In addition, and most importantly, the current preemptive strategy leads in many instances to excessive and prolonged duration of therapy among patients who might be able to spontaneously clear a low level of CMV reactivation if CMV-specific immune reconstitution is achieved. The ability of CMV-specific ELISPOT assay to identify patients who probably do not need therapy or further monitoring should be examined. Finally, the role of the CMV-specific ELISPOT assay response as a prognostic factor for clinical outcomes such as CMV-associated end-organ disease, graft outcome, and all-cause mortality in HCT recipients should be determined in future studies.In conclusion, the CMV-specific ELISPOT assay appears to be an important diagnostic tool for identifying patients no longer at risk for CMV infection, following allo-HCT. The findings from this study should be confirmed in a multicenter trial.Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.


The aim of this work is to evaluate perioperative hemodynamic monitoring for major abdominal surgery using the estimated continuous cardiac output (esCCO) technique available with the NIHON KOHDEN Vismo monitor as compared with the LiDCOrapid hemodynamic monitoring system.The esCCO technique is a novel noncalibrated non-invasive method of cardiac output monitoring. It is based on the estimation of pulse pressure from the speed of pulse wave propagation, which is measured as a time delay between the ECG R-wave and the corresponding peripheral pulse wave detected by pulse oximetry - the delay being called the pulse wave transit time (PWTT) . LiDCOraTen patients were monitored with a total of 141 esCCO-nCO measurement pairs. The correlation coefficient between esCCO and nCO was 0.65 and in the Bland-Altman difference analysis bias was + 1.2 l/minute and 95% limits of agreement were ± 2.6 l/minute. The change of cardiac output between two consecutive measurements detected by LiDCOrapid was detected by esCCO in 80% of cases.Hemodynamic monitoring with esCCO yields cardiac output values different from those measured by LiDCOrapid. The reliability of PWTT is questionable when systemic vascular resistance changes , but it can still trace the trends of CO changes. Due to its non-invasiveness, esCCO might be the monitoring of choice for high-risk patients undergoing low-risk surgery.


In accordance with the Theory of Structural Dissociation of the Personality (TSDP), studies of dissociative identity disorder (DID) have documented that two prototypical dissociative subsystems of the personality, the “Emotional Part” (EP) and the “Apparently Normal Part” (ANP), have different biopsychosocial reactions to supraliminal and subliminal trauma-related cues and that these reactions cannot be mimicked by fantasy prone healthy controls nor by actors.Arterial spin labeling perfusion MRI was used to test the hypotheses that ANP and EP in DID have different perfusion patterns in response to rest instructions, and that perfusion is different in actors who were instructed to simulate ANP and EP. In a follow-up study, regional cerebral blood flow of DID patients was compared with the activation pattern of healthy non-simulating controls.Compared to EP, ANP showed elevated perfusion in bilateral thalamus. Compared to ANP, EP had increased perfusion in the dorsomedial prefrontal cortex, primary somatosensory cortex, and motor-related areas. Perfusion patterns for simulated ANP and EP were different. Fitting their reported role-play strategies, the actors activated brain structures involved in visual mental imagery and empathizing feelings. The follow-up study demonstrated elevated perfusion in the left temporal lobe in DID patients, whereas non-simulating healthy controls had increased activity in areas which mediate the mental construction of past and future episodic events.DID involves dissociative part-dependent resting-state differences. Compared to ANP, EP activated brain structures involved in self-referencing and sensorimotor actions more. Actors had different perfusion patterns compared to genuine ANP and EP. Comparisons of neural activity for individuals with DID and non-DID simulating controls suggest that the resting-state features of ANP and EP in DID are not due to imagination. The findings are consistent with TSDP and inconsistent with the idea that DID is caused by suggestion, fantasy proneness, and role-playing. Consistent clinical observations and retrospective findings indicate that dissociative identity disorder (DID) Socioculture contexts affect any mental disorder Several studies have compared psychobiological reactions of different dissociative parts in DID, but advances in the field critically depend on theoretical predictions with respect to the kind of biopsychosocial differences that exist among different types of dissociative subsystems or “parts” of the personality as a whole biopsychosocial system TSDP is supported by clinical and empirical evidence. For example, in a Positron Emission Tomography (PET) study, DID patients listened as ANP and as EP (subtype active defense) to audiotaped descriptions of a neutral autobiographical memory that these dissociative parts had in common, as well as to a description of a traumatic memory that was only autobiographical for EP With main effect and conjunction analyses The study of resting-state neural activity has recently become an important area of neuroimaging. Of special interest is the so called default mode network (DMN), a set of brain areas consisting of the medial prefrontal cortex (MPFC), posterior cingulate (PCC) in addition to midline parietal structures, lateral parietal regions, and medial and lateral temporal lobes The main goal of the current study was to examine and compare brain perfusion patterns for ANP and EP (subtype active defense) in DID patients following rest instructions. Rest instructions, that is, instructions to relax and close the eyes, do not imply that the participants are actually resting. The term resting-state thus merely refers to the state that rest instructions elicit.No study to date investigated so called resting-state perfusion differences in ANP and EP in DID. However, a Single-Photon Emission Computed Tomography (SPECT) study yielded bilateral orbitofrontal hypoperfusion and left lateral hyperperfusion during rest for a type of dissociative part in DID described as the “host” compared to healthy volunteers In addition to the comparison of brain perfusion patterns for ANP and EP in DID patients, the current study also investigated neural activation patterns for ANP and EP in healthy simulating controls (SIM) in order to test the idea that DID involves suggestion and role-playing rather than a trauma-related condition Considerations based on TSDP and the mentioned empirical foundation lead us to hypothesize that (i) there are perfusion differences for DID patients and DID simulating controls. In particular, we predicted that (ia) compared to the actors, DID patients show relatively higher activation in areas which commonly exhibit increased neural activity following rest instructions (default mode activity), i.e. MPFC, PCC/precuneus, medial and lateral parietal areas, and medial and lateral temporal regions. Looking at the comparison from the other side, we hypothesized that (ib) actors compared to DID patients elicit less default mode activity because simulating an ANP and EP involves a goal-directed task. We furthermore hypothesized that in response to the described rest instructions, (ii) ANP and EP in DID have different patterns of brain perfusion and that (iii) comparisons of ANP and EP simulating controls yield different neural reactivity patterns than comparisons of ANP and EP in DID patients.Fifteen female DID patients were included in the current study, which was part of a larger study in DID patients and DID simulating controls The DID simulating control group (SIM) consisted of 15 female actors, who were instructed and motivated to simulate ANP and EP during the resting-state experiment. They were carefully informed about the characteristics of ANP and EP using written information on TSDP. They also watched a video showing a DID patient, who alternates between ANP and EP. The actors were instructed to create an ANP and EP using a list of properties . Of particular importance was that ANP should be a dissociative part without personalized memories of traumatizing events and EP a dissociative part with personalized traumatic memories. The actors were requested to practice simulating ANP and EP as often as they deemed necessary to effectively simulate ANP and EP, but at least three times before the MRI measurement.There were no significant differences between the DID and SIM group in age  = 0.019, p>0.05) and educational level  = −1.341, p>0.05; the educational level was assessed by a 7-point Likert scale based on the common European educational system). To ensure that none of the actors had a PTSD and/or major depression, the controls completed the German version of the Posttraumatic Diagnostic Scale (PDS) Each subject was informed about risks and inconveniences associated with the experiment. All subjects gave written informed consent. The local ethics committee  approved the study in compliance with the Helsinki Declaration.The subjects were instructed to relax with their eyes closed and to stay motionless during the fMRI measurement e.g., . All par2 in-plane resolution. During a single run, 35 pairs of control/label image volumes were measured over a total scan time of 5 minutes. An additional M0 image was acquired for measurement of the magnetization of arterial blood . A 3D MPRAGE T1-weighted anatomical scan was acquired for anatomical reference and post-processing.The data of the DID and SIM group were obtained at the University Hospital of Zurich with a 3-T Philips Achieva whole-body magnetic resonance imaging equipped with an eight-channel Philips SENSE head coil. Resting regional cerebral perfusion (rCBF) images were acquired with a pseudo-continuous ASL (p-CASL) sequence with background suppression (saturation of the imaging slice preceding the labeling and inversion pulses 1680 ms and 2760 ms after the saturation pulse) and a single shot echo-planar imaging (EPI) readout  The data of three actors and one patient were excluded due to huge movement artefacts and low signal quality. One patient had fallen asleep during the ANP and had switched several times during the EP run. One patient was not able to undergo the MRI measurement, and the data of one patient were lost due to a storage failure at the MRI center. The final brain imaging statistical analysis was performed with data of 11 participants in the DID group and 12 in the SIM group.http://www.fil.ion.ucl.ac.uk/spm). rCBF maps were normalized to the EPI template 3). The normalized rCBF maps were spatially smoothed with an 8-mm full width at half-maximum (FWHM) Gaussian kernel.Resting-state rCBF maps were calculated using in-house programmed MATLAB scripts performing a simple pair-wise subtraction of control and label images Type in the rest of the article. In order to correct for biological variation in total cerebral blood flow, the mean gray matter (GM) CBF was included in the analysis as a covariate of no interest. The mean GM signal per subject was calculated over a GM mask obtained from the segmentation of the 3D T1 image by thresholding the GM probability images at 0.5. Only the GM signal was taken into account, as a previous study revealed that GM perfusion showed most variability between sessions The preprocessed data were analyzed using a flexible factorial design that consisted of two independent variables resulting in a 2×2 ANOVA with repeated measures on the second factor: Group (two levels: DID/SIM), Type of dissociative part of the personality (two levels: ANP/EP). The second factor will be referred to as We performed a whole-brain voxel-wise analysis. The study design allows the calculation of various effects, i.e. main effect of Group, main effect of Type, and interaction effect Group by Type. An uncorrected statistical threshold  for multiple testing) of p<0.005 was set for the main effects and interaction effect. The minimum cluster-size was set at 10 voxels.Our main hypotheses were tested using one-sided t-tests. The participants were measured as ANP and EP in the patient group (DIDanp/DIDep) and in the control group (SIManp/SIMep). Group differences between the patients (DID) and actors (SIM) were assessed with a two-sample t-test based on the mean perfusion map of ANP and EP of every single participant (DID-SIM). Two planned comparisons consisting of Type effects between groups  and two planned comparisons consisting of Type effects within groups  were performed. All five t-tests were one-sided, thus were performed twice to assess positive rCBF differences in one and in the inverse contrast. To keep statistical thresholding of a priori defined regions conservative enough, we used a Bonferroni correction for these subsequent tests (p<0.005/5 = p<0.001). Thus, all t-tests were fixed to a p<0.005 and corrected for the number of tests. A similar approach has been used in Schlumpf et al. (2013).http://www.fmrib.ox.ac.uk/fsl) was applied at the level of the statistical interference to remove extracranial voxels. The mask was normalized to MNI space and had the same dimension and voxel size as the rCBF maps. Only the most significant finding of a brain area and first peak of a cluster are reported in the An explicit binary mask provided by FSL . In SIM compared to DID, we observed increased perfusion in the middle frontal gyrus and the occipital fusiform gyrus.Between-group comparisons of Type  are listed in Significant rCBF changes for both ANP and EP between the groups are in accordance with our first hypothesis.Compared to SIManp, DIDanp was associated with more activation in the angular gyrus, temporal pole of the middle temporal gyrus, dorsal posterior cingulate cortex (dPCC), and precuneus (DIDanp-SIManp). In the inverse contrast (SIManp-DIDanp), we found a higher perfusion in the middle frontal gyrus. Increased activation in the precuneus, DMPFC, angular gyrus, and temporal pole of the middle temporal gyrus was found in the contrast DIDep-SIMep. In SIMep compared to DIDep (SIMep-DIDep), we observed increased perfusion in the thalamus and in several occipital-temporal regions . The inverse contrast (DIDep-DIDanp) showed increased perfusion in the primary somatosensory cortex and in several motor-related brain areas including the primary motor cortex and higher-order motor areas , premotor cortex). DMPFC hyperperfusion was also observed Figure 1Comparing SIManp to SIMep (SIManp-SIMep) revealed higher brain activation in the right thalamus and in the extrastriate cortex. In the inverse contrast (SIMep-SIManp), we observed a higher perfusion in insular-opercular regions  and in inferior frontal areas ).Simulating a resting DID patient is a paradoxical situation because resting and simulating are opposite actions. Resting instructions invite relaxed, internally directed attention, whereas instructions to simulate a dissociative part elicit effortful goal-directed action. This paradox did not exist in a follow-up study in which the mentally healthy control group (NS) only received resting instructions. The NS group consisted of 15 female participants. There were no significant differences between the DID and NS group in age  = 1.428, p>0.05) and educational level  = −2.018, p>0.05).Atypical default mode activity has been observed in a number of mental disorders Text S1 in Information S1). The data set of 15 participants of the NS group were compared with the data set of the DID group (n = 11). For the pre- and postprocessing of the NS data set, the same strategy was used as for the other two groups. The following three two-sample t-tests were performed: DID-NS, DIDanp-NS, DIDep-NS. All three t-tests were one-sided, and were performed twice to assess positive rCBF differences in one and in the inverse contrast. Bonferroni corrected p-values were used (p<0.005/3 = p<0.00167) and cluster-size threshold was set at 10 voxels.For the follow-up study, the data of the NS group were obtained on the same scanner which has been used for the DID and SIM group, but after an upgrade from the Achieva to the Ingenia system (different MRI system). The involved system changes are described in the Supporting DID and NS showed a different resting-state activity pattern within the DMN see Table 5.NS compared to DID (NS-DID) had increased perfusion in posterior parietal regions , in several occipital areas , in the frontal polar cortex, in the right superior and inferior temporal gyrus, and in the hippocampus. Increased rCBF in posterior parietal regions, occipital areas, frontal polar regions, and hippocampus were also present for NS compared to DIDanp (NS-DIDanp) and for NS compared to DIDep (NS-DIDep).This is the first fMRI perfusion study measuring brain perfusion following rest instructions in DID patients. As hypothesized, we found differences between DID patients and DID simulating actors, as well as between two different prototypes of dissociative parts of the personality (ANP and EP) in DID patients. The follow-up study demonstrated different perfusion patterns for DID and controls who did not simulate ANP and EP.Compared to actors, DID patients showed higher resting-state metabolism in several areas belonging to the DMN  The between-group Type effects fit to these interpretations. Of special interest is the increased activity in the precuneus and angular gyrus for ANP and EP in DID patients when contrasted with the corresponding simulated ANP and EP . Both brain areas are part of the DMN In line with our second hypothesis, we found different patterns of resting-state perfusion for ANP and EP in the patients. Compared to EP, ANP showed more metabolism in the bilateral thalamus (DIDanp-DIDep), and right thalamus activity was higher in controls simulating EP than in authentic EP (SIMep-DIDep). However, controls simulating ANP also had more bilateral thalamus metabolism than controls simulating EP (SIManp-SIMep). Whereas relatively high right thalamus activity for ANP in DID patients may not be a DID-specific finding, our result parallels prior PTSD studies conducted under rest However, at the same time, they may have become self-aware, focused on internally alarming bodily and emotional cues, and prone to reactivate painful memories. Indeed, comparing EP to ANP in DID patients (DIDep-DIDanp), we found increased rCBF in the primary somatosensory cortex, in several motor-related brain areas, and in the DMPFC see Figure 1In line with our third hypothesis, comparisons of ANP and EP in controls yielded different neural reactivity patterns than comparisons of ANP and EP in DID patients. The actors reported that they used two major strategies to fulfill their simulation task: 1) imagining being another person and 2) trying to experience the other person's feelings. According to cognitive and social neuroscience, the first strategy can be described as visual mental imagery The follow-up study revealed the involvement of the temporal lobe in DID patients compared to non-simulating controls .Based on these interpretations and empirical foundations, our follow-up study suggest that under resting instructions as ANP and EP, DID patients activate left temporal regions more than mentally healthy controls who do not simulate ANP and EP. These controls may have been less involved in the task and scanner environment allowing them to let their mind wander to their personal past and imagined future. Of note: further analysis suggests that these results are not explained by a time or scanner effect  are associated with different patterns of brain activity following rest instructions in a challenging environment. The study also demonstrates for the first time that in this context and in contrast to DID simulating actors, particularly but not exclusively as EP, DID patients activated brain structures involved in self-consciousness. Following these instructions, neural activation associated with motivated role-playing of ANP and EP by mentally healthy controls was different from neural activation associated with being a genuine ANP and EP. The study adds to the evidence from supraliminal and subliminal neuroimaging studies of ANP and EP in DID We would like to thank the colleagues of Prof. Jäncke's lab for their helpful comments and Franz Liem for his technical support. We are indebted to Ekaterina Weder and Eva Zimmermann for their collaboration as research clinicians. Special thanks go to the patients and their therapists for participating in the study.Information S1(PDF)Click here for additional data file.


Cognitive dysfunction is amongst the most conspicuous symptoms in Gulf War illness (GWI). Combined exposure to the nerve gas antidote pyridostigmine bromide (PB), pesticides and stress during the Persian Gulf War-1 (PGW-1) are presumed to be among the major causes of GWI. Indeed, our recent studies in rat models have shown that exposure to GWI-related (GWIR) chemicals and mild stress for 4 weeks engenders cognitive impairments accompanied with several detrimental changes in the hippocampus. In this study, we tested whether reduced numbers of hippocampal gamma-amino butyric acid (GABA)-ergic interneurons are among the pathological changes induced by GWIR-chemicals and stress. Animals were exposed to low doses of GWIR-chemicals and mild stress for 4 weeks. Three months after this exposure, subpopulations of GABA-ergic interneurons expressing the calcium binding protein parvalbumin (PV), the neuropeptide Y (NPY) and somatostatin (SS) in the hippocampus were stereologically quantified. Animals exposed to GWIR-chemicals and stress for 4 weeks displayed reduced numbers of PV-expressing GABA-ergic interneurons in the dentate gyrus and NPY-expressing interneurons in the CA1 and CA3 subfields. However, no changes in SS+ interneuron population were observed in the hippocampus. Furthermore, GABA-ergic interneuron deficiency in these animals was associated with greatly diminished hippocampus neurogenesis. Because PV+ and NPY+ interneurons play roles in maintaining normal cognitive function and neurogenesis, and controlling the activity of excitatory neurons in the hippocampus, reduced numbers of these interneurons may be one of the major causes of cognitive dysfunction and reduced neurogenesis observed in GWI. Hence, strategies that improve inhibitory neurotransmission in the hippocampus may prove beneficial for reversing cognitive dysfunction in GWI. Almost 25–30% of 700,000 troops deployed to the Persian Gulf War-1 (PGW-1) are diagnosed with Gulf War illness (GWI), which is a chronic multi-symptom illness affecting multiple systems including the central nervous system  chemicals for extended periods are considered perilous for CNS function because of several reasons. The drug PB is a reversible acetylcholinesterase (AChE) inhibitor and does not cross the blood brain barrier (BBB) under normal circumstances. However, in situations when BBB gets leaky (such as after exposure to stress or pesticides), PB can enter the brain -ergic interneurons can promote hippocampus hyperexcitability as well as worsen cognitive function via decreased inhibitory synaptic transmission . Animals in GWI group received daily exposure to GWIR-chemicals PB, DEET and PM and mild stress (5 min of restraint stress) for 4 weeks. From here onwards, these rats will be referred to as “GWI-rats”.Young adult (~3-months old) male Sprague-Dawley rats purchased from Harlan  were used in this study. After 2 weeks of acclimatization to the vivarium, animals were randomly assigned to either the naïve control group or the GWI group  was dissolved in 500 μl of sterile water and administered via oral gavage. The chemicals DEET and PM were administered dermally. For these, solutions of DEET  and PM  were prepared, the fur on the back of the neck and the upper thoracic region were shaved off and the exposed skin was gently wiped with 70% alcohol. Following this, using a short plastic pipette, DEET and PM (200 μl each) were successively spread over the shaved skin. A rat restrainer  was used for the induction of 5 min of restraint stress, as described in our previous study .Three months after the completion of 4 weeks of exposure to GWIR-chemicals PB, DEET and PM and 5 min of restraint stress, GWI-rats and age-matched naive control rats were subjected to terminal anesthesia with isoflurane and trans-cardiac perfusion with 4% paraformaldehyde solution in phosphate buffer (PB). Following this, the brain was carefully removed from the skull of each animal and post-fixed in 4% paraformaldehyde for ~16 h at 4°C. The brain tissues were next treated with 30% sucrose solution in PB until it sank to the bottom, and thirty-micrometer thick cryostat sections were cut coronally through the entire septo-temporal axis of the hippocampus. The sections were collected serially in 24-well plates filled with PB. Every 20th section through the entire hippocampus was then selected from six naive control rats and six GWI-rats and processed for PV immunohistochemistry, which visualized the PV+ interneurons in different subfields of the hippocampus. Additional series of sections were processed for NPY, SS (every 20th) and DCX (every 15th) immunohistochemistry for visualization of NPY+ and SS+ interneurons in different regions of the hippocampus, and DCX+ newly born neurons in the SGZ-GCL of the DG , numbers of PV+, NPY+ and SS+ interneurons were stereologically counted for the DG and the CA1 and CA3 subfields of the hippocampus. On the other hand, the DCX+ newly born neurons were stereologically counted for the SGZ-GCL region. All counts utilized every 15th or 20th section through the entire septo-temporal axis of the hippocampus and the optical fractionator method available in the StereoInvestigator system (Microbrightfield Inc.). The StereoInvestigator system consisted of a color digital video camera (Optronics Inc.) interfaced with a Nikon E600 microscope.In every animal belonging to naive control and GWI groups . The ellipsis hsf stands for the height sampling fraction, which was measured by dividing the height sampled  with the actual section depth. EQ- denotes the total count of neurons sampled for each subfield. The software program also facilitated the computation of data as density per mm3 volume of the tissue sampled.A detailed protocol employed for counting using the optical fractionator method is described in our preceding publications  for the two groups. The PV+, NPY+, SS+ total interneuron counts and densities for different hippocampal regions and DCX+ newly born neuron numbers and densities in the SGZ-GCL region of age-matched naive control rats (n = 6) and GWI-rats (n = 6) were then compared through two-tailed, unpaired Student’s t-test.The total numbers as well as densities per mmp < 0.01, Figure p > 0.05, Figure p < 0.05, Figure 3 volume of tissue also revealed 50% reduction in the DG  per hippocampus in naive control animals. However, GWI-rats demonstrated a clearly reduced number of newly born neurons per hippocampus . Thus, a clear association was found between the decreased neurogenesis and PV+ interneuron loss in the DG of GWI-rats.Because of the suggested links between the integrity of PV+ and NPY+ interneurons and neurogenesis in the DG, we investigated the status of neurogenesis from both naive control and GWI-rats via stereological counting of DCX+ newly born neurons in the SGZ-GCL. Immunostaining for DCX visualized newly born neurons in both age-matched naive control rats and GWI-rats Figures . StereolThis study provides new evidence that combined exposure to low doses of GWIR chemicals and mild stress for 4 weeks reduces the overall number as well as the density of PV-expressing GABA-ergic interneurons in the DG and NPY-expressing interneurons in the CA1 and CA3 subfields of the hippocampus. This study also demonstrates that PV and NPY interneuron deficiency in the hippocampus co-exists with greatly decreased neurogenesis in GWI-rats. The mechanism by which PV+ and NPY+ interneuron numbers decline after exposure to GWIR chemicals and stress remains to be determined however. Persistent inhibition of AChE in the exposure and early post-exposure periods may be involved because the GWIR chemicals are AChE inhibitors and persistent exposure to even low doses of AChE inhibitors can alter the regulation of cholinergic function and increase oxidative stress Golomb, . AlthougIn the hippocampus, GABA-ergic interneurons expressing the calcium binding protein PV are mainly observed in the dentate GCL, the dentate hilus, and strata oriens and pyramidale of CA1–CA3 subfields  occasionally divide to generate a highly proliferative intermediate progenitor cell population (transient amplifying cells), which give rise to large numbers of DCX+ immature neurons. Ambient GABA in the microenvironment tonically activates these immature neurons, which is then followed successively by depolarizing GABA-ergic and glutamatergic synaptic inputs (Song et al., Our analyses showed that the overall number and density of NPY+ interneuron population remained stable in the DG but decreased in the CA1 and CA3 subfields of GWI-rats. This may have several consequences because NPY+ interneurons in the hippocampus play an important role in regulating the activity of hippocampal circuitry. The NPY released from these interneurons modulates the activity of excitatory neurons by consistently hyperpolarizing and reducing their spike frequency (Fu and van den Pol, Our findings establish for the first time that PV+ interneurons in the DG and NPY+ interneurons in CA1 and CA3 subfields of the hippocampus decline in total number as well as density per unit volume of the tissue in a model of GWI. The results also uncover that decreased interneuron numbers are coupled with greatly decreased neurogenesis in the hippocampus of GWI-rats. Because PV+ and NPY+ interneurons play roles in maintaining normal cognitive function, regulating the extent of neurogenesis, and controlling the activity of excitatory neurons, their deficiency is likely among the major factors contributing to impaired cognitive function seen in GWI.Tarick Megahed contributed to stereological counts of interneurons, data analyses, the preparation of figures and figure composites, and prepared an initial draft of the manuscript text. Bharathi Hattiangady gave input to the experimental design, and immunohistochemistry, performed some stereological counts and data analyses, prepared figure composites and contributed to manuscript writing. Bing Shuai performed chemical and stress exposures to animals, tissue processing and immunohistochemistry. Ashok K. Shetty conceptualized the experimental design, analyzed and interpreted data, revised figure composites and wrote the final version of the manuscript text. All authors gave input to the manuscript text and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.