CatFISH did not reveal significant changes in Arc mRNA expression. Similar results were obtained with radioactive in situ hybridization. We have shown that while rats reinstate drug seeking in response to temporally discrete presentations of distinct drug-associated cues, such reinstatement is not associated with increased transcriptional activation of Arc or zif268 mRNAs, suggesting that expression of these genes may not be necessary for cue-induced reinstatement of drug-seeking behavior.Drug-seeking behavior elicited by drug-associated cues contributes to relapse in addiction; however, whether relapse elicited by drug-associated conditioned reinforcers (CR) versus discriminative stimuli (DS) involves distinct or overlapping neuronal populations is unknown. To address this question, we developed a novel cocaine self-administration and cue-induced reinstatement paradigm that exposed the same rats to distinct cocaine-associated CR and DS. Rats were trained to self-administer cocaine in separate sessions. In one, a DS signaled cocaine availability; in the other, cocaine delivery was paired with a different CR. After extinction training and reinstatement testing, where both cues were presented in separate sessions, rats were sacrificed and processed for cellular analysis of temporal activity by fluorescent  Current theories posit that addiction reflects the strong association of previously neutral stimuli with drug-seeking and -taking behaviors, and the subsequent ability of these stimuli to elicit such behavior Drug addiction is defined as uncontrollable, compulsive drug seeking and use in the face of negative consequences (At least two types of associations are involved in stimulus-associated learning. Discriminative stimuli (DS) indicate the opportunity (DS+), or lack thereof (DS−), to obtain a reinforcer, whereas conditioned reinforcers (CR) come to serve as reinforcers themselves due to their repeated pairing with primary reinforcement. Through the course of drug use and drug-seeking behavior, people are repeatedly exposed to both types of drug-stimulus associations. There are likely similarities and differences in the neural circuits underlying drug-seeking and drug-taking behavior mediated by exposure to CRs and DS Other studies have examined neural circuits underlying DS-induced reinstatement of drug-seeking behavior per se that is resulting in IEG activation. Also, prior studies examining IEG expression in the context of cue-induced reinstatement of drug-seeking behavior have not afforded the opportunity to examine whether discrete types of drug-associated cues activate overlapping populations of neurons. Cellular analysis of temporal activity by fluorescent in situ hybridization (catFISH) for activity-regulated cytoskeleton-associated protein (Arc) mRNA is a labeling technique that allows a tight temporal relation to be drawn between a behavioral event and resulting gene expression profile. CatFISH further allows one to determine whether the same neuronal populations within a brain region are activated by two temporally discrete behavioral events Arc mRNA transcription can be detected within 3–5 min of activation in forebrain regions, including hippocampus Arc mRNA is subsequently transported into the cytoplasm and dendrites within 30 min of transcriptional activation Arc mRNA transcriptional activation returns to basal levels within 60 min Arc mRNA in the nucleus are those activated 3–5 min prior to sacrifice of the animal; cells with Arc mRNA in the cytoplasm are those activated approximately 30 min prior to sacrifice; and cells with Arc mRNA expression in both nuclear foci and cytoplasm are activated at both time points relative to sacrifice via catFISH analysis of Arc mRNA expression neuronal populations activated during reinstatement of cocaine-seeking behavior induced by discrete drug-associated CRs and DS.The expression of IEGs has been used to map neural circuits involved in particular behaviors Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee at the University of Utah (approved protocol #08–04004). All surgery was performed under ketamine/xylazine anesthesia, and all efforts were made to minimize suffering.This study was conducted in strict accordance with the Adult male Sprague-Dawley rats  were food restricted (∼15 g chow/day) for two days and then trained to lever press (>300 presses/16 hrs) on a free operant schedule (FR1) for food reinforcement (45-mg pellet) in operant training chambers (Coulbourn Instruments) housed within light- and sound-attenuating boxes. Subsequently, rats were randomly assigned to treatment groups. Five cohorts were run, consisting of one animal in each of the following groups: experimental , stimulus novelty control , stimulus control , and drug control . During training, experimental rats engaged in lever pressing behavior for intravenous cocaine  in response to a DS and for response-contingent exposure to a complex CR in distinct segments of daily sessions. Novelty control animals were placed in chambers, but not exposed to stimuli or given infusions. Drug controls received cocaine infusions simultaneously with EXP rats, but were not exposed to sensory stimuli. Stimulus controls received exposure to the DS and CR stimuli simultaneously with EXP rats, but received saline  infusions. An additional cohort (n = 10) consisted entirely of EXP animals. After the EXP rat had achieved self-administration stability criterion (<15 coefficient of variation in cocaine infusions across three consecutive sessions), all rats in a cohort then received identical extinction training and exposure to the CR and DS on the reinstatement test-day. Finally, age-matched, caged-control animals  were maintained for the duration of training and extinction of each cohort and were sacrificed on test day immediately prior to EXP animals.Rats  were implanted with jugular vein catheters constructed from 22-gauge guide cannulae, silastic tubing, and monofilament mesh under ketamine/xylazine anesthesia . Rats recovered for four days with free access to food and water. On the 5th day rats were food restricted as described above, and on the 6th day, cocaine self-administration training began.Training occurred during the dark phase of the light-dark cycle in chambers containing one active and one inactive lever, tricolor LEDs above each lever, and a wall-mounted house light and speaker. Each session began with a 5-min delay and lasted for four hr and 35 min. EXP rats had a two-hr DS or CR session, a 30-min time out, and the other two-hr DS or CR session. DS/CR session order was balanced across subjects.Initially, the DS (house light) remained illuminated until the EXP rat pressed the active lever  were placed in the chambers for 14 daily, 4.5-hr sessions. Neither the DS nor the CR was given to any rat during extinction. Lever pressing had no consequence.After the 14th extinction session, all rats remained in the chambers overnight to avoid handling effects on IEG expression (2 for one min and then decapitated. Brains were rapidly removed (∼90 sec) and frozen in isopentane (−80°C). Brains were cryosectioned at 12 µm and thaw mounted on Superfrost Plus slides . Slides for direct comparison were processed together through all steps, as previously described After the final DS exposure, all rats were sacrificed via exposure to COArc mRNA) fluorescence, respectively. Sections from dorsal striatum and nucleus accumbens were also scanned at 633-nm to resolve Cy5 (PPE mRNA) fluorescence for phenotypic identification of striatal efferent neurons.Brain sections were visualized using a scanning confocal microscope . Images were captured with a 60X, 1.2-NA oil-immersion objective. A three-by-three montage of 235 µm by 235 µm fields was compiled for dorsomedial  mRNA expression. However, analysis revealed no differences in Arc mRNA expression in PPE-positive and PPE-negative cells, and thus Arc mRNA-positive cells were not phenotypically differentiated for the final analysis. Data were statistically analyzed via one-way ANOVA for treatment group for each gene-expression profile in each brain region using JMP10 (SAS Institute Inc.).An experimenter blinded to treatment condition counted the number of neurons in each field with one or two intranuclear foci of Brains were cryosectioned at 12 µm and thaw mounted on Superfrost Plus slides . Slides for direct comparison were processed together through all steps, as previously described http://rsbweb.nih.gov/ij), yielding the product of area sampled and mean gray value for pixels in the field with signal above threshold. These measurements were summed across the 6 samples analyzed in each region for each animal. Values are thus reported as mean ± SEM of integrated density for signal above threshold for each brain region for each treatment group. Data were statistically analyzed via one-way ANOVA for treatment group for each brain region using JMP10 (SAS Institute Inc.). Post-hoc analysis of significant ANOVAs was accomplished via the Tukey-Kramer honestly significant difference (HSD) test. Statistical significance was set at p<0.05.Digitized images of 705 µm by 705 µm fields  of prelimbic . Planned comparisons via Bonferroni-corrected student's t-tests revealed significant increases in active- vs. inactive-lever pressing for the EXP group during both the CR  and DS  exposure segments of the reinstatement test session. EXP active-lever pressing was also significantly increased relative to the previous extinction session ; DS vs. EXT ), as well as relative to the 20-min delay for DS active-lever pressing . EXP active-lever pressing was further significantly increased when compared to all control groups within reinstatement session segments , SC , DC ; DS: EXP vs. NC , SC , DC ). These data demonstrate that EXP rats acquired stable self-administration of cocaine in the context of two distinct sensory-cue associations, extinguished responding to the context of the self-administration chamber over 14 days, and then reinstated drug-seeking behavior in response to temporally discrete presentations of the specific, drug-associated sensory cues.Of the 10 EXP rats reaching criterion on the dual training paradigm, 6 reinstated drug-seeking behavior in response to both the DS and CR on the reinstatement test day, with reinstatement being defined as ≥1 lever press per minute during each of the CR and DS segments. EXP rats that did not meet this criterion during both stimulus-exposure segments of the reinstatement test were excluded from the subsequent gene expression analysis. Of the 6 EXP rats reinstating drug-seeking behavior in response to both the DS and CR on test day, four were run with control cohorts and two were from the additional cohort consisting of only EXP animals. Analysis of the reinstatement test session with a two-within  by one-between (treatment group) mixed-factor ANOVA for lever pressing revealed a significant three-way interaction between treatment group, session segment, and lever  were considered “DS-associated,” neurons expressing only cytoplasmic Arc mRNA (i.e. activated 30 min prior to sacrifice) were considered “CR-associated,” and neurons expressing both intranuclear foci and cytoplasmic Arc mRNA expression (i.e. activated both 5 and 30 min prior to sacrifice) were considered “dual-associated.” Caged control subjects were never exposed to the reinstatement test stimuli and are not showing transcription related to stimuli exposure, rather these animals demonstrate basal levels of Arc mRNA expression. The tissue from one cohort was not available and was not analyzed via catFISH for the DMS, DLS, NAc, and NAs. Tissue from this cohort was analyzed for CA1, CA3, and BLA.The behavioral data presented establish that distinct cocaine-associated CRs and DS support reinstatement of cocaine-seeking behavior when presented in a temporally distinct manner to individual rats. We then asked whether it was possible to use catFISH to assess the transcriptional activation in neural populations associated with this reinstatement. For clarity, neurons determined to be Arc mRNA-positive neurons activated at the time of the DS session (DS-associated), the CR session (CR-associated), or both segments of the reinstatement test session  in the DMS, DLS, NAc or NAs, CA1 or CA3 subregions of the hippocampus, or the BLA (Arc mRNA expression (i.e. those with nuclear foci and cytoplasmic Arc mRNA expression) was very low across all brain regions examined in the EXP, as well as all other control groups. One-way ANOVAs failed to show significant effects of treatment group on the proportion of neurons with dual Arc mRNA expression for all brain regions except the NAs. Analysis of the NAs revealed a main effect of treatment group . Post-hoc analysis by Tukey-Kramer HSD (p<0.05) revealed that the percentage of Arc mRNA-positive cells with dual expression in the SC group was significantly greater than in the CC, EXP, and NC groups. Notably, however, the robust reinstatement of cocaine-seeking behavior elicited by temporally discrete exposure to CR and DS cues in the EXP animals was not associated with significant increases in the numbers of Arc mRNA-positive neurons in any of these brain regions, nor a change in the proportion of neurons  showing dual (nuclear plus cytoplasmic) Arc mRNA expression.One-way ANOVAs for each brain region analyzed revealed no significant effects of treatment group on the number of  the BLA Table 1.Arc mRNA-positive neurons in EXP animals relative to controls in any subcellular compartment, we became concerned that the amplification steps in the catFISH assay might be masking differences in the relative amount of Arc mRNA expression per cell across treatment groups, as several studies using radioactive in situ hybridization to examine gene expression in the context of cue-induced reinstatement of cocaine-seeking behavior have reported increases in Arc and zif268 mRNA expression in situ hybridization histochemistry in which the signal detected is directly related to the amount of probe bound.Given the lack of increase in the numbers of Arc mRNA expression in CG1, NAc, NAs, DLS, DMS  towards significance.A one-way ANOVA revealed no significant effect of treatment group on the density of S, DMS , 7, or CArc mRNA expression in OFC  confirmed increased density of Arc mRNA in experimental animals relative to caged controls, but this increase was not significantly different from other operant chamber exposed groups .A one-way ANOVA did reveal a significant main effect of treatment group on the density of if268 mRNA expression in OFC, PRL, CG1, NAc, NAs, DLS, DMS, or CA3  confirmed a significant decrease in the expression of zif268 mRNA in experimental animals in the opposite direction of predicted results.One-way ANOVA did reveal a significant main effect of treatment group on Arc mRNA or changes in the proportions of neurons expressing Arc mRNA in response to dual DS/CR cue-induced reinstatement of cocaine seeking in experimental rats. Subsequent autoradiographic analysis of the expression of Arc and zif268 mRNAs also failed to reveal increases in the expression of these genes associated with the reinstatement of drug-seeking behavior induced by exposure to drug-associated cues in EXP animals relative to controls. These data demonstrate that drug-seeking behavior evoked by brief exposure to discrete sensory cues is not associated with detectable increases in the transcriptional activation of Arc or zif268 mRNAs in multiple brain regions, suggesting that neither Arc nor zif268 mRNA expression, in the regions analyzed, is necessary for discrete cue-induced reinstatement of drug-seeking behavior and that brief exposure to reinstatement may not be sufficient to drive such expression.The first goal of these experiments was to determine the feasibility of training rats to self-administer cocaine in a segregated DS/CR model. The results show that rats can learn to stably self-administer cocaine in such a dual paradigm Figure 3Rats learned and reinstated cocaine-seeking behavior in response to two distinct types of stimuli, however the proportion of animals completing training and reinstating to both the CR and the DS was low. Two factors likely contribute to this outcome. First, the number of days required for rats to reach criterion was relatively high. Rats in the present study completing dual DS/CR training required 24.9±2.6 sessions to reach criterion. Other labs utilize 10 consecutive training sessions with ≥10 infusions per session The second factor likely contributing to high attrition in the dual DS/CR paradigm is general task difficulty. Two rats reinstated to only one stimulus, and their performance during training was better for the corresponding stimulus. Although both DS and CRs likely contribute to reinstatement and maintenance of drug-seeking in addicted subjects Arc and zif268 mRNA expression across multiple brain regions in the EXP group relative to other control groups was quite surprising, especially in light of reports of significant increases in the expression of these genes in striatum Arc and zif268 mRNA expression in learning, memory, and reconsolidation of previously acquired associations As noted above, the second goal of the present study was to determine whether catFISH could be used to map neuronal ensembles activated by reinstatement of cocaine-seeking behavior by either or both of the cocaine-associated cues (DS and/or CR). The lack of significant increases in the numbers of cells with expression or in the general levels of Arc and zif268 mRNA transcriptional activation in EXP animals in the present work. This reinstatement model is different from those used in other studies showing reinstatement-induced increases in gene expression in that extreme care was taken in the present work to isolate the specific, discrete sensory cues most proximally eliciting reinstatement of cocaine-seeking behavior and to present them for necessarily brief periods of time to capitalize on the strength of the catFISH technique. Thus, the two major differences across the studies reporting increases in IEG expression al. exposed rats to a full hour of reinstatement testing al. There are several possible explanations for the lack of specific Arc mRNA anti-sense oligonucleotides into the striatum failed to alter context-induced reinstatement of cocaine-seeking behavior. However, the following day, rats so treated showed greater reinstatement behavior, suggesting that the Arc mRNA anti-sense had impaired consolidation of the extinction learning occurring on the first day of reinstatement testing. Although IEG expression has often been used to map activated neural circuitry, given the role of IEGs in learning, and its consolidation and reconsolidation, careful consideration needs to be given when interpreting the meaning of changes in IEG expression induced during reinstatement of drug-seeking behavior when the contingencies between drug-seeking behavior and drug delivery change on the test day. The new learning occurring under such altered contingencies clearly becomes a significant confound in interpreting the meaning of the associated IEG expression. The use of second-order schedules of reinforcement therefore may be more useful for mapping, via IEG expression, brain circuitry activated during drug seeking, as such schedules engender significant drug-seeking behavior in the absence of primary reinforcement and without altering existing contingencies.Exposure to drug-associated cues for a longer period of time may be necessary to induce gene expression in these types of models not because such expression is required for the activation of a learned association, but rather because the gene expression may in fact reflect new extinction learning, or neural plasticity related to alteration of the encoded associations in the context of longer sessions in which cues are no longer paired with drug delivery. In this regard, the data of Hearing and colleagues Arc or zif268 mRNA expression specific to EXP animals in the present study. There was a significant increase in the level of Arc mRNA expression analyzed by radioactive in situ hybridization in the OFC of EXP animals compared to the CC group, and a trend towards significance in PRL. The OFC has been shown to be important for drug context-induced reinstatement of drug seeking Arc mRNA in the OFC of EXP animals observed herein may be evidence that new learning is taking place in these structures, as the reinstatement test is, in effect, an extinction session. However, the fact that Arc mRNA expression in the EXP group was not significantly different from that in the other control groups exposed to the discrete sensory cues on the test day  suggests that increases in Arc mRNA expression in OFC may simply reflect general behavioral activation induced by exposure to environmental stimuli, independent of any association those cues have with prior drug taking. A recent analysis of Arc mRNA expression in mice undergoing CR-induced reinstatement of cocaine-seeking behavior found increased levels of Arc mRNA only in prelimbic cortex zif268 mRNA expression, with post-hoc analysis showing zif268 mRNA expression to be significantly lower in the EXP group relative to the caged controls. This decrease was unexpected. Our data are thus the first to suggest suppression of zif268 mRNA expression in a reinstatement model, although the functional significance of this decrease remains to be determined.Generally, there were limited differences in Arc and zif268 across multiple brain regions heavily implicated in cue-induced reinstatement of drug-seeking behavior. These data therefore emphasize the need to carefully consider not only the role of drug-seeking behavior, but also that of new learning that is likely occurring in drug-reinstatement paradigms, when interpreting changes in IEG expression associated with reinstatement of drug-seeking behavior.In conclusion, the experiments described herein show that individual rats can be trained with discrete associative mechanisms and then reinstate drug-seeking behavior in response to each type of drug-associated cue. The data further suggest that such reinstatement of drug-seeking behavior is not necessarily associated with elevated transcriptional activation of the IEGs


DNA methylation changes have been implicated in many common chronic diseases leading to the hypothesis that environmental and age-related DNA methylation changes within individuals are involved in disease etiology. Few studies have examined DNA methylation changes within an individual over time and all of these studies have been conducted in adults. Here, we aim to characterize how global DNA methylation changes from birth to age three within a longitudinal birth cohort study and to determine whether there are consistent predictors of DNA methylation levels measured three years apart. We measured global DNA methylation in the same children at birth (cord blood) and again at three years of age among 165 children, using an immunoassay. We found that on average, DNA methylation was significantly higher in blood at age 3-years than in cord blood (p<0.01). However, for any individual child, the difference was less than would be expected by chance. We found that pre-pregnancy BMI was negatively predictive of both cord and three-year DNA methylation, even after statistical adjustment to account for the correlation between cord blood and three-year DNA methylation. The biologic implications of small changes in global DNA methylation are unknown. However, the observation that global DNA methylation levels persist within an individual from birth to age three supports the belief that factors that influence global DNA methylation, including pre-pregnancy BMI, may confer long-term effects. DNA methylation is an epigenetic process by which non-sequence-based regulatory information can be mitotically transferred from mother to daughter cell. Involving the addition of a methyl group at the 5-position of a cytosine-guanine (CpG) dinucleotide, DNA methylation inhibits gene expression by interfering with transcription binding proteins and is associated with chromatin remodeling DNA methylation patterns are reset during early embryogenesis, allowing for tissue differentiation. Thereafter, DNA methylation is thought to be conserved, albeit labile, in somatic cells, allowing the organism to adjust to changing developmental and environmental conditions Relatively few studies have examined DNA methylation changes within an individual over time and all of these studies have been conducted in adults In this report, we aim to characterize how global DNA methylation changes from birth to age three within our longitudinal birth cohort study and to determine whether there are consistent predictors of DNA methylation levels measured three years apart. Additionally, we sought to confirm the findings concerning DNA methylation, pre-pregnancy BMI and birth weight reported previously by Michels et al. Further, given the theory that DNA methylation levels may persist throughout childhood, we sought to determine whether maternal pre-pregnancy BMI is also associated with global DNA methylation measured in the offspring at three years of age.th week of pregnancy, free of reported diabetes, hypertension and HIV, and had resided in the Washington Heights, Central Harlem or the South Bronx areas of New York City for at least one year. Eligible women who gave informed consent were considered ‘initially enrolled’. Women who completed the prenatal questionnaire and provided maternal and/or umbilical cord blood were considered ‘fully enrolled’  is a longitudinal cohort study of African American and Dominican women who were recruited in the prenatal clinics of New York-Presbyterian Medical Center, Harlem Hospital or their satellite clinics, as previously described TM Global DNA Methylation Quantification Kit . This method quantifies the methylated fraction of DNA using an ELISA-like reaction. The proportion of methylated DNA in the full sample is determined by plotting the intensity of the optical density generated from the reaction in comparison to the linear range of a standard curve produced using a methylated DNA control. Using this assay, DNA methylation is represented as the ratio of methylated DNA per 100 nanograms (ng) of total DNA. In humans, with the exception of stem cells, which make up a very small proportion of cord blood, DNA methylation occurs exclusively at cytosine-guanine dinucleotides DNA isolated from umbilical cord blood leukocytes (WBC) was analyzed for global DNA methylation using the MethylampResearch workers abstracted information about birth outcomes including gestational age at delivery, birth weight, birth length, and head circumference from maternal and infant medical records after delivery. We also collected additional information about important covariates including infant sex, maternal height, pre-pregnancy weight, total gestational weight gain (weight closest to delivery minus pre-pregnancy weight), and complications of pregnancy and delivery. Standardized pre-pregnancy body mass index (BMI) categories were grouped according to the recommendations of the CDC (2011) as underweight, normal weight, overweight and obese Descriptive statistics were calculated for DNA methylation in cord and three-year blood samples. Cord and three-year DNA methylation levels were initially compared statistically using Pearson’s and Spearman correlation coefficients. To test the hypothesis that the difference between DNA methylation levels in cord and three-year samples from the same individuals was significantly smaller than the difference between DNA methylation levels in cord and three-year samples from different individuals, we conducted permutation analyses. More specifically, with 165 children that have both cord and three-year DNA methylation samples, we held the cord DNA methylation data intact and shuffled the three-year DNA methylation to generate cord and three-year pairing from different individuals, which we call random pairs. We repeated the permutation 10,000 times, which generated a distribution of the average difference in DNA methylation between 165 randomly paired cord and three-year samples from different children. We then compared the average difference in DNA methylation between the 165 observed cord and three-year pairs from the same individuals to the 10,000 average differences in DNA methylation between the 165 randomly paired cord and three-year samples from different individuals.Univariate associations between demographic, socioeconomic, and other potential covariates and cord and three-year DNA methylation, respectively, were explored using Student’s t-test and simple linear regression, as appropriate. In linear regressions, DNA methylation measures were natural log-transformed to meet normality assumptions. Change in DNA methylation was calculated as the absolute difference in DNA methylation from birth to three years. To further determine which factors predicted cord and three-year DNA methylation or the change in DNA methylation over time, respectively, we used multiple linear regression models where natural log-transformed DNA methylation (either cord or three-year or the change in DNA methylation) was the dependent variable. In the model where three-year natural log-transformed DNA methylation was the dependent variable, we constructed a separate model that included cord natural log-transformed DNA methylation as a covariate to determine the independent influence of other covariates on DNA methylation measured at three years. We used principal components analysis to deconstruct the variation associated with plate-to-plate variation associated with technical variability 3 x 100birth weight in g/(length in cm)), a measure of body proportions at birth. Global DNA methylation measured in cord blood, an independent variable, was log transformed to stabilize the variance. Covariates were included in the birth outcome models if they were associated with the outcome in bivariate analysis with p<0.10 and changed the beta coefficient representing the relationship between cord blood DNA methylation and each birth outcome more than 5%.Finally, we evaluated the relationships between cord blood DNA methylation and birth outcomes, including gestational age at delivery, birth weight, length, head circumference and ponderal index , The average change in DNA methylation measured in cord blood and again in blood at age three in the same individuals was 0.88 ng/100ng total DNA (standard deviation  = 4.32). The range was −12.62 to 31.11, such that two-thirds of individuals had global DNA methylation that increased from cord to three years and one-third of individuals had DNA methylation that decreased during this same time period. However, the majority of individuals (62%) had DNA methylation that changed very little  from birth to age 3 while 12% had a negative change greater than 1 ng/100 ng total DNA and 27% had a positive change greater than 1 ng/100 ng total DNA). In the permutation analyses to assess whether the difference between DNA methylation levels in cord and three-year samples from the same individuals were significantly smaller than the difference between DNA methylation levels in cord and three-year samples from different individuals, we compared the 10,000 permuted mean difference from random pairs and one mean difference from the observed pairs.  and The factors that predict cord and three year DNA methylation are shown in months was (non-significantly) associated with higher three year DNA methylation (p = 0.07) compared with not having breast fed at all. Previously, we reported an association between prenatal PAH and lower cord blood DNA methylation At three years, we found that having a smoker in the home at three years was (non-significantly) associated with lower three year DNA methylation (p = 0.08) and having breast fed more than 3Figure S1A-D in File S1, we present the partial regression plots (reflecting the regression coefficient from the multiple linear regression models) presenting the association of pre-pregnancy BMI and DNA methylation in cord and three-year DNA. Being a former smoker and concurrent ETS exposure (defined as having a smoker in the home at three years) were also predictors of lower global DNA methylation in the offspring at three years .In multiple linear regression models Table 4,Table S2 in File S1 ). Pre-pregnancy BMI predicted a smaller absolute change in DNA methylation from birth to three years. Breast feeding duration during the first year was associated with a larger absolute change in DNA methylation.We explored the factors that influence the absolute value of the difference between DNA methylation measured in cord blood and DNA methylation measured in blood collected from the same children at age 3  with birth outcomes.The goals of this analysis were three-fold. First, we explored the relationship between global DNA methylation measured in the same children at birth (cord blood) and again at three years of age. We found that on average, DNA methylation was significantly higher in blood at age 3Changes in epigenetic marks have been proposed to be a biologic mechanism by which exposures affect health and disease across the life course Because global DNA hypomethylation is a hallmark of many age-related diseases Here, we report changes in global DNA methylation measured at birth and age three. This is an important life stage to examine, as DNA methylation marks may be programmed during development – a fluid process that does not end at birth – and maintained thereafter. Because much of development, including tissue differentiation, is epigenetically driven The observed protracted relation of pre-pregnancy BMI to global DNA methylation measured in both cord and three-year blood in the offspring is of particular note. A sub-optimal intrauterine environment resulting from maternal obesity could predispose the newborn infant and child to a variety of adverse health outcomes The World Health Organization has estimated that one third of U.S. women of reproductive age (20 to 44 years) are obese In contrast to the report by Michels et al. There are a few aspects of our study that warrant a cautious interpretation of the findings. It is possible that underlying health conditions present at the time the blood sample was collected might influence the cellular constituency of the blood, influencing DNA methylation measured; however, these conditions  are likely to be random rather than systematic. It should also be noted that variation in DNA methylation between birth and three years is likely a function of heritability as well as additional exposures occurring during these early years of life. While we were able to evaluate the impact of some of the non-heritable factors , there are many postnatal exposures that we were not able to measure; whether any of these exposures confound observed associations is not known. Additionally, our pre-pregnancy maternal weight and height measures were self-reported, which may result in under-estimating weight and BMI and over-estimating height Global DNA methylation is a non-specific biomarker that is a broad indicator of early biological changes that may affect an array of functions in the offspring. The biologic implications of small changes in global DNA methylation are unknown. However, the observation that global DNA methylation levels persist within an individual from birth to age three supports the belief that factors that influence global DNA methylation may confer long-term effects. Therefore, the effect of these factors (including pre-pregnancy BMI) on global DNA methylation at birth and at three years deserves further study. Specifically, examining the DNA methylation of specific genes or regions of the genome that are influenced by pre-pregnancy BMI could contribute to the development of hypotheses about how the offspring of overweight women may be affected across the life course.File S1Contains: Table S1 Comparison of those with and without cord DNA methylation and with and without three year DNA methylation. Table S2 Factors predicting absolute value of the change in DNA methylation over time (change  =  |log-three year meth – log-cord meth|). Figure S1 A–D: Partial regression plots from multiple regression analysis that test the association between DNA methylation  and pre-pregnancy maternal body mass index (BMI).(DOCX)Click here for additional data file.


The role of structural brain magnetic resonance imaging (MRI) is becoming more and more emphasized in the early diagnostics of Alzheimer's disease (AD). This study aimed to assess the improvement in classification accuracy that can be achieved by combining features from different structural MRI analysis techniques. Automatically estimated MR features used are hippocampal volume, tensor-based morphometry, cortical thickness and a novel technique based on manifold learning. Baseline MRIs acquired from all 834 subjects , 238 stable mild cognitive impairment (S-MCI), 167 MCI to AD progressors (P-MCI), 198 AD) from the Alzheimer's Disease Neuroimaging Initiative (ADNI) database were used for evaluation. We compared the classification accuracy achieved with linear discriminant analysis (LDA) and support vector machines (SVM). The best results achieved with individual features are 90% sensitivity and 84% specificity (HC/AD classification), 64%/66% (S-MCI/P-MCI) and 82%/76% (HC/P-MCI) with the LDA classifier. The combination of all features improved these results to 93% sensitivity and 85% specificity (HC/AD), 67%/69% (S-MCI/P-MCI) and 86%/82% (HC/P-MCI). Compared with previously published results in the ADNI database using individual MR-based features, the presented results show that a comprehensive analysis of MRI images combining multiple features improves classification accuracy and predictive power in detecting early AD. The most stable and reliable classification was achieved when combining all available features. Alzheimer's disease (AD) is the most common cause of dementia globally and one of the major healthcare issues of the future. It has been estimated that during the next four decades the prevalence of AD will quadruple from 27 to 106 million by which time 1 in 85 persons worldwide will be living with the disease The search for reliable biomarkers of AD-type pathology and predictors of disease progression among MCI subjects is ongoing. AD is characterized by neurofibrillary tangles and amyloid plaques in the brain It has been shown that the early diagnostics of AD can be improved by using multiple different biomarkers simultaneously. Usually these studies have combined MRI-based markers with biomarkers based on positron emission tomography (PET) In this paper we use the ADNI database to evaluate the ability of the combination of different MR-based features to increase classification accuracy. We evaluate the power of hippocampal volume (HV), cortical thickness (CTH), tensor-based morphometry (TBM) and features extracted from a recently proposed manifold-based learning (MBL) framework to discriminate healthy controls from subjects with AD and to predict conversion from MCI to AD. For evaluation we used all 834 ADNI baseline images that were available from the ADNI webpage. Compared to previous work this paper aims at establishing the improvement in accuracy and stability that can be achieved by combining more than one MR-based feature. To the best of our knowledge it is the first comprehensive study that analyzes MRI-derived features for the full ADNI dataset. For direct comparison with the work by Cuingnet et al. To test the influence of the classification method used, we utilized both support vector machines (SVMs) and a linear discriminant analys (LDA) to evaluate classification accuracy (CCR), sensitivity (SEN) and specificity (SPE) in each experiment.In the ADNI study, brain MR images were acquired at regular intervals after an initial baseline scan from approximately 200 cognitively normal older subjects (HC), 400 subjects with mild cognitive impairment (MCI), and 200 subjects with early AD. Detailled inclusion/exclusion criteria used for the different subject groups in ADNI are defined in All 834 ADNI subjects  for which a 1.5T T1-weighted MRI scan at baseline was available were included in this study. 167 subjects in the MCI group converted to AD as of July 2011. We therefore independently analysed progressive MCI (P-MCI) subjects and subjects with a stable diagnosis of MCI (S-MCI).Standard 1.5T screening/baseline T1-weighted images obtained using volumetric 3D MPRAGE protocol with resolutions ranging from 0.9 mmAll fully automated feature extraction methods described below were applied to images that were preprocessed by the ADNI pipeline.Baseline hippocampal volumes were measured using an approach based on fast and robust multi-atlas segmentation http://www2.bic.mni.mcgill.ca/) CTH is measured in the baseline T1-weighted structural MR images by using an automated computational surface-based method developed at the McConnell Brain Imaging Centre, Montreal Neurological Institute, McGill University, Montreal, Canada  information, either on cortical thickness or the volume. Thus, the number of original features is enormous, and to make the classification more efficient and robust, the number of features has to be reduced. We evaluated both features in a statistical region of interest (ROI) defined as detailed in We used two subsets to perform classification:All 834 available baseline images described in the subjects section509 baseline images used by Cuingnet et al. The following sections describe the definition of the statistical ROIs and evaluation strategy used for the two datasets respectively.In order to perform the study using cross-validation in the full dataset, it was divided into three equally sized parts. One part was used to perform the statistical tests for the CTH and TBM features, and the remaining two parts were used to evaluate the classification accuracy. This was repeated three times so that each part was once used to perform the statistical tests. Afterwards, the results of the three repetitions were averaged. The classification accuracy was evaluated using leave-N-out cross validation on those subjects not included in the statistical tests. Five percent of the evaluation subjects were regarded as the test set, and the remaining 95% of the subjects were used to train a classifier which was then applied to the test set. This was repeated table-1-caption100 times, each time selecting randomly the test set subjects. Finally, the results of the 100 repetitions were averaged. Consequently, in overall, the classification evaluation was performed using 300 .Linear discriminant analysis (LDA) is a widely used technique to find a linear combination of features to best separate several classes http://www.csie.ntu.edu.tw/cjlin/libsvm).Support vector machines use training data to find a separating hyperplane in the n-dimensional training space that best separates two subject groups We used both classification methods to measure classification accuracy based on individual features as well as the combination of all features. The results for the comparisons HC vs AD, HC vs P-MCI and S-MCI vs P-MCI in the full ADNI database are presented in For direct comparison with work presented by Cuingnet et al. In this study we assessed the automatic diagnostic capabilities of 4 structural MRI features  separately and combined in 834 baseline images acquired in the ADNI study. When applied separately, TBM provided the overall best results, closely followed by MBL. Combining all features improved the results in all study experiments. Our results show how a combination of different MRI-based features can improve results based on only one measurement, resulting in a more powerful and stable classifier. The most significant improvement of the combination over the best individual feature can be observed for HC vs P-MCI with 5% units followed by 3 and 2% units for S-MCI vs P-MCI and HC vs AD, respectively. These improvements lead to 20, 12 and 9 subjects more being correctly classified respectively when using the combined feature set as compared to the best single feature for every comparison. Comparing two classification approaches based on LDA and SVMs resulted in a clear advantage of the former.Several studies reported classification results using single MRI methods for the HC/AD classification . Liu et Varying results concerning AD prediction (S-MCI/P-MCI classification using baseline measurements) have been published : QuerbesThere can be several explanations for the variation in the reported results. A majority of the studies in this field have used different statistical methods and MRI feature extraction strategies on different datasets, which makes a comparison of the results complicated. Also the variation in the size of the study samples and the use (or ignoring) of cross-validation or separate training/testing sets are important factors, which both have crucial impact on the reliability and generalizability of the results. In Lötjönen et al. A recent study with a subset of ADNI subjects assessed the classification performance of several structural MRI methods in experiments comparable to our investigation Some studies have also combined different biomarkers  with the idea of measuring different aspects of AD pathology and thus improve the classification accuracy. Hinrichs et al. Considering solely the classification accuracies of the present study and those reported in literature, it seems questionable if the collection of several biomarkers is worth the effort and resource. A combination of different features extracted from a single MRI seems to provide results that are comparable or better than those obtained with other or multiple biomarkers. In a clinical point of view, this is interesting since it means that a single MRI scan provides not only aid to differential diagnostics of cognitive impairment, but also reliably describes a persons phase in the HC/AD continuum. MRI is also widely available, non-invasive and often useful in the differential diagnostics of memory problems thus making it a compelling option as the first biomarker that would be obtained from a patient with mild memory problems. However, a comprehensive differential diagnostics between AD and non-AD cognitive impairments will still require assessment of various different biomarkers. Also, it should be noted that the computational techniques used in this paper are not widely available in the clinical environment and thus limit their usage in the clinical work at present.Strengths of the presented study are i) the use of multiple features extracted from one imaging modality, ii) large groups, iii) rigorous validation process of the results using cross-validation, and iv) results comparable or better than the ones published so far.Our study has also some limitations that should be mentioned. The results are obtained from a single  cohort and should be also validated in other cohorts. A longer clinical follow-up time would be needed to see if the classification results of S-MCI/P-MCI experiment changed when more of the MCI subjects converted to AD. Furthermore, the ADNI study does not provide postmortem pathological confirmation of the clinical status. With this limitation, individual subjects might be wrongly categorized. Although a rigorous validation process was used, optimally we need to establish standardized cut-offs that would be well generalizable to other cohorts outside ADNI. That is, however, beyond the possibilities of this study and will require vast standardization and validation procedures. Also, the CTH pipeline had problems especially with severely atrophied brains or MRI scans with poor image quality. A more robust pipeline would be desirable in order to guarantee a more reliable feature extraction.Appendix S1The Alzheimer's Disease Neuroimaging Initiative.(DOCX)Click here for additional data file.Appendix S2ROI-wise features for CTH and TBM.(DOCX)Click here for additional data file.


Condylar geometry, in addition to post-cam geometry, played a significant role in minimizing engagement velocity and forces and stresses in the post. This analysis guides selection and design of PS implants that facilitate smooth post-cam engagement and reduce edge loading of the post.Posterior-stabilized (PS) total knee arthroplasty (TKA) components employ a tibial post and femoral cam mechanism to guide anteroposterior knee motion in lieu of the posterior cruciate ligament. Some PS TKA patients report a clicking sensation when the post and cam engage, while severe wear and fracture of the post; we hypothesize that these complications are associated with excessive impact velocity at engagement. We evaluated the effect of implant design on engagement dynamics of the post-cam mechanism and resulting polyethylene stresses during dynamic activity. In vitro simulation of a knee bend activity was performed for four cadaveric specimens implanted with PS TKA components. Post-cam engagement velocity and flexion angle at initial contact were determined. The experimental data were used to validate computational predictions of PS mechanics using the same loading conditions. A lower limb model was subsequently utilized to compare engagement mechanics of eight TKA designs, relating differences between implants to geometric design features. Flexion angle and post-cam velocity at engagement demonstrated considerable ranges among designs . Post-cam velocity was correlated ( The posterior cruciate ligament (PCL) is a primary contributor to anterior knee stability. During total knee arthroplasty (TKA), the PCL is often sacrificed. Posterior-stabilizing (PS) TKA components, which are designed to guide anteroposterior (AP) motion and rollback, are employed in lieu of the PCL. PS designs typically include a tibial post and femoral cam that engage as the knee flexes. Many patients receiving PS TKAs report a clicking sensation when the post and cam engage, likely due to excessive post-cam impact velocity. In a retrieval study of 23 components, seven posts (30%) exhibited severe wear, while two required early revision due to wear.A number of investigations into post-cam engagement mechanics were previously reported. The majority evaluated the influence of post-cam geometry on post wear, contact mechanics, or tibiofemoral (TF) kinematics.Several studies employed in vivo imaging techniques. Fluoroscopy, typically integrated with 3D models of the TKA components and used to track the relative poses between components during dynamic activity, has been used to calculate the timing of post-cam engagement or describe the effect of post-cam engagement on tibiofemoral kinematics.The dynamics of post-cam engagement are critical to joint stability, insert wear and fracture risk; the engagement velocity impacts the forces, stresses and potential longevity of the post. Excessive impact velocity likely causes the clicking sensation felt by many PS TKA patients at engagement. Thus far, however, engagement velocity has not been reported for PS TKAs. Also, investigations into the influence of design on post-cam mechanics have focused mostly on the post-cam designA TKA knee system  was tested in vitro in a knee simulator. TKA was performed on four fresh-frozen, healthy cadaveric knees by an experienced surgeon. Each implanted specimen was mounted into the Kansas knee simulator (KKS), and a deep knee bend was performed from 10° to 110° flexion. The KKS is a dynamic simulator, with loads applied at the hip, ankle, and quadriceps; the knee remains unconstrained in all six degrees-of-freedom (6-DOF)Combining the position data from the motion system with computer-aided-design (CAD) models from the implant manufacturer, the 3D kinematics between the femoral component and tibial insert were calculated throughout the activity. Relative 3D kinematics were used to estimate post-cam velocity and the flexion angle at the point of engagement. Post-cam velocity was calculated by identifying the points on the post and cam that came into initial contact, calculating the distance between these points throughout the cycle, and reporting the change in distance as a function of change in flexion angle. A flexion-extension cycle took ∼20 s, so velocity was measured as mm/° to normalize for flexion rate of (°/s).E = 572 MPa, ν = 0.45).A previously developed FE model of the KKS was virtually implanted with the same TKA components as the cadaveric tests. The model included femur, tibia, and patella, TKA components, TF ligaments, extensor mechanism, quadriceps muscles, and mechanical actuators To overcome some of the limitations of experimental simulators in reproducing in vivo dynamic activities, the model’s boundary conditions were adapted to better represent physiological loading at the knee.Post-cam velocity and engagement angle were calculated for each device throughout the knee bend simulation. Additionally, contact mechanics  on the post and medial and lateral condyles, internal stress in the tibial post, forces acting on the post, and AP femoral translations with respect to the tibia were recorded for each design. Medial and lateral AP translations were calculated by identifying the lowest medial and lateral points, respectively, on the femoral articular geometry along the SI axis of the tibia.R). The center of the radius at engagement (C) was found by calculating the tangent (T) to the femoral articular geometry at the lowest point on the femur . C was perpendicular to T and a distance R from the point of tangency (PoT). The distance from C to the point of first contact on the cam was also calculated  with the initial distance between the post and the cam (at the start of the squat cycle) and to a lesser non-significant extent the AP position of the posterior surface of the post  and also with the initial distance between the post and the cam , in addition to the change in slope .Post-cam velocity at engagement varied considerably among implants, ranging from 0.05 to 0.22 mm/° . Post-car = 0.78) with the ML spacing between the post and femoral box; designs with a wider gap  had lower ML force. An association between AP position of the posterior post surface and peak AP force on the post was found, but did not reach significance . The SI position (relative to the insert dwell point) at which initial contact occurred was correlated with flexion angle at engagement (r = −0.73) and post-cam velocity at engagement (−0.72); superior contact position on the post was related to an earlier engagement and slower velocity.External femoral torque imposed by the loading condition resulted in loading of the medial edge of the post and high ML force in Vanguard, Journey, Scorpio, and Sigma; r = 0.54), larger distance from the center of the radius at engagement to the point of first contact on the cam (r = 0.51), and narrower ML post-femoral gap (r = −0.59), although none of these relationships reached a level of significance (p > 0.05).Peak von Mises stress in the post ranged from 20 to 33 MPa, with Attune, Sigma, and Journey maintaining the lowest, and Genesis II and Vanguard creating the highest stress. The 90th percentile value ranged from 4 (Attune) to 9.5 MPa (Vanguard) in deep flexion . Higher Prior studies investigating post-cam mechanics under dynamic conditions measured sagittal plane angle of the patellar tendonA number of studies investigated the impact of component design on post-cam mechanics, comparing flat-on-flat and curve-on-curve geometriesOur study also reiterates the influence of post design features that have been described in prior studies. Some devices are designed with an ultra-constraining post to resist VV deformity and limit tibial rotation. Nakayama et al.The computational model demonstrated excellent agreement in predicting flexion angle of engagement and engagement velocity, while prior work demonstrated agreement in prediction of six-DOF TF and patellofemoral kinematics.We assessed engagement mechanics of these devices under a limited set of conditions. The analysis was performed for a single activity; alternative activities would test these components under different kinematic and loading conditions. However, a squat activity was chosen as an activity of daily living that required deep enough flexion to initiate post-cam engagement. Components were evaluated with a single set of representative soft-tissues. As illustrated by the standard deviation of the flexion angle at engagement for the four cadaveric specimens, substantial interspecimen variability exists in ligament integrity, and the level of constraint the soft-tissues provide to the knee influence kinematics and engagement mechanics. The anatomy and material properties used in the model were developed from another cadaveric specimen that was run in the KKS and exhibited kinematic behavior representative of a normal knee. Additional laxity tests were preformed on this specimen to calibrate ligament properties  in the model.In conclusion, we verified the importance of condylar geometry, in addition to geometry of the post-cam mechanism itself, in controlling post-cam engagement mechanics during dynamic activity and compared post-cam engagement velocity for a variety of commercial TKA devices. This knowledge may guide implant design to incorporate condylar and post-cam geometric features that reduce edge loading and facilitate smooth post-cam engagement.


Xist RNA which coats in cis the chromosome and triggers epigenetic reprogramming that will prevent chromosome-wide transcription. Xist transcription is controlled, directly and/or indirectly, by several ncRNA  and factors . However, mechanisms leading to Xist monoallelic regulation remain poorly understood.Dosage compensation for X-linked genes in female mammals relies on X-chromosome inactivation. This process involves monoallelic up-regulation of the non-coding Xist locus of the inactive X-chromosome. We show that this monoallelic binding is controlled by DNA methylation. To gain further insights into YY1 functional role on the Xist locus, we conducted YY1 knockdown experiments. We show by RNA-FISH that depletion of YY1 in female somatic cells impairs the accumulation of Xist on the inactive X-chromosome. This is likely due to a transcriptional effect as we observe a drastic reduction of both spliced and unspliced Xist RNA levels. This hypothesis is further reinforced by the in vitro analysis Xist promoter activity, which displays strict dependency on the YY1 binding sites. Importantly, YY1 is also necessary for the upregulation of Xist that triggers X-chromosome inactivation. Taken together, these results suggest a strong requirement for YY1 in the upregulation and maintenance of Xist transcription. Importantly, we demonstrate that the function of YY1 in the control of Xist expression is conserved in humans and predicted in other mammalian species.Using mouse ES and differentiating cells, we identified specific YY1 and CTCF interacting sites on the Xist at the exit of pluripotency and in the maintenance of its expression in somatic cells. Taken together with previous studies, we propose that through its dual action on Tsix and Xist, YY1 acts as a bimodal transcriptional regulator of X-inactivation.These results highlight the importance of YY1 both in the monoallelic upregulation of


The Department of Orán is located on the North of Argentina and is populated by native settlements and urbanized areas. During the last twelve years, an increasing number of HIV infections have been detected. We previously reported a highly limited HLA diversity with a reduced number of HLA-A and B alleles present at high prevalence. The objective of the present study was to characterize the HIV epidemic through full-length genome sequencing of the virus in this population.Samples from 147 HIV positive individuals collected in three campaigns to Orán were analyzed. Viral load, CD4-count and drug resistance were assessed and reported to the clinicians. Limiting-dilution full genome sequencing was performed from plasma on 65 of the samples. Viral diversity was analyzed by recombination (SimPlot) and Neighbor-Joining trees with bootstrap. HLA-A, B and C were characterized by SBT.HLA typing showed a limited genetic diversity even at the four-digits high-resolution typing. This allele distribution resembles the one reported previously for this community with two-digits and confirms that the individuals included were in fact native. SimPlot analysis of the 65 complete and nearly-complete genomes showed a high prevalence of BF recombinants(70.77%) and the presence of subtype B(23.08%), C(3.08%) and F(3.08%) virus. 7 main recombination structures were found repeatedly in not-epidemiologically-linked individuals within highly supported monophyletic clades suggesting transmission of these particular viral strains within the community.This HIV epidemic seems to be characterized by a few introductions of viral variants that had spread on the community. Most of them have been BF recombinants with particular recombination structures. This work will allow us to further investigate the impact of the genetic features of this native community on viral evolution but also has facilitated the access of these individuals to clinical studies and counseling.


Memory and mood deficits are the enduring brain-related symptoms in Gulf War illness (GWI). Both animal model and epidemiological investigations have indicated that these impairments in a majority of GW veterans are linked to exposures to chemicals such as pyridostigmine bromide , permethrin  and DEET (a mosquito repellant) encountered during the Persian Gulf War-1. Our previous study in a rat model has shown that combined exposures to low doses of GWI-related (GWIR) chemicals PB, PM, and DEET with or without 5-min of restraint stress (a mild stress paradigm) causes hippocampus-dependent spatial memory dysfunction in a water maze test (WMT) and increased depressive-like behavior in a forced swim test (FST). In this study, using a larger cohort of rats exposed to GWIR-chemicals and stress, we investigated whether the memory deficiency identified earlier in a WMT is reproducible with an alternative and stress free hippocampus-dependent memory test such as the object location test (OLT). We also ascertained the possible co-existence of hippocampus-independent memory dysfunction using a novel object recognition test (NORT), and alterations in mood function with additional tests for motivation and depression. Our results provide new evidence that exposure to low doses of GWIR-chemicals and mild stress for 4 weeks causes deficits in hippocampus-dependent object location memory and perirhinal cortex-dependent novel object recognition memory. An open field test performed prior to other behavioral analyses revealed that memory impairments were not associated with increased anxiety or deficits in general motor ability. However, behavioral tests for mood function such as a voluntary physical exercise paradigm and a novelty suppressed feeding test (NSFT) demonstrated decreased motivation levels and depression. Thus, exposure to GWIR-chemicals and stress causes both hippocampus-dependent and hippocampus-independent memory impairments as well as mood dysfunction in a rat model. Gulf War illness (GWI) afflicts ~30% of the 700,000 military personnel who served in the Persian Gulf War-1 (PGW-1). Central nervous system (CNS) impairments are the most ubiquitous among the various symptoms of GWI  receiving exposure to GWIR-chemicals PB, PM, and DEET and 5 min of restraint stress for 4 weeks (here after referred to as GWI-rats). A separate vehicle (VEH) group undergoing oral gavage, shaving and handling was not included in this study, as our earlier study showed that such VEH-treated rats do not differ from age-matched naive control rats in terms of behavior or hippocampal cytoarchitecture . Following their arrival, animals were housed for 2 weeks in an environmentally controlled room (~23°C) with a 12:12-h light–dark cycle, and were given food  and water The chemical PB  was administered through an oral gavage (500 μl in sterile water). Solutions of PM  and DEET  and were applied sequentially to shaved skin areas located on the back of neck and between scapulae. A rat restrainer was used for the induction of 5 min of restraint stress, as described in our previous study  was divided into 16 squares (25 × 25 cm each) on the computer tracking system (Noldus-Ethovision). The resulting grid appeared on the computer screen as illustrated in Figure This test comprised three successive trials with an inter-trial interval of 60 min. The first trial comprised placing the rat in the center of an empty open field box [100 cm (L) × 100 cm (W) × 60 cm (H)] and allowing the rat to freely explore the box for 5 min  for 4 continuous weeks. The equipment comprised a freely running activity wheel incorporated into the home cage and connected to a wheel counter having digital display to show the distance run by the rat . Food and water were provided This test provides a sensitive and reliable measure of depression and motivation level in animals that resemble those in humans  demonstrated that GWI-rats did not exhibit increased anxiety-like behavior, in comparison to naive control rats Figures . Furtherp < 0.0001, Figures p > 0.05, Figures p > 0.05, Figure p > 0.05, Figures In the OLT, naïve rats displayed a clear preference for the object moved to a novel place in comparison to the object that remained in the same (familiar) place, as these rats spent 71% of their object exploration time in the test phase with the object that was moved to a novel place . There was no interaction between treatment and time (p>0.05). Both groups exhibited an increased motivation for running over the course of 4 weeks . Comparison of distances ran for the entire 4-week of running period demonstrated significantly reduced running activity in GWI-rats  analyses in veterans with GWI have suggested hippocampus dysfunction  where animals treated with GWIR-chemicals and stress exhibited signs of anxiety in the form of reduced entries to open arms and reduced dwell time in open arms (Parihar et al., The results of this study reinforce that exposure to GWIR-chemicals and stress causes both hippocampus-dependent and hippocampus-independent memory dysfunction as well as mood impairments. From this viewpoint, this animal model recapitulates the major CNS symptoms seen in veterans with GWI, as memory and mood deficits being the most common of symptoms (Odegard et al., Bharathi Hattiangady and Vikas Mishra contributed equally to this work. Bharathi Hattiangady contributed to the experimental design for the various behavioral studies, analyzed and interpreted data, prepared figures and wrote the first version of manuscript text. Vikas Mishra performed several behavioral experiments, analyzed and interpreted data, and contributed toward the preparation of figures. Maheedhar Kodali performed some of the behavioral studies, analyzed data and contributed to preparation of figures. Bing Shuai and Xiolan Rao performed chemical and stress exposures to animals and contributed to behavioral experiments and collection of data. Ashok K. Shetty conceived the study, conceptualized behavioral paradigms, interpreted data, and prepared the final version of 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.


There is now considerable interest in the clinical practice of EUS. Over the decades, EUS has evolved from diagnostic imaging and tissue acquisition to EUS-guided interventions. Technological advances have improved the EUS image quality and in recent years, image enhancement using contrast and elastography have also become more widely available. In this special issue that focuses on EUS, five papers explored various diagnostic and therapeutic aspects of EUS. Three papers examined the impact of EUS in cancer T-staging. Two other papers dealt with the issue of EUS-guided therapeutics. One explored the role of EUS-guided radiofrequency ablation (RFA) in an animal model while the other are concerned with EUS-guided biliary access and drainage. Accuracy of endoscopic ultrasonography for determining the treatment method for early gastric cancer,” K. Mandai and K. Yasuda examined the value of EUS in determining the therapeutic strategy for early gastric cancer. They found excellent correlation (92.8%) between EUS assessment of mucosa/sm1 involvement and histology. However the accuracy was reduced in the context of ulcerated lesions and lesions larger than 2 cm.  In the first paper entitled “Comparison of diagnostic accuracies of various endoscopic examination techniques for evaluating the invasion depth of colorectal tumours,” S. Haruki et al. assessed the clinical value of magnifying endoscopy combined with EUS for estimating the invasion depth of colorectal tumours. They concluded that when it was difficult to evaluate the invasion depth of colorectal tumours on conventional endoscopy alone, the additional use of EUS may enhance diagnostic accuracy. In the second paper entitled “Endoscopic ultrasound-guided radiofrequency ablation (eus-rfa) of the pancreas in a porcine model,” S. Gaidhane et al. evaluated the use of a prototype monopolar probe inserted through the EUS-needle to perform EUS-RFA in the porcine pancreas. They found that EUS-guided RFA of the pancreatic head was well tolerated with minimal amount of pancreatitis. Recent studies have shown the feasibility of RFA in patients with stage III pancreatic cancer in open, percutaneous, or laparoscopic setting. This study offers the promise of a less invasive means of performing RFA of unresectable pancreatic cancer. In the third paper entitled “Evaluation of endoscopic ultrasound image quality is necessary in endosonographic assessment of early gastric cancer invasion depth,” S. Yamamoto et al. evaluated whether EUS image quality affected the accuracy of diagnosing the vertical invasion depth of early gastric cancer. They reported that low-quality EUS images led to an incorrect diagnosis of invasion depth of early gastric cancer, independent of tumour location and size. In the fourth paper entitled “The spectrum of endoscopic ultrasound intervention in biliary diseases: a single centre's experience in 31 cases,” S. Attasaranya et al. described the spectrum and experience of EUS-guided interventions in biliary diseases in a single-tertiary center. The overall technical success for EUS-guided biliary drainage was 77%; this was considerably higher in the last 2 years compared to the first 3 years (89% versus 61.5%), reflecting the effect of a learning curve in this challenging procedure, which may be associated with significant morbidity. In the fifth paper entitled “


Allergen-specific immunotherapy (SIT) offers a disease specific causative treatment by modifying the allergen-specific immune response allowing tolerance to higher doses of allergen and preventing progression of allergic diseases. It may be considered in patients allergic to furry animals. Current mammalian allergy vaccines are still prepared from relatively poorly defined allergen extracts and may induce immediate and late phase side effects. Although the mechanisms of SIT are still not fully understood, the more recent approaches report different strategies to reduce both allergen-specific IgE as well as T cell reactivity. The availability of recombinant allergens and synthetic peptides from the mammalian species has contributed to formulating new allergy vaccines to improve SIT for furry animal allergy. The majority of studies have focused on the major cat allergen Fel d 1 due to its extensive characterization in terms of IgE and T cell epitopes and to its dominant role in cat allergy. Here we review the most recent approaches, e.g., synthetic peptides, recombinant allergen derivatives, different hypoallergenic molecules, and recombinant allergens coupled to virus-like particles or immunomodulatory substances as well as strategies targeting the allergen to Fcγ receptors and the MHC class II pathway using a new route for administration. Many of the new vaccines hold promise but only a few of them have been investigated in clinical trials which will be the gold standard for evaluation of safety and efficacy in allergic patients. Allergen-specific immunotherapy (SIT) offers a disease specific causative treatment by inducing tolerance to the allergen and preventing progression of allergic diseases, for example development of asthma . Hence, The mechanisms of SIT are still not fully understood. The clinical efficacy is associated with the production of allergen-specific blocking immunoglobulin (Ig)G antibodies and the generation of T regulatory cell (T regs) responses and secretion of immunoregulatory cytokines such as interleukin 10 , 6; howeAllergy vaccines of today are still made from crude allergen extracts, which are not well-defined. Since the structures of the most common allergen molecules are now available, well-defined recombinant and synthetic allergy vaccines can be generated allowing targeting specific mechanisms of allergic disease without increasing their allergenicity –11. Many-Strategies targeting allergen-specific T cells by using T cell epitope-containing peptides , which give no IgE-mediated activation of effector cells.-Strategies reducing allergen-specific IgE reactivity, but conserving T cell reactivity.-Strategies based on carrier-bound fusion proteins using peptides. These allergen derived peptides are obtained from the IgE-binding sites of the allergen but contain no or reduced allergen-specific T cell epitopes and exhibit no or strongly reduced IgE reactivity. The peptides are fused to an allergen-unrelated viral carrier molecule and expressed as recombinant proteins. This technology was promoted by the Vienna group and a grass-pollen allergy vaccine based on carrier-bound peptides of the four major grass-pollen allergens is now under evaluation in a clinical phase II trial , 13–15.Side effects are related to the production of allergen-specific IgE antibodies but can also be mediated by the allergen-specific T cells. Several studies during the last years have used an allergen-unrelated carrier molecule, which provides T cell help for the production of IgG antibodies but avoid the activation of allergen-specific T cells. This approach would reduce T cell mediated side effects. Different strategies to reduce both IgE as well as allergen-specific T cell epitopes have been reported. These approaches will lead to less IgE and T cell-mediated side effects , 13.-StSeveral other strategies for improving SIT have been described in the studies published within the last years: e.g., targeting the allergen to Fcγ receptors , targetiTable ®, also known as cat PAD, Circassia Ltd, Oxford, UK), which were selected on the basis of MHC-binding characteristics. This approach allowed reducing the number of peptides without substantially compromising population coverage. The safety and tolerability of the vaccine was evaluated in a phase IIa study  compared to wild-type Fel d 1 . The theOther hypoallergenic mutants of rFel d 1 have been generated by the introduction of random mutated allergen sequences and the selection of derivatives with a maintained tertiary structure by phage display using IgE antibodies from cat-allergic patients. The mutants had a lower IgE-binding and T cell activation capacity and could induce strong IgG-anti Fel d 1 protective responses by mouse immunization experiments. Thus they should be good candidates for safe alternatives for SIT .Furthermore, in search of a vaccine for mouse allergy a structure-guided single point mutation has been performed for Mus m 1, the major mouse allergen which is an urinary protein. This mutation induced a spatial rearrangement of aromatic side chains and a lower allergenic activity although the T cell reactivity was preserved .One strategy to improve SIT has been to couple allergens to the bacteriophage Qß-derived virus-like particles. In mice, one injection of Qß coupled to Fel d 1 induced a Fel d 1-specific IgG response, and reduced anaphylactic reactions after rFel d 1 challenge . HoweverAnother carrier protein, the Pre-S domain of hepatitis B virus has been evaluated more recently . The catA chimeric human–cat fusion protein composed of a truncated human IgG Fc gamma 1 and Fel d 1 has been proposed for a new approach of SIT . This coA new technology called modular antigen translocation (MAT) has been applied to Fel d 1 to enhance immunogenicity and safety of SIT. By attaching a truncated invariant chain peptide, and a transactivator of transcription peptide to allergens, they are converted into cytoplasmic proteins targeting the MHC class II pathway . MAT fus1,25-Dihydrovitamin D3 has been shown to induce dendritic cells with tolerogenic properties, thus, increasing regulatory T cell responses . In an arFel d 1 has also been covalently coupled to carbohydrate-based particles (CBP) for targeting of dendritic cells and enhanced adjuvanticity in SIT . This apBetter characterization of recombinant allergens from mammalian species has contributed to formulate new allergy vaccines to improve SIT for patients allergic to furry animals. The identification of the major allergens in allergen sources is essential for generating vaccines, which may replace the natural allergen extract , 46. ThiThe 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.


Chronic pulmonary diseases are a major cause of morbidity and mortality and their impact is expected to increase in the future. Respiratory viruses are the most common cause of acute respiratory infections and it is increasingly recognized that respiratory viruses are a major cause of acute exacerbations of chronic pulmonary diseases such as asthma, chronic obstructive pulmonary disease and cystic fibrosis. There is now increasing evidence that the host response to virus infection is dysregulated in these diseases and a better understanding of the mechanisms of abnormal immune responses has the potential to lead to the development of new therapies for virus-induced exacerbations. The aim of this article is to review the current knowledge regarding the role of viruses and immune modulation in chronic pulmonary diseases and discuss avenues for future research and therapeutic implications. Chronic diseases are the leading cause of death worldwide and the third most common group of chronic diseases are chronic pulmonary diseases that account for an estimated four million deaths annually . The mosDespite the differences between COPD, asthma and CF, all three have in common that respiratory virus infections are a major trigger of acute exacerbations. An important mechanism underlying this may be impaired host immune responses to virus infection and a better understanding of these mechanisms has the potential to lead to the development of new therapies that may be beneficial in different chronic pulmonary diseases. The aim of this article is to review the current knowledge regarding the role of viruses and host immune responses in asthma, COPD and CF, and discuss avenues for future research and therapeutic interventions.Although this article primarily focuses on acute exacerbations of chronic respiratory diseases, virus infection has also been implicated in the induction of asthma. Asthma is strongly related to a genetic predisposition to develop allergic reactions to aeroallergens. However, not all individuals with atopy develop asthma and, therefore, it has been proposed that other environmental factors may act as 'triggers' to the development of asthma in genetically susceptible individuals. One such factor that has attracted much research interest is respiratory virus infections, in particular infection with respiratory syncytial virus (RSV). In the majority of cases RSV causes a self-limiting upper respiratory tract infection, but in infants under the age of one year it can cause a more serious infection of the lower respiratory tract - bronchiolitis - and studies have linked RSV bronchiolitis with an increased frequency of subsequent wheezing and asthma . Recentl2-agonists and inhaled corticosteroids, respectively. These are highly effective in relieving symptoms and reduce exacerbations by approximately 50% in clinical trials [Asthma is the most common chronic respiratory disease affecting up to 10% of adults and 30% of children in the western world . The Glol trials . Howeverl trials .It has long been recognized that viral respiratory tract infections are triggers for exacerbations of asthma in both adults and children but early studies reported low detection rates of viruses in asthma exacerbations casting doubt on this association. The development of highly sensitive and specific molecular diagnostic techniques using polymerase chain reaction (PCR) technology led to a reappraisal of the role of virus infections in asthma. Studies using PCR detected viruses in approximately 80% to 85% of asthma exacerbations in school-aged children and 60% to 80% of exacerbations in adults. Although respiratory virus infection can be detected in stable asthma patients detection rates are consistently lower than in exacerbated patients ,14. TherFollowing discovery of the role of RV in asthma exacerbations research attention has focused on the mechanisms of susceptibility to virus infection in asthmatics. RV infection in healthy individuals results in a predominantly upper respiratory symptom syndrome ('common cold'), whereas in asthmatics infection results in lower respiratory symptoms and airflow obstruction ('acute exacerbation'). A study of co-habiting partners discordant for the presence of asthma demonstrated that asthmatics do not have a higher frequency of RV infections but have more severe lower respiratory symptoms and changes in airway physiology . Similarin vitro and in vivo. RVs primarily enter and replicate in epithelial cells in the respiratory tract and trigger a cascade of immune and inflammatory responses. Following viral entry into a cell, uncoating of the virus leads to the release of viral RNA that is recognized by pattern recognition receptors including toll-like receptors (TLR)-3, -7 and -8, and the cytosolic RNA helicases, retinoic acid inducible gene I (RIG-I) and melanoma differentiation-associated protein-5 (MDA-5) [Most research into virus-induced asthma exacerbations has focused on RV as these are the most common viruses detected in asthma exacerbations and well-characterized models of RV infection exist both  (MDA-5) ,23. The  (MDA-5) -28. IFN- (MDA-5) , up-regu (MDA-5) . The IFNThe pro-inflammatory mediators and cytokines induced by RV infection lead to chemoattraction of inflammatory cells such as neutrophils, lymphocytes and eosinophils. This inflammatory response contributes to virus clearance but is also responsible for the pathology induced by RV infections. The balance between antiviral and inflammatory responses following virus infection is likely to determine the clinical outcome of the infection. An effective antiviral response rapidly controls viral replication with a minimal inflammatory response and limited clinical illness. If antiviral responses are inadequate this is likely to result in uncontrolled viral replication, greater inflammatory response and more severe clinical illness Figure . The eviet al. examined the kinetics of virus replication in bronchial epithelial cells obtained from asthmatics and healthy volunteers and reported that viral replication is increased in cells from asthmatics compared to non-asthmatic subjects [In 2005 Wark subjects . This wasubjects . Deficiesubjects , as wellsubjects -36 and dsubjects  has alsosubjects . Howeversubjects ,40. In esubjects ,41. TherIf interferon production in response to virus infection is impaired in asthmatics what are the possible molecular mechanisms underlying this? The discovery that IFN-α, IFN-β and IFN-λ are all deficient suggests that it is not a genetic defect as IFN-α and IFN-β are on different genetic loci than IFN-λ. A key family of proteins regulating both interferon production and allergic inflammation are the suppressor of cytokine signaling family (SOCS), and one member of this family, SOCS1, is a potent negative regulator of type I and type II interferons and of Th2 inflammation ,43. SOCSIn vitro infection of airway epithelial cells with RV induces the production of inflammatory mediators and this has also been reported in vivo in both experimental and naturally-acquired viral infections. Chemokines and cytokines such as IL-8, IL-6 and regulated on activation, normal T-cell expressed and secreted (RANTES) have been detected during virus infections in asthmatic patients [patients -49. Howepatients  but not patients . Increaspatients . These cMany of the inflammatory mediators produced are chemoattractants and, therefore, following virus infection inflammatory cells are recruited to the lungs. A number of different inflammatory cells have been identified in both naturally-occurring and experimental virus infections in asthma. Although stable asthma is characterized by eosinophilic inflammation, a number of studies have identified neutrophils as the key inflammatory cell in virus-induced asthma exacerbations ,50-52. NIncreased lymphocyte numbers in bronchoalveolar lavage (BAL) and bronchial biopsies in experimental RV infection in asthmatics has been reported, with increases in CD4+, CD8+ and NK cells ,54. AbnoThe identification of impaired innate immunity in asthma suggests a possible mechanism not only for virus-induced asthma exacerbations but also for the link between respiratory virus infections and the subsequent development of asthma. It is possible that infants who will develop asthma in later life have impaired immune responses from birth and, therefore, are more likely to develop more severe disease manifestations  following respiratory virus infection. Most studies to date have focused on the role of the acquired immune system and identified reduced IFN-γ production as a significant risk factor both for subsequent wheezing illness and allergic sensitization -61. No sIn conclusion there is evidence that both innate and acquired immune responses in asthmatics are impaired and this may be a key mechanism underlying virus-induced asthma exacerbations and the link between virus infections and the subsequent development of asthma in infants. Further studies are needed to determine whether these deficiencies are common to all asthmatics, whether they represent a specific asthma phenotype and how they relate to conventional measures of asthma control. Another important research question is whether new interventions targeting the interferon pathways can prevent asthma exacerbations and even potentially prevent the development of asthma in infants.Chronic obstructive pulmonary disease (COPD) is the most common chronic respiratory condition in adults. The Global Initiative for Obstructive Lung Disease (GOLD), a collaboration between the World Health Organization and the National Heart Lung and Blood Institute, defines COPD as 'a preventable and treatable disease with some significant extrapulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lung to noxious inhaled particles or gases' . The maiAlthough viruses are frequently detected in COPD exacerbations, their presence during exacerbations does not prove a definite causative role. Experimental infection using RV provides a novel tool for investigating relationships between virus infection and exacerbations. Such studies have been previously conducted in asthma and yielded important insights into the mechanisms linking virus infection to exacerbations in asthma. A recent study from our group reported the first experimental RV infection study in COPD . COPD paet al. reported that elevated sputum eosinophils were only seen in exacerbations in which a virus was present [Much less is known regarding mechanisms of virus-induced exacerbations in COPD compared to asthma. In the experimental infection study, symptoms, airflow obstruction and airways inflammation were more severe in the COPD subjects compared to non-obstructed controls . Therefo present . Others  present  and that present . From thThere is evidence from animal models that the inflammatory response to virus infection may be exaggerated in COPD. In a mouse model of COPD utilizing intranasal administration of lipopolysaccharide and elastase, infection with RV resulted in increased levels of TNF-α and IL-13 compared to control mice . This waIn vitro studies have shown that cigarette smoke impairs release of IFN-β and IFN-α [ex vivo with RV demonstrated deficient induction of IFN-β with similar trends for deficient induction of IFNs-α and -λ, associated with deficiency of the interferon stimulated gene CXCL10 [in vitro RV infection of epithelial cells from COPD patients resulted in higher virus load and increased inflammatory mediators, but no differences in interferon production compared to cells from control subjects [nd IFN-α . BAL cele CXCL10 . Similare CXCL10 . Howeversubjects . Furthersubjects ,88 and, The majority of studies have detected viruses at a greater frequency during acute exacerbations compared to the stable state. One study indicated that RSV is detected in nasal lavage at a similar frequency of around 25% in the stable state and during exacerbations . This waLatent infection by adenovirus has also been proposed to be involved in the pathogenesis of COPD. Lung tissue from COPD patients has been demonstrated to carry more group C adenoviral DNA than matched non-obstructed smokers . Latent Pseudomonas aeruginosa the most common organism detected. There has been relatively little research on the role of virus infections in CF but recent studies have suggested that viruses have a significant impact on the CF patient.Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene for the cystic fibrosis transmembrane regulator (CFTR) protein. Defective CFTR function leads to abnormal transport of chloride and sodium across the pulmonary epithelium, resulting in viscous secretions in the lungs, recurrent bacterial infections and progressive loss of lung function. Pulmonary involvement is the most common manifestation of the disease and respiratory failure the most common cause of death. Respiratory infections are the leading cause of morbidity, decline in lung function and hospitalizations due to acute exacerbations. The major cause of infectious complications in CF has always been considered to be bacterial infection, with The role of respiratory viruses in CF exacerbations is likely to have been under-appreciated in the past because older studies investigated only one virus type and the detection methods used were not sufficiently sensitive. Newer PCR techniques have helped to improve detection and it is now becoming clear that viruses are implicated in exacerbations in CF. Previous studies using serology, culture and immunoflourescence detected viruses in 10% to 28% of exacerbations in CF patients -98. In cThe mechanisms of viral-induced CF exacerbations and increased clinical illness are poorly understood with conflicting results from published studies. Some authors have reported increased production of pro-inflammatory cytokines and chemokines by epithelial cells obtained from CF patients compared to healthy controls ,104. HowIn vitro studies have reported that RV infection increases bacterial adhesion to epithelial cells [et al. reported that RV infection of CF cells liberates planktonic bacteria from biofilm [Haemophilus influenzae (a common organism in COPD) increases susceptibility to infection by RV, possibly by up-regulation of ICAM-1 [P.aeruginosa and then with RV produced less IFN and viral loads were higher compared to cells infected with the RV alone [Pseudomonas and was related to the inhibition of Akt phosphorylation and IRF-3 activation - both pre-requisites for the IFN response to RV infection.Both bacterial and virus infections are common in CF and COPD and, therefore, co-infections are likely to be common. There is now increasing evidence that both viral and bacterial infections can modulate host immune responses and increase susceptibility to subsequent infection. There is abundant evidence from both human studies and animal models that influenza infection impairs antibacterial immunity and this can result in secondary bacterial pneumonia ,110. Howal cells -113 and al cells . We have biofilm . Planktof ICAM-1 . CF cellRV alone . This efIt is widely acknowledged that the main infectious cause of asthma exacerbations is virus infection and it is believed that bacteria play only a minor role. However, a recent study using culture-independent molecular methods for bacterial detection reported that the bacterial flora in the airways of asthmatics is closer to that of COPD patients than of non-asthmatics . The rolOur knowledge of the interactions between respiratory viruses and bacteria, and how these influence host immune responses in pulmonary diseases, is still at an early stage. Further research is required to understand better these complex relationships and to explore the implications they may have for the development of new therapies.There is now convincing data implicating respiratory viruses as a major cause of acute exacerbations in asthma, COPD and CF. In all these conditions there is evidence that host immune responses to virus infection are impaired, but whether this occurs through a common mechanism, or whether mechanisms differ between the different diseases is unclear. Further research is needed to elucidate the exact mechanisms of increased susceptibility to virus infection in pulmonary diseases, the interactions between viruses and bacteria and how these impact on host immune responses. A better understanding of these mechanisms has the potential to lead to the development of novel therapies that will reduce the impact of acute exacerbations in chronic pulmonary diseases.ATF: activating transcription factor; BAL: bronchoalveolar lavage; CF: cystic fibrosis; CFTR: cystic fibrosis transmembrane regulator; COPD: chronic obstructive pulmonary disease; ENA-78: epithelial-derived neutrophil-activating peptide 78; ICAM-1: intercellular adhesion molecule; IFN-α: interferon-alpha; IFN-β: interferon-beta; IFN-λ: interferon-lambda; IFN-γ: interferon-gamma; IL: interleukin; IP-10: IFN-γ-induced protein-10; IRF: interferon regulatory factor; ISG: interferon stimulated genes; MDA-5: melanoma differentiation-associated protein-5; NF-κB: nuclear factor-kappa B; NO: nitric oxide; NOS2: nitric oxide synthase 2; PCR: polymerase chain reaction; PEF: peak expiratory flow; PIV: parainfluenza virus; RANTES: regulated on activation: normal T-cell expressed and secreted; RIG-I: retinoic acid inducible gene I; RSV: respiratory syncytial virus; RV: rhinovirus; SLPI: secretory leukoprotease inhibitor; SOCS: suppressor of cytokine signaling family; Th1/2: T helper 1/2; TLR: toll-like receptors; TNF-α: tumor necrosis factor-alpha -1.SLJ has patents on the use of interferons as a treatment for asthma exacerbations and COPD exacerbations and share options in Synairgen. AS, PVJ, and PM declare that they have no competing interests.All the authors contributed equally to writing the manuscript. All authors read and approved the final manuscript.SLJ heads a group working on mechanisms of asthma and chronic obstructive pulmonary disease and the role of respiratory viral infections in these diseases, with a particular interest in acute exacerbations and the role of rhinovirus infections. PM and AS work in his group with a particular focus on human and mouse models of virus infection in COPD. PVJ works in the Department of Respiratory Medicine at Imperial College NHS Healthcare Trust.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/10/27/prepub