Generalized multi-SNP mediation intersection–union test

To elucidate the molecular mechanisms underlying genetic variants identified from genome-wide association studies (GWAS) for a variety of phenotypic traits encompassing binary, continuous, count, and survival outcomes, we propose a novel and flexible method to test for mediation that can simultaneously accommodate multiple genetic variants and different types of outcome variables. Specifically, we employ the intersection–union test approach combined with the likelihood ratio test to detect mediation effect of multiple genetic variants via some mediator (e.g., the expression of a neighboring gene) on outcome. We fit high-dimensional generalized linear mixed models under the mediation framework, separately under the null and alternative hypothesis. We leverage Laplace approximation to compute the marginal likelihood of outcome and use coordinate descent algorithm to estimate corresponding parameters. Our extensive simulations demonstrate the validity of our proposed methods and substantial, up to 97%, power gains over alternative methods. Applications to real data for the study of Chlamydia trachomatis infection further showcase advantages of our methods. We believe our proposed methods will be of value and general interest in this post-GWAS era to disentangle the potential causal mechanism from DNA to phenotype for new drug discovery and personalized medicine

Characterization of the Horizontal and Vertical Sexual Transmission of Chlamydia Genital Infections in a New Mouse Model.

Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide, and there is a need to control this epidemic. So far there is no established animal model in which both the horizontal and the vertical transmission of Chlamydia can be studied. To implement a horizontal sexual transmission model, male mice were inoculated in the meatus urethra with Chlamydia muridarum and they were caged with naive female mice. Urine and vaginal swab specimens were collected for culture. To study vertical transmission, newborns were euthanized and specimens were cultured. As controls, females were mated with sham-infected male mice. All C. muridarum-inoculated male mice had positive urine cultures. As determined by serology, all females caged with C. muridarum-inoculated males became infected, and 93% of them had positive vaginal swab specimen cultures. More females mated with C. muridarum-infected male mice (35%) than females mated with sham-infected male mice (0%) were infertile (P < 0.05). Also, C. muridarum-infected females delivered significantly fewer pups (3.8 ± 3.2/mouse) than control females (6.3 ± 1.6/mouse) (P < 0.05). Of the newborn mice, 32% were C. muridarum positive either in the lungs or in the intestines. Female mice housed with sham-infected males had no positive vaginal swab specimen cultures or C. muridarum-positive pups. This new mouse model of horizontal and vertical sexual transmission of Chlamydia closely parallels C. trachomatis sexual transmission in humans and may be a good model system to better understand the pathogenesis of these infections

Plasmid-Cured Chlamydia caviae Activates TLR2-Dependent Signaling and Retains Virulence in the Guinea Pig Model of Genital Tract Infection

Loss of the conserved “cryptic” plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL) in C. caviae. However, 2-deoxyglucose (2DG) treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains

Human Female Genital Tract Infection by the Obligate Intracellular Bacterium Chlamydia trachomatis Elicits Robust Type 2 Immunity

While Chlamydia trachomatis infections are frequently asymptomatic, mechanisms that regulate host response to this intracellular Gram-negative bacterium remain undefined. This investigation thus used peripheral blood mononuclear cells and endometrial tissue from women with or without Chlamydia genital tract infection to better define this response. Initial genome-wide microarray analysis revealed highly elevated expression of matrix metalloproteinase 10 and other molecules characteristic of Type 2 immunity (e.g., fibrosis and wound repair) in Chlamydia-infected tissue. This result was corroborated in flow cytometry and immunohistochemistry studies that showed extant upper genital tract Chlamydia infection was associated with increased co-expression of CD200 receptor and CD206 (markers of alternative macrophage activation) by endometrial macrophages as well as increased expression of GATA-3 (the transcription factor regulating TH2 differentiation) by endometrial CD4+ T cells. Also among women with genital tract Chlamydia infection, peripheral CD3+ CD4+ and CD3+ CD4- cells that proliferated in response to ex vivo stimulation with inactivated chlamydial antigen secreted significantly more interleukin (IL)-4 than tumor necrosis factor, interferon-γ, or IL-17; findings that repeated in T cells isolated from these same women 1 and 4 months after infection had been eradicated. Our results thus newly reveal that genital infection by an obligate intracellular bacterium induces polarization towards Type 2 immunity, including Chlamydia-specific TH2 development. Based on these findings, we now speculate that Type 2 immunity was selected by evolution as the host response to C. trachomatis in the human female genital tract to control infection and minimize immunopathological damage to vital reproductive structures. © 2013 Vicetti Miguel et al

Inferring Regulatory Networks From Mixed Observational Data Using Directed Acyclic Graphs

Construction of regulatory networks using cross-sectional expression profiling of genes is desired, but challenging. The Directed Acyclic Graph (DAG) provides a general framework to infer causal effects from observational data. However, most existing DAG methods assume that all nodes follow the same type of distribution, which prohibit a joint modeling of continuous gene expression and categorical variables. We present a new mixed DAG (mDAG) algorithm to infer the regulatory pathway from mixed observational data containing both continuous variables (e.g. expression of genes) and categorical variables (e.g. categorical phenotypes or single nucleotide polymorphisms). Our method can identify upstream causal factors and downstream effectors closely linked to a variable and generate hypotheses for causal direction of regulatory pathways. We propose a new permutation method to test the conditional independence of variables of mixed types, which is the key for mDAG. We also utilize an L1 regularization in mDAG to ensure it can recover a large sparse DAG with limited sample size. We demonstrate through extensive simulations that mDAG outperforms two well-known methods in recovering the true underlying DAG. We apply mDAG to a cross-sectional immunological study of Chlamydia trachomatis infection and successfully infer the regularity network of cytokines. We also apply mDAG to a large cohort study, generating sensible mechanistic hypotheses underlying plasma adiponectin level. The R package mDAG is publicly available from CRAN at https://CRAN.R-project.org/package=mDAG

Enhanced Virulence of Chlamydia muridarum Respiratory Infections in the Absence of TLR2 Activation

Chlamydia trachomatis is a common sexually transmitted pathogen and is associated with infant pneumonia. Data from the female mouse model of genital tract chlamydia infection suggests a requirement for TLR2-dependent signaling in the induction of inflammation and oviduct pathology. We hypothesized that the role of TLR2 in moderating mucosal inflammation is site specific. In order to investigate this, we infected mice via the intranasal route with C. muridarum and observed that in the absence of TLR2 activation, mice had more severe disease, higher lung cytokine levels, and an exaggerated influx of neutrophils and T-cells into the lungs. This could not be explained by impaired bacterial clearance as TLR2-deficient mice cleared the infection similar to controls. These data suggest that TLR2 has an anti-inflammatory function in the lung during Chlamydia infection, and that the role of TLR2 in mucosal inflammation varies at different mucosal surfaces

Role of Secreted Conjunctival Mucosal Cytokine and Chemokine Proteins in Different Stages of Trachomatous Disease

Trachoma, a disease of antiquity dating back to the 16th century B.C.E., predominates among developing countries, where it remains the primary cause of preventable blindness worldwide. In trachoma, recurrent Chlamydia trachomatis bacterial infections during childhood are thought to result in inflammation and subsequent conjunctival scarring that can progress to trichiasis (TT; chronic trachoma; inversion of ≥1 eyelash that touches the globe of the eye). The trachomatous follicular grade (TF; active disease) is a self-limiting disease, suggesting the coexistence of protective inflammatory proteins. The trachomatous inflammatory grade (TI; active disease) is more likely to progress to trachomatous scarring (TS; chronic trachoma). To date, there are only a handful of studies that have examined the immune response in trachoma, and these were primarily based on gene expression. Characterizing quantified conjunctival mucosal immune differences for secreted proteins among individuals with no, active, and chronic trachoma may identify protein biomarkers associated with protection versus disease, which would greatly aid our understanding of the immunopathogenesis of trachoma. In this study, we characterized 25 cytokine and chemokine proteins for all trachoma grades. We identified eight cytokines and chemokines as risk factors for chronic trachoma and four as protective. Together, these findings further characterize the immunopathologic responses involved during trachoma, which will likely aid in the design of a vaccine and immunomodulating therapeutics for trachoma

Chlamydial Pre-Infection Protects From Subsequent Herpes Simplex Virus-2 Challenge in a Murine Vaginal Super-Infection Model

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singlyinfected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans

Incidence of Respiratory Virus-Associated Pneumonia in Urban Poor Young Children of Dhaka, Bangladesh, 2009–2011

Pneumonia is the leading cause of childhood death in Bangladesh. We conducted a longitudinal study to estimate the incidence of virus-associated pneumonia in children aged <2 years in a low-income urban community in Dhaka, Bangladesh.We followed a cohort of children for two years. We collected nasal washes when children presented with respiratory symptoms. Study physicians diagnosed children with cough and age-specific tachypnea and positive lung findings as pneumonia case-patients. We tested respiratory samples for respiratory syncytial virus (RSV), rhinoviruses, human metapneumovirus (HMPV), influenza viruses, human parainfluenza viruses (HPIV 1, 2, 3), and adenoviruses using real-time reverse transcription polymerase chain reaction assays.Between April 2009-March 2011, we followed 515 children for 730 child-years. We identified a total of 378 pneumonia episodes, 77% of the episodes were associated with a respiratory viral pathogen. The overall incidence of pneumonia associated with a respiratory virus infection was 40/100 child-years. The annual incidence of pneumonia/100 child-years associated with a specific respiratory virus in children aged < 2 years was 12.5 for RSV, 6 for rhinoviruses, 6 for HMPV, 4 for influenza viruses, 3 for HPIV and 2 for adenoviruses.Young children in Dhaka are at high risk of childhood pneumonia and the majority of these episodes are associated with viral pathogens. Developing effective low-cost strategies for prevention are a high priority

The Interplay between NF-kappaB and E2F1 Coordinately Regulates Inflammation and Metabolism in Human Cardiac Cells

Pyruvate dehydrogenase kinase 4 (PDK4) inhibition by nuclear factor-κB (NF-κB) is related to a shift towards increased glycolysis during cardiac pathological processes such as cardiac hypertrophy and heart failure. The transcription factors estrogen-related receptor-α (ERRα) and peroxisome proliferator-activated receptor (PPAR) regulate PDK4 expression through the potent transcriptional coactivator PPARγ coactivator-1α (PGC-1α). NF-κB activation in AC16 cardiac cells inhibit ERRα and PPARβ/δ transcriptional activity, resulting in reduced PGC-1α and PDK4 expression, and an enhanced glucose oxidation rate. However, addition of the NF-κB inhibitor parthenolide to these cells prevents the downregulation of PDK4 expression but not ERRα and PPARβ/δ DNA binding activity, thus suggesting that additional transcription factors are regulating PDK4. Interestingly, a recent study has demonstrated that the transcription factor E2F1, which is crucial for cell cycle control, may regulate PDK4 expression. Given that NF-κB may antagonize the transcriptional activity of E2F1 in cardiac myocytes, we sought to study whether inflammatory processes driven by NF-κB can downregulate PDK4 expression in human cardiac AC16 cells through E2F1 inhibition. Protein coimmunoprecipitation indicated that PDK4 downregulation entailed enhanced physical interaction between the p65 subunit of NF-κB and E2F1. Chromatin immunoprecipitation analyses demonstrated that p65 translocation into the nucleus prevented the recruitment of E2F1 to the PDK4 promoter and its subsequent E2F1-dependent gene transcription. Interestingly, the NF-κB inhibitor parthenolide prevented the inhibition of E2F1, while E2F1 overexpression reduced interleukin expression in stimulated cardiac cells. Based on these findings, we propose that NF-κB acts as a molecular switch that regulates E2F1-dependent PDK4 gene transcription