Transcriptomics in lung tissue upon respiratory syncytial virus infection reveals aging as important modulator of immune activation and matrix maintenance.

Aging poses an increased risk of severe infection by respiratory syncytial virus (RSV). The many different biological pathways comprising the response to infection in lungs that are influenced by aging are complex and remain to be defined more thoroughly. Towards finding new directions in research on aging, we aimed to define biological pathways in the acute response to RSV that are affected in the lungs by aging. We therefore profiled the full transcriptome of lung tissue of mice prior to and during RSV infection both at young and old age. In the absence of RSV, we found aging to downregulate genes that are involved in constitution of the extracellular matrix. Moreover, uninfected old mice showed elevated expression of pathways that resemble injury, metabolic aberrations, and disorders mediated by functions of the immune system that were induced at young age only by an exogenous trigger like RSV. Furthermore, infection by RSV mounted stronger activation of anti-viral type-I interferon pathways at old age. Despite such exaggerated anti-viral responses, old mice showed reduced control of virus. Altogether, our findings emphasize important roles in aging-related susceptibility to respiratory disease for extracellular matrix dysfunctions and dysregulated immune activation in lungs

Analysis of Lipid Metabolism, Immune Function, and Neurobehavior in Adult C57BL/6JxFVB Mice After Developmental Exposure to di (2-ethylhexyl) Phthalate

Background: Developmental exposure to di (2-ethylhexyl) phthalate (DEHP) has been implicated in the onset of metabolic syndrome later in life. Alterations in neurobehavior and immune functions are also affected by phthalate exposure and may be linked to the metabolic changes caused by developmental exposure to DEHP.Objectives: Our goal was to study the effects of developmental exposure to DEHP in the context of metabolic syndrome by integrating different parameters to assess metabolic, neurobehavioral, and immune functions in one model.Methods: Female C57BL/6J mice were exposed to DEHP through the diet during gestation and lactation at doses ranging from 3.3 to 100,000 μg/kg body weight/day (μkd). During a 1-year follow-up period, a wide set of metabolic parameters was assessed in the F1 offspring, including weekly body weight measurements, food consumption, physical activity, glucose homeostasis, serum lipids, and endocrine profile. In addition, neurobehavioral and immune functions were assessed by sweet preference test, object recognition test, acute phase protein, and cytokines production. Animals were challenged with a high fat diet (HFD) in the last 9 weeks of the study.Results: Increased free fatty acids (FFA) and, high density lipoprotein (HDL-C) were observed in serum, together with a decrease in glycated hemoglobin levels in blood of 1-year old male DEHP-exposed offspring after HFD challenge. For the most sensitive endpoint measured (FFA), a lower bound of the 90%-confidence interval for benchmark dose (BMD) at a critical effect size of 5% (BMDL) of 2,160 μkd was calculated. No persistent changes in body weight or fat mass were observed. At 33,000 μkd altered performance was found in the object recognition test in males and changes in interferon (IFN)γ production were observed in females.Conclusions: Developmental exposure to DEHP combined with HFD in adulthood led to changes in lipid metabolism and neurobehavior in male offspring and cytokine production in female offspring. Our findings contribute to the evidence that DEHP is a developmental dyslipidemic chemical, however, more research is needed to further characterize adverse health outcomes and the mechanisms of action associated with the observed sex-specific effects

Genome-wide association study of polymorphisms predisposing to bronchiolitis

Bronchiolitis is a major cause of hospitalization among infants. Severe bronchiolitis is associated withlater asthma, suggesting a common genetic predisposition. Genetic background of bronchiolitis is notwell characterized. To identify polymorphisms associated with bronchiolitis, we conducted a genomewideassociation study (GWAS) in which 5,300,000 single nucleotide polymorphisms (SNPs) were testedfor association in a Finnish–Swedish population of 217 children hospitalized for bronchiolitis and 778controls. The most promising SNPs (n = 77) were genotyped in a Dutch replication population of 416cases and 432 controls. Finally, we used a set of 202 Finnish bronchiolitis cases to further investigatecandidate SNPs. We did not detect genome-wide significant associations, but several suggestiveassociation signals (p were nominally associated (p (eQTL) for KCND3, previously shown to be associated with occupational asthma. In the additionalset of Finnish cases, the association for another SNP (rs9591920) within a noncoding RNA locus wasfurther strengthened. Our results provide a first genome-wide examination of the genetics underlyingbronchiolitis. These preliminary findings require further validation in a larger sample size.</p

Systemic Signature of the Lung Response to Respiratory Syncytial Virus Infection

Respiratory Syncytial Virus is a frequent cause of severe bronchiolitis in children. To improve our understanding of systemic host responses to RSV, we compared BALB/c mouse gene expression responses at day 1, 2, and 5 during primary RSV infection in lung, bronchial lymph nodes, and blood. We identified a set of 53 interferon-associated and innate immunity genes that give correlated responses in all three murine tissues. Additionally, we identified blood gene signatures that are indicative of acute infection, secondary immune response, and vaccine-enhanced disease, respectively. Eosinophil-associated ribonucleases were characteristic for the vaccine-enhanced disease blood signature. These results indicate that it may be possible to distinguish protective and unfavorable patient lung responses via blood diagnostics

Development and Comparison of Two Assay Formats for Parallel Detection of Four Biothreat Pathogens by Using Suspension Microarrays

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95 % confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for applicatio

Local interleukin-10 production during respiratory syncytial virus bronchiolitis is associated with post-bronchiolitis wheeze

Abstract Background Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants. Following RSV bronchiolitis, 50% of children develop post-bronchiolitis wheeze (PBW). Animal studies have suggested that interleukin (IL)-10 plays a critical role in the pathogenesis of RSV bronchiolitis and subsequent airway hyperresponsiveness. Previously, we showed that ex vivo monocyte IL-10 production is a predictor of PBW. Additionally, heterozygosity of the single-nucleotide polymorphism (SNP) rs1800872 in the IL10 promoter region was associated with protection against RSV bronchiolitis. Methods This study aimed to determine the in vivo role of IL-10 in RSV pathogenesis and recurrent wheeze in a new cohort of 235 infants hospitalized for RSV bronchiolitis. IL-10 levels in nasopharyngeal aspirates (NPAs) were measured at the time of hospitalization and the IL10 SNP rs1800872 genotype was determined. Follow-up data were available for 185 children (79%). Results Local IL-10 levels during RSV infection turned out to be higher in infants that later developed physician diagnosed PBW as compared to infants without PBW in the first year after RSV infection (958 vs 692 pg/ml, p = 0.02). The IL10 promoter SNP rs1800872 was not associated with IL-10 concentration in NPAs. Conclusion The relationship between high local IL-10 levels during the initial RSV infection and physician diagnosed PBW provides further evidence of the importance of the IL-10 response during RSV bronchiolitis.</p

Detection limits of the DH and TSPE-UH suspension array formats.

a<p>Values displayed represent the lowest DNA concentration at which 95% of the positive samples are detected, as calculated by using probit analysis. ND = not determined.</p

Typical results from DH and TSPE-UH suspension microarrays detecting select pathogens.

<p>Two 17-plex bead arrays were developed for the detection of <i>B. anthracis</i> (Ba), <i>F. tularensis</i> (Ft), <i>Y. pestis</i> (Yp), <i>C. burnetii</i> (Cb) and an internal control for DNA extraction and microarray detection (Bt). The microarrays were based on (<b>A</b>) direct hybridization (DH), or (<b>B</b>) target specific primer extension combined with universal microarray hybridization (TSPE-UH) assay formats. Both microarrays make use of identical amplification products from a 16-plex asymmetric PCR. Mean fluorescence intensity (MFI) is displayed for the different probes that are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031958#pone-0031958-t001" target="_blank">Table 1</a>.</p

Oligonucleotides used for amplification and labeling of signature sequences and as fixed probes.

a<p>Excess primer = X, Limiting primer = L.</p>b<p><i>F. tularensis</i> subspecies <i>tularensis</i> yields amplicon of 307 bp, subspecies <i>novicida</i> and <i>mediasiatica</i> of 451 bp.</p