HLA gene expression is altered in whole blood and placenta from women who later developed preeclampsia

Preeclampsia is a multi-system disease that significantly contributes to maternal and fetal morbidity and mortality. In this study, we used a non-biased microarray approach to identify dysregulated genes in maternal whole blood samples which may be associated with the development of preeclampsia. Whole blood samples were obtained at 28 weeks of gestation from 5 women who later developed preeclampsia (cases) and 10 matched women with normotensive pregnancies (controls). Placenta samples were obtained from an independent cohort of 19 women with preeclampsia matched with 19 women with normotensive pregnancies. We studied gene expression profiles using Illumina microarray in blood and validated changes in gene expression in whole blood and placenta tissue by qPCR. We found a transcriptional profile differentiating cases from controls; 236 genes were significantly dysregulated in blood from women who developed preeclampsia. Functional annotation of microarray results indicated that most of the genes found to be dysregulated were involved in inflammatory pathways. Whilst general trends were preserved, only HLA-A was validated in whole blood samples from cases using qPCR (2.30 ± 0.9 fold change) whereas in placental tissue HLA-DRB1 expression was found to be significantly increased in samples from women with preeclampsia (5.88 ± 2.24 fold change). We have identified that HLA-A is up-regulated in the circulation of women who went on to develop preeclampsia. In placenta of women with preeclampsia we identified that HLA-DRB1 is up-regulated. Our data provide further evidence for involvement of the HLA gene family in the pathogenesis of preeclampsia

In vivo safety and efficacy testing of a thermally triggered injectable hydrogel scaffold for bone regeneration and augmentation in a rat model

Bone loss resulting from degenerative diseases and trauma is a significant clinical burden which is likely to grow exponentially with the aging population. In a number of conditions where pre-formed materials are clinically inappropriate an injectable bone forming hydrogel could be beneficial. The development of an injectable hydrogel to stimulate bone repair and regeneration would have broad clinical impact and economic benefit in a variety of orthopedic clinical applications. We have previously reported the development of a Laponite® crosslinked pNIPAMco- DMAc (L-pNIPAM-co-DMAc) hydrogel delivery system, loaded with hydroxyapatite nanoparticles (HAPna), which was capable of inducing osteogenic differentiation of mesenchymal stem cells (MSCs) without the need for additional growth factors in vitro. However to enable progression towards clinical acceptability, biocompatibility and efficacy of the L-pNIPAM-co-DMAc hydrogel to induce bone repair in vivo must be determined. Biocompatibility was evaluated by subcutaneous implantation for 6 weeks in rats, and efficacy to augment bone repair was evaluated within a rat femur defect model for 4 weeks. No inflammatory reactions, organ toxicity or systemic toxicity were observed. In young male rats where hydrogel was injected, defect healing was less effective than sham operated controls when rat MSCs were incorporated. Enhanced bone healing was observed however, in aged exbreeder female rats where acellular hydrogel was injected, with increased deposition of collagen type I and Runx2. Integration of the hydrogel with surrounding bone was observed without the need for delivered MSCs; native cell infiltration was also seen and bone formation was observed within all hydrogel systems investigated. This hydrogel can be delivered directly into the target site, is biocompatible, promotes increased bone formation and facilitates migration of cells to promote integration with surrounding bone, for safe and efficacious bone repai

Differential expression of microRNA-206 and its target genes in pre-eclampsia

Objectives: Pre-eclampsia is a multi-system disease that significantly contributes to maternal and fetal morbidity and mortality. In this study, we used a non-biased microarray approach to identify novel circulating miRNAs in maternal plasma that may be associated with pre-eclampsia. Methods: Plasma samples were obtained at 16 and 28 weeks of gestation from 18 women who later developed pre-eclampsia (cases) and 18 matched women with normotensive pregnancies (controls). We studied miRNA expression profiles in plasma and subsequently confirmed miRNA and target gene expression in placenta samples. Placental samples were obtained from an independent cohort of 19 women with pre-eclampsia matched with 19 women with normotensive pregnancies. Results: From the microarray, we identified 1 miRNA that was significantly differentially expressed between cases and controls at 16 weeks of gestation and 6 miRNAs that were significantly differentially expressed at 28 weeks. Following qPCR validation only one, miR-206, was found to be significantly increased in 28 week samples in women who later developed pre-eclampsia (1.4 fold change ± 0.2). The trend for increase in miR-206 expression was mirrored within placental tissue from women with pre-eclampsia. In parallel, IGF-1, a target gene of miR-206, was also found to be down-regulated (0.41 ± 0.04) in placental tissue from women with pre-eclampsia. miR-206 expression was also detectable in myometrium tissue and trophoblast cell lines. Conclusions: Our pilot study has identified miRNA-206 as a novel factor up-regulated in pre-eclampsia within the maternal circulation and in placental tissue

Acute exacerbations of chronic obstructive pulmonary disease are associated with decreased CD4+ & CD8+ T cells and increased growth & differentiation factor-15 (GDF-15) in peripheral blood

Abstract Background Although T cells, especially CD8+, have been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, their role during acute exacerbations (AE-COPD) is uncertain. Methods We recruited subjects with COPD and a history of previous AE-COPD and studied them quarterly to collect blood and spontaneously expectorated sputum while stable. During exacerbations (defined by a change in symptoms plus physician diagnosis and altered medications), we collected blood and sputum before administering antibiotics or steroids. We used flow cytometry to identify leukocytes in peripheral blood, plus Luminex® analysis or ELISA to determine levels of inflammatory biomarkers in serum and sputum supernatants. Results Of 33 enrolled subjects, 13 participated in multiple stable visits and had ≥1 AE-COPD visit, yielding 18 events with paired data. Flow cytometric analyses of peripheral blood demonstrated decreased CD4+ and CD8+ T cells during AE-COPD (both absolute and as a percentage of all leukocytes) and significantly increased granulocytes, all of which correlated significantly with serum C-reactive protein (CRP) concentrations. No change was observed in other leukocyte populations during AE-COPD, although the percentage of BDCA-1+ dendritic cells expressing the activation markers CD40 and CD86 increased. During AE-COPD, sICAM-1, sVCAM-1, IL-10, IL-15 and GDF-15 increased in serum, while in sputum supernatants, CRP and TIMP-2 increased and TIMP-1 decreased. Conclusions The decrease in CD4+ and CD8+ T cells (but not other lymphocyte subsets) in peripheral blood during AE-COPD may indicate T cell extravasation into inflammatory sites or organized lymphoid tissues. GDF-15, a sensitive marker of cardiopulmonary stress that in other settings independently predicts reduced long-term survival, is acutely increased in AE-COPD. These results extend the concept that AE-COPD are systemic inflammatory events to which adaptive immune mechanisms contribute. Trial registration NCT00281216 , ClinicalTrials.gov.http://deepblue.lib.umich.edu/bitstream/2027.42/112169/1/12931_2015_Article_251.pd

Smoking decreases the response of human lung macrophages to double-stranded RNA by reducing TLR3 expression

Abstract Background Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mø) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production. Methods We collected alveolar macrophages (AMø) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules (n = 16) and of volunteers who were current or former smokers (n = 7) or never-smokers (n = 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMø and in the human Mø cell line THP-1 following phorbol myristate acetate/vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mø from subjects undergoing clinically-indicated lung resections (n = 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMø production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay. Results Relative to AMø of never-smokers, AMø of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mø, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMø of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMø of smokers. Conclusions Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mø expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers. Trial registration ClinicalTrials.gov NCT00281190 , NCT00281203 and NCT00281229 .http://deepblue.lib.umich.edu/bitstream/2027.42/134585/1/12931_2012_Article_1336.pd

Acute exacerbations of chronic obstructive pulmonary disease are associated with decreased CD4+ & CD8+ T cells and increased growth & differentiation factor-15 (GDF-15) in peripheral blood

Abstract Background Although T cells, especially CD8+, have been implicated in chronic obstructive pulmonary disease (COPD) pathogenesis, their role during acute exacerbations (AE-COPD) is uncertain. Methods We recruited subjects with COPD and a history of previous AE-COPD and studied them quarterly to collect blood and spontaneously expectorated sputum while stable. During exacerbations (defined by a change in symptoms plus physician diagnosis and altered medications), we collected blood and sputum before administering antibiotics or steroids. We used flow cytometry to identify leukocytes in peripheral blood, plus Luminex® analysis or ELISA to determine levels of inflammatory biomarkers in serum and sputum supernatants. Results Of 33 enrolled subjects, 13 participated in multiple stable visits and had ≥1 AE-COPD visit, yielding 18 events with paired data. Flow cytometric analyses of peripheral blood demonstrated decreased CD4+ and CD8+ T cells during AE-COPD (both absolute and as a percentage of all leukocytes) and significantly increased granulocytes, all of which correlated significantly with serum C-reactive protein (CRP) concentrations. No change was observed in other leukocyte populations during AE-COPD, although the percentage of BDCA-1+ dendritic cells expressing the activation markers CD40 and CD86 increased. During AE-COPD, sICAM-1, sVCAM-1, IL-10, IL-15 and GDF-15 increased in serum, while in sputum supernatants, CRP and TIMP-2 increased and TIMP-1 decreased. Conclusions The decrease in CD4+ and CD8+ T cells (but not other lymphocyte subsets) in peripheral blood during AE-COPD may indicate T cell extravasation into inflammatory sites or organized lymphoid tissues. GDF-15, a sensitive marker of cardiopulmonary stress that in other settings independently predicts reduced long-term survival, is acutely increased in AE-COPD. These results extend the concept that AE-COPD are systemic inflammatory events to which adaptive immune mechanisms contribute. Trial registration NCT00281216 , ClinicalTrials.gov.http://deepblue.lib.umich.edu/bitstream/2027.42/134660/1/12931_2015_Article_251.pd

Disgust sensitivity is not associated with health in a rural Bangladeshi sample.

Disgust can be considered a psychological arm of the immune system that acts to prevent exposure to infectious agents. High disgust sensitivity is associated with greater behavioral avoidance of disease vectors and thus may reduce infection risk. A cross-sectional survey in rural Bangladesh provided no strong support for this hypothesis. In many species, the expression of pathogen- and predator-avoidance mechanisms is contingent on early life exposure to predators and pathogens. Using childhood health data collected in the 1990s, we examined if adults with more infectious diseases in childhood showed greater adult disgust sensitivity: no support for this association was found. Explanations for these null finding and possible directions for future research are discussed

The induction of macrophage gene expression by LPS predominantly utilizes Myd88-independent signaling cascades

Myeloid differentiation protein-88 (MyD88) is a signal adaptor protein required for cytokine production following engagement of Toll-like receptors (TLRs) by their cognate ligands. Activation of both TLR-3 and TLR-4, however, can engage signaling events independent of MyD88 expression. The relative importance of these MyD88-dependent and -independent signaling pathways in the macrophage response to lipopolysaccharide (LPS) is unknown. Here we define these events using microarray expression profiling of LPS-stimulated macrophages taken from MyD88-null and wild-type mice. Of the 1,055 genes found to be LPS responsive, only 21.5% were dependent on MyD88 expression, with MyD88-independent genes constituting 74.7% of the genetic response. This MyD88-independent gene expression was predominantly transcriptionally regulated, as it was unaffected by cycloheximide blockade of new protein synthesis. A previously undescribed group of LPS-regulated genes (3.8%), whose induction or repression was significantly greater in the absence of MyD88, was also identified by these studies. The regulation of these genes suggested that MyD88 could serve as a molecular brake, constraining gene activity in a subset of LPS-responsive genes. The findings generated with LPS stimulation were recapitulated by exposure of macrophages to live Escherichia coli. These expression-profiling studies redefine the current dogma of TLR-4 signaling and establish that MyD88, although essential for some of the best-characterized macrophage responses to LPS, is not required for the regulation of the majority of genes engaged by macrophage exposure to endotoxin or live bacteria

Lung CD8+ T cells in COPD have increased expression of bacterial TLRs

Abstract Background Toll-like receptors (TLRs) on T cells can modulate their responses, however, the extent and significance of TLR expression by lung T cells, NK cells, or NKT cells in chronic obstructive pulmonary disease (COPD) is unknown. Methods Lung tissue collected from clinically-indicated resections (n = 34) was used either: (a) to compare the expression of TLR1, TLR2, TLR2/1, TLR3, TLR4, TLR5, TLR6 and TLR9 on lung CD8+ T cells, CD4+ T cells, NK cells and NKT cells from smokers with or without COPD; or (b) to isolate CD8+ T cells for culture with anti-CD3ε without or with various TLR ligands. We measured protein expression of IFN-γ, TNF-α, IL-13, perforin, granzyme A, granzyme B, soluble FasL, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL9 in supernatants. Results All the lung subsets analyzed demonstrated low levels of specific TLR expression, but the percentage of CD8+ T cells expressing TLR1, TLR2, TLR4, TLR6 and TLR2/1 was significantly increased in COPD subjects relative to those without COPD. In contrast, from the same subjects, only TLR2/1 and TLR2 on lung CD4+ T cells and CD8+ NKT cells, respectively, showed a significant increase in COPD and there was no difference in TLR expression on lung CD56+ NK cells. Production of the Tc1 cytokines IFN-γ and TNF-α by lung CD8+ T cells were significantly increased via co-stimulation by Pam3CSK4, a specific TLR2/1 ligand, but not by other agonists. Furthermore, this increase in cytokine production was specific to lung CD8+ T cells from patients with COPD as compared to lung CD8+ T cells from smokers without COPD. Conclusions These data suggest that as lung function worsens in COPD, the auto-aggressive behavior of lung CD8+ T cells could increase in response to microbial TLR ligands, specifically ligands against TLR2/1.http://deepblue.lib.umich.edu/bitstream/2027.42/112427/1/12931_2012_Article_1320.pd

Early-Life Farm Exposures and Eczema Among Adults in the Agricultural Lung Health Study

Background Several studies conducted in Europe have suggested a protective association between early-life farming exposure and childhood eczema or atopic dermatitis; however, few studies have examined associations in adults. Objectives We investigated associations between early-life exposures and eczema among 3217 adult farmers and farm spouses (mean age, 62.8 years) in a case–control study nested within an US agricultural cohort. Methods We used sampling-weighted logistic regression to estimate odds ratios and 95% confidence intervals for associations between early-life exposures and self-reported doctor-diagnosed eczema (273 cases) and polytomous logistic regression to estimate odds ratios (95% confidence intervals) for a 4-level outcome combining information on eczema and atopy (specific IgE ≥ 0.35). Additionally, we explored genetic and gene–environment associations with eczema. Results Although early-life farming exposures were not associated with eczema overall, several early-life exposures were associated with a reduced risk of having both eczema and atopy. Notably, results suggest stronger protective associations among individuals with both eczema and atopy than among those with either alone. For example, odds ratios (95% confidence intervals) for having a mother who did farm work while pregnant were 1.01 (0.60, 1.69) for eczema alone and 0.80 (0.65, 0.99) for atopy alone, but 0.54 (0.33, 0.80) for having both. A genetic risk score based on previously identified atopic dermatitis variants was strongly positively associated with eczema, and interaction testing suggested protective effects of several early-life farming exposures only in individuals at lower genetic risk. Conclusions In utero and childhood farming exposures are associated with decreased odds of having eczema with atopy in adults