Rural and urban exposures shape early life immune development in South African children with atopic dermatitis and nonallergic children.

BACKGROUND: Immunological traits and functions have been consistently associated with environmental exposures and are thought to shape allergic disease susceptibility and protection. In particular, specific exposures in early life may have more significant effects on the developing immune system, with potentially long-term impacts. METHODS: We performed RNA-Seq on peripheral blood mononuclear cells (PBMCs) from 150 children with atopic dermatitis and healthy nonallergic children in rural and urban settings from the same ethnolinguistic AmaXhosa background in South Africa. We measured environmental exposures using questionnaires. RESULTS: A distinct PBMC gene expression pattern was observed in those children with atopic dermatitis (132 differentially expressed genes [DEGs]). However, the predominant influences on the immune cell transcriptome were related to early life exposures including animals, time outdoors, and types of cooking and heating fuels. Sample clustering revealed two rural groups (Rural_1 and Rural_2) that separated from the urban group (3413 and 2647 DEGs, respectively). The most significantly regulated pathways in Rural_1 children were related to innate activation of the immune system (e.g., TLR and cytokine signaling), changes in lymphocyte polarization (e.g., TH17 cells), and immune cell metabolism (i.e., oxidative phosphorylation). The Rural_2 group displayed evidence for ongoing lymphocyte activation (e.g., T cell receptor signaling), with changes in immune cell survival and proliferation (e.g., mTOR signaling, insulin signaling). CONCLUSIONS: This study highlights the importance of the exposome on immune development in early life and identifies potentially protective (e.g., animal) exposures and potentially detrimental (e.g., pollutant) exposures that impact key immunological pathways.

Fluorescence Assisted Capillary Electrophoresis of Glycans Enabled by the Negatively Charged Auxochromes in 1-Aminopyrenes.

A compact and negatively charged acceptor group, N-(cyanamino)sulfonyl, is introduced for dye design and its influence on the absorption and emission spectra of the "push-pull" chromophores is demonstrated with 1,3,6-tris[(cyanamino)sulfonyl]-8-aminopyrene. The new sulfonamides, including O-phosphorylated (3-hydroxyazetidine)-N-sulfonyl, are negatively charged electron acceptors and auxochromes. 1-Aminopyrenes decorated with the new sulfonamides have three or six negative charges (pH ≥8), low m/z ratios, high mobilities in an electric field, and yellow to orange emission. We labeled maltodextrin oligomers by reductive amination, separated the products by electrophoresis, and demonstrated their high brightness in a commercial DNA analyzer and the distribution of the emission signal among the detection channels.

Der p 1-specific regulatory T-cell response during house dust mite allergen immunotherapy.

BACKGROUND: Allergen-specific immunotherapy (AIT) is the only available treatment for allergic diseases that can induce specific immune tolerance to allergens. The key mechanisms involved in this process include changes in allergen-specific regulatory T (Treg) cells. METHODS: We studied 25 allergic rhinitis patients undergoing subcutaneous house dust mite-specific immunotherapy. Peripheral blood mononuclear cells were studied before and after 10, 30 weeks, and 3 years of AIT. Der p 1-specific T regulatory cell responses were investigated by characterization of Der p 1-MHC class II tetramer-positive cells and correlated with nasal symptom score. RESULTS: Twelve of 25 AIT patients matched with their MHC class II expression to the Der p 1 peptide-MHC class II tetramers. A significant increase in the numbers of Der p 1-specific FOXP3+ Helios+ CD25+ CD127- Treg cells after 30 weeks was observed, which slightly decreased after 3 years of AIT. In contrast, Der p 1-specific immunoglobulin-like transcript 3 (ILT3)+ CD25+ Treg cells decreased substantially from baseline after 3 years of AIT. ILT3+ Treg cells displayed compromised suppressive function and low FOXP3 expression. In addition, Der p 1-specific IL-10 and IL-22 responses have increased after 30 weeks, but only IL-10+ Der p 1-specific Treg cells remained present at high frequency after 3 years of AIT. Increased number of FOXP3+ Helios+ and IL-10+ and decreased ILT3+ Treg cell responses correlated with improved allergic symptoms. CONCLUSION: The results indicate that AIT involves upregulation of the activated allergen-specific Treg cells and downregulation of dysfunctional allergen-specific Treg cell subset. Correction of dysregulated Treg cells responses during AIT is associated with improved clinical response.

Regulation of bronchial epithelial barrier integrity by type 2 cytokines and histone deacetylases in asthmatic patients.

BACKGROUND: Tight junctions (TJs) form a barrier on the apical side of neighboring epithelial cells in the bronchial mucosa. Changes in their integrity might play a role in asthma pathogenesis by enabling the paracellular influx of allergens, toxins, and microbes to the submucosal tissue. OBJECTIVE: The regulation of bronchial epithelial TJs by TH2 cells and their cytokines and their involvement in epigenetic regulation of barrier function were investigated. METHODS: The expression, regulation, and function of TJs were determined in air-liquid interface (ALI) cultures of control and asthmatic primary human bronchial epithelial cells (HBECs) by means of analysis of transepithelial electrical resistance, paracellular flux, mRNA expression, Western blotting, and immunofluorescence staining. RESULTS: HBECs from asthmatic patients showed a significantly low TJ integrity in ALI cultures compared with HBECs from healthy subjects. TH2 cell numbers and levels of their cytokines, IL-4 and IL-13, decreased barrier integrity in ALI cultures of HBECs from control subjects but not in HBECs from asthmatic patients. They induced a physical separation of the TJs of adjacent cells in immunofluorescence staining of the TJ molecules occludin and zonula occludens-1. We observed that expression of histone deacetylases (HDACs) 1 and 9, and Silent information regulator genes (sirtuins [SIRTs]) 6 and 7 were significantly high in HBECs from asthmatic patients. IL-4 and IL-13 significantly increased the expression of HDACs and SIRTs. The role of HDAC activation on epithelial barrier leakiness was confirmed by HDAC inhibition, which improved barrier integrity through increased synthesis of TJ molecules in epithelium from asthmatic patients to the level seen in HBECs from control subjects. CONCLUSION: Our data demonstrate that barrier leakiness in asthmatic patients is induced by TH2 cells, IL-4, and IL-13 and HDAC activity. The inhibition of endogenous HDAC activity reconstitutes defective barrier by increasing TJ expression.

Defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by IFN-γ and IL-4.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common disease with still unclear pathophysiologic mechanisms. Epithelial tight junctions (TJs) have been shown to be involved in different chronic disorders, including bronchial asthma, inflammatory bowel diseases, and skin disorders. The regulation of epithelial barrier function and TJ expression has not been extensively studied in patients with CRS and in the paranasal sinus epithelium thus far. OBJECTIVE: We sought to elucidate the TJ expression pattern in the epithelium of the sinonasal mucosa and its regulation in patients with CRS. METHODS: Trans-tissue resistance was measured in biopsy specimens from healthy control subjects and patients with CRS with and without nasal polyps. TJ protein expression was determined by using immunofluorescence, Western blotting, and real-time PCR. Primary epithelial cell cultures from patients with CRS and control subjects were used in air-liquid interface (ALI) cultures for the measurement of transepithelial resistance (TER) and TJ expression. The effect of IFN-γ, IL-4, and IL-17 on ALI cultures was assessed. RESULTS: A decreased trans-tissue resistance was found in biopsy specimens from patients with CRS with nasal polyps along with an irregular, patchy, and decreased expression of the TJ molecules occludin and zonula occludens 1. TER was reduced in ALI cultures from patients with CRS with nasal polyps. The cytokines IFN-γ and IL-4 decreased TER, whereas IL-17 did not have any influence on epithelial integrity. CONCLUSION: A defective epithelial barrier was found in patients with CRS with nasal polyps along with a decreased expression of TJ proteins. The disruption of epithelial integrity by IFN-γ and IL-4 in vitro indicates a possible role for these proinflammatory cytokines in the pathogenesis of patients with CRS.

Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. OBJECTIVE: The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. METHODS: Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. RESULTS: Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1ß (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1ß in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. CONCLUSION: Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

The expression of gingival epithelial junctions in response to subgingival biofilms.

Periodontitis is an infectious inflammatory disease that destroys the tooth-supporting tissues. It is caused by the formation of subgingival biofilms on the surface of the tooth. Characteristic bacteria associated with subgingival biofilms are the Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, collectively known as the "red complex" species. Inter-epithelial junctions ensure the barrier integrity of the gingival epithelium. This may however be disrupted by the biofilm challenge. The aim of this in vitro study was to investigate the effect of subgingival biofilms on the expression of inter-epithelial junctions by gingival epithelia, and evaluate the relative role of the red complex. Multi-layered human gingival epithelial cultures were challenged with a 10-species in vitro subgingival biofilm model, or its variant without the red complex, for 3 h and 24 h. A low-density array microfluidic card platform was then used for analyzing the expression of 62 genes encoding for tight junctions, gap junctions, adherens junctions, and desmosomes. Although there was a limited effect of the biofilms on the expression of tight, adherens and gap junctions, the expression of a number of desmosomal components was affected. In particular, Desmoglein-1 displayed a limited and transient up-regulation in response to the biofilm. In contrast, Desmocollin-2, Desmoplakin and Plakoglobin were down-regulated equally by both biofilm variants, after 24 h. In conclusion, this subgingival biofilm model may down-regulate selected desmosomal junctions in the gingival epithelium, irrespective of the presence of the "red complex." In turn, this could compromise the structural integrity of the gingival tissue, favoring bacterial invasion and chronic infection.