Assel Biyasheva

Cascade pathway of filopodia formation downstream of SCAR

The protrusion of two distinct actin-containing organelles, lamellipodia and filopodia, is thought to be regulated by two parallel pathways: from Rac1 through Scar/WAVEs to lamellipodia, and from Cdc42 through N-WASP to filopodia. We tested this hypothesis in Drosophila, which contains a single gene for each WASP subfamilies, SCAR and WASp. We performed targeted depletion of SCAR or WASp by dsRNA-mediated interference in two Drosophila cultured cell lines expressing lamellipodial and filopodial protrusion. Knockdown was verified by laser capture microdissection and RT-PCR, as well as western blotting. Morphometrical, kinetic and electron microscopy analyses of the SCAR-depleted phenotype in both cell types revealed strong inhibition of lamellipodial formation and cell spreading, as expected. More importantly, filopodia formation was also strongly inhibited, which is not consistent with the parallel pathway hypothesis. By contrast, depletion of WASp did not produce any significant phenotype, except for a slight inhibition of spreading, showing that both lamellipodia and filopodia in Drosophila cells are regulated predominantly by SCAR. We propose a new, cascade pathway model of filopodia regulation in which SCAR signals to lamellipodia and then filopodia arise from lamellipodia in response to additional signal(s).

Airway epithelial cells activate TH2 cytokine production in mast cells through IL-1 and thymic stromal lymphopoietin

BACKGROUND Airway epithelial cells are important regulators of innate and adaptive immunity. Although mast cells are known to play a central role in manifestations of allergic inflammation and are found in the epithelium in patients with T(H)2-related diseases, their role is incompletely understood. OBJECTIVES The objective of this study was to investigate the role of airway epithelial cells in the production of T(H)2 cytokines in mast cells. METHODS Normal human bronchial epithelial (NHBE) cells were stimulated with TNF, IL-4, IFN-γ, IL-17A, and double-stranded RNA (dsRNA) alone or in combination. Human mast cells were stimulated with epithelial cell-derived supernatants or cocultured with NHBE cells. T(H)2 cytokine responses were blocked with neutralizing antibodies. RESULTS Supernatants from IL-4- and dsRNA-stimulated NHBE cells significantly enhanced T(H)2 cytokine production from mast cells. The combination of IL-4 and dsRNA itself or supernatants from NHBE cells stimulated with other cytokines did not activate mast cells, suggesting that mast cell responses were induced by epithelial cell factors that were only induced by IL-4 and dsRNA. Epithelial supernatant-dependent T(H)2 cytokine production in mast cells was suppressed by anti-IL-1 and anti- thymic stromal lymphopoietin (TSLP) and was enhanced by anti-IL-1 receptor antagonist. Similar results were observed in coculture experiments. Finally, we found dsRNA-dependent production of IL-1, TSLP, and IL-1 receptor antagonist in NHBE cells was regulated by T(H) cytokines, and their ratio in NHBE cells correlated with T(H)2 cytokine production in mast cells. CONCLUSIONS Pathogens producing dsRNA, such as respiratory viral infections, might amplify local T(H)2 inflammation in asthmatic patients through the production of TSLP and IL-1 by epithelial cells and subsequent activation of T(H)2 cytokine production by mast cells in the airways.

Asthmatics with exacerbation during acute respiratory illness exhibit unique transcriptional signatures within the nasal mucosa

BackgroundAcute respiratory illness is the leading cause of asthma exacerbations yet the mechanisms underlying this association remain unclear. To address the deficiencies in our understanding of the molecular events characterizing acute respiratory illness-induced asthma exacerbations, we undertook a transcriptional profiling study of the nasal mucosa over the course of acute respiratory illness amongst individuals with a history of asthma, allergic rhinitis and no underlying respiratory disease.MethodsTranscriptional profiling experiments were performed using the Agilent Whole Human Genome 4X44K array platform. Time point-based microarray and principal component analyses were conducted to identify and distinguish acute respiratory illness-associated transcriptional profiles over the course of our study. Gene enrichment analysis was conducted to identify biological processes over-represented within each acute respiratory illness-associated profile, and gene expression was subsequently confirmed by quantitative polymerase chain reaction.ResultsWe found that acute respiratory illness is characterized by dynamic, time-specific transcriptional profiles whose magnitudes of expression are influenced by underlying respiratory disease and the mucosal repair signature evoked during acute respiratory illness. Most strikingly, we report that people with asthma who experience acute respiratory illness-induced exacerbations are characterized by a reduced but prolonged inflammatory immune response, inadequate activation of mucosal repair, and the expression of a newly described exacerbation-specific transcriptional signature.ConclusionFindings from our study represent a significant contribution towards clarifying the complex molecular interactions that typify acute respiratory illness-induced asthma exacerbations.

Evidence for Association between Polycystic Ovary Syndrome (PCOS) and TCF7L2 and Glucose Intolerance in Women with PCOS and TCF7L2

CONTEXT AND OBJECTIVE Of the recently identified type 2 diabetes mellitus (T2D) susceptibility loci, transcription factor 7-like 2 (TCF7L2) confers the greatest relative risk for T2D and significantly predicts conversion to T2D in persons with impaired glucose tolerance. TCF7L2 is, therefore, also a strong candidate gene for polycystic ovary syndrome (PCOS), a common endocrine disorder characterized by androgen excess and menstrual irregularities and associated with insulin resistance and a 7-fold increased risk for T2D. RESEARCH DESIGN AND METHODS We tested for association between 58 single nucleotide polymorphisms mapping to TCF7L2 and PCOS in 624 index (PCOS) cases and 553 control women of European ancestry. Furthermore, in the women with PCOS, we tested for association with seven reproductive and metabolic quantitative traits. RESULTS Although we did not detect evidence for association between the previously described TCF7L2 T2D locus, the proinsulin:insulin molar ratio, a marker of pancreatic beta-cell dysfunction, was strongly associated with this locus (P = 2.1 x 10(-4)). We also observed evidence for association between PCOS and two single nucleotide polymorphisms, rs11196236 (P = 9.0 x 10(-4)) and rs11196229 (P = 0.0027) mapping more than 100 kb centromeric to the previously published T2D susceptibility loci. CONCLUSIONS We have observed evidence of association with two independent TCF7L2 loci in a PCOS cohort: 1) association between the proinsulin:insulin molar ratio and the T2D locus; and 2) association with reproductive PCOS phenotype and a novel locus. This study suggests that variation in different regions of a susceptibility gene contributes to distinct phenotypes.

Neutrophils are a major source of the epithelial barrier disrupting cytokine oncostatin M in patients with mucosal airways disease

Background We have previously shown that oncostatin M (OSM) levels are increased in nasal polyps (NPs) of patients with chronic rhinosinusitis (CRS), as well as in bronchoalveolar lavage fluid, after segmental allergen challenge in allergic asthmatic patients. We also showed in vitro that physiologic levels of OSM impair barrier function in differentiated airway epithelium. Objective We sought to determine which hematopoietic or resident cell type or types were the source of the OSM expressed in patients with mucosal airways disease. Methods Paraffin‐embedded NP sections were stained with fluorescence‐labeled specific antibodies against OSM, GM‐CSF, and hematopoietic cell‐specific markers. Live cells were isolated from NPs and matched blood samples for flow cytometric analysis. Neutrophils were isolated from whole blood and cultured with the known OSM inducers GM‐CSF and follistatin‐like 1, and OSM levels were measured in the supernatants. Bronchial biopsy sections from control subjects, patients with moderate asthma, and patients with severe asthma were stained for OSM and neutrophil elastase. Results OSM staining was observed in NPs, showed colocalization with neutrophil elastase (n = 10), and did not colocalize with markers for eosinophils, macrophages, T cells, or B cells (n = 3‐5). Flow cytometric analysis of NPs (n = 9) showed that 5.1% ± 2% of CD45+ cells were OSM+, and of the OSM+ cells, 56% ± 7% were CD16+Siglec‐8−, indicating neutrophil lineage. Only 0.6 ± 0.4% of CD45+ events from matched blood samples (n = 5) were OSM+, suggesting that increased OSM levels in patients with CRS was locally stimulated and produced. A majority of OSM+ neutrophils expressed arginase 1 (72.5% ± 12%), suggesting an N2 phenotype. GM‐CSF levels were increased in NPs compared with those in control tissue and were sufficient to induce OSM production (P < .001) in peripheral blood neutrophils in vitro. OSM+ neutrophils were also observed at increased levels in biopsy specimens from patients with severe asthma. Additionally, OSM protein levels were increased in induced sputum from asthmatic patients compared with that from control subjects (P < .05). Conclusions Neutrophils are a major source of OSM‐producing cells in patients with CRS and severe asthma.