搜索结果: 'gmp t cell activator'

BACKGROUND & AIMS The etiology of abdominal pain in postinfectious,diarrhea-predominant irritable bowel syndrome (PI-IBS-D) is unknown,and few treatment options exist. Catechol-O-methyltransferase (COMT),an enzyme that inactivates and degrades biologically active catecholamines,plays an important role in numerous physiologic processes,including modulation of pain perception. Our objective was to determine the mechanism(s) of how decreased colonic COMT in PI-IBS-D patients contributes to the chronic abdominal pain phenotype after enteric infections. METHODS Colon neurons,epithelial cells,and macrophages were procured with laser capture microdissection from PI-IBS-D patients to evaluate cell-specific colonic COMT,microRNA-155 (miR-155),and tumor necrosis factor (TNF) ? expression levels compared to recovered patients (infection cleared: did not develop PI-IBS-D) and control individuals. COMT-/-,colon-specific COMT-/-,and miR-155-/- mice and human colonoids were used to model phenotypic expression of COMT in PI-IBS-D patients and to investigate signaling pathways linking abdominal pain. Citrobacter rodentium and trinitrobenzene sulfonic acid animal models were used to model postinflammatory changes seen in PI-IBS-D patients. RESULTS Colonic COMT levels were significantly decreased and correlated with increased visual analog scale abdominal pain ratings in PI-IBS-D patients compared to recovered patients and control individuals. Colonic miR-155 and TNF-? were increased in PI-IBS-D patients with diminished colonic COMT. COMT-/- mice had significantly increased expression of miR-155 and TNF-? in both colon tissues and dorsal root ganglia. Introduction of cV1q antibody (anti-TNF-?) into mice reversed visceral hypersensitivity after C rodentium and trinitrobenzene sulfonic acid. CONCLUSIONS Decreased colonic COMT in PI-IBS-D patients drives abdominal pain phenotypes via the COMT/miR-155/TNF-? axis. These important findings will allow new treatment paradigms and more targeted and personalized medicine approaches for gastrointestinal disorders after enteric infections. View Publication

The combination therapy of lumacaftor and ivacaftor (Orkambi®) is approved for patients bearing the major cystic fibrosis (CF) mutation: ΔF508 It has been predicted that Orkambi® could treat patients with rarer mutations of similar theratype"; however a standardized approach confirming efficacy in these cohorts has not been reported. Here we demonstrate that patients bearing the rare mutation: c.3700 A>G causing protein misprocessing and altered channel function-similar to ΔF508-CFTR are unlikely to yield a robust Orkambi® response. While in silico and biochemical studies confirmed that this mutation could be corrected and potentiated by lumacaftor and ivacaftor respectively this combination led to a minor in vitro response in patient-derived tissue. A CRISPR/Cas9-edited bronchial epithelial cell line bearing this mutation enabled studies showing that an "amplifier" compound effective in increasing the levels of immature CFTR protein augmented the Orkambi® response. Importantly this "amplifier" effect was recapitulated in patient-derived nasal cultures-providing the first evidence for its efficacy in augmenting Orkambi® in tissues harboring a rare CF-causing mutation. We propose that this multi-disciplinary approach including creation of CRISPR/Cas9-edited cells to profile modulators together with validation using primary tissue will facilitate therapy development for patients with rare CF mutations. View Publication

While all forms of tobacco exposure have negative health effects,the significance of exposure to electronic cigarettes (eCig) is not fully understood. Here,we studied the global effects of eCig on the micro RNA (miRNA) transcriptome in human lung epithelial cells. Primary human bronchial epithelial (NHBE) cells differentiated at air-liquid interface were exposed to eCig liquid. Exposure of NHBE to any eCig liquid resulted in the induction of oxidative stress-response genes including GCLM,GCLC,GPX2,NQO1 and HO-1. Vaporization of,and/or the presence of nicotine in,eCig liquid was associated with a greater response. We identified 578 miRNAs dysregulated by eCig exposure in NHBE,and 125 miRNA affected by vaporization of eCig liquid. Nicotine containing eCig vapor displayed the most profound effects upon miRNA expression. We selected 8 miRNAs (29A,140,126,374A,26A-2,147B,941 and 589) for further study. We validated increased expression of multiple miRNAs,including miR126,following eCig exposure. We also found significant reduction in the expression of two miR126 target genes,MYC and MRGPRX3,following exposure. These data demonstrated that eCig exposure has profound effects upon gene expression in human lung epithelial cells,some of which are epigenetically programmed at the level of miRNA regulation. View Publication

To better characterize biological responses to atmospheric organic aerosols,the efficient delivery of aerosol to in vitro lung cells is necessary. In this study,chamber generated secondary organic aerosol (SOA) entered the commercialized exposure chamber (CULTEX® Radial Flow System Compact) where it interfaced with an electrostatic precipitator (ESP) (CULTEX® Electrical Deposition Device) and then deposited on a particle collection plate. This plate contained human lung cells (BEAS-2B) that were cultured on a membrane insert to produce an air-liquid interface (ALI). To augment in vitro assessment using the ESP exposure device,the particle dose was predicted for various sampling parameters such as particle size,ESP deposition voltage,and sampling flowrate. The dose model was evaluated against the experimental measured mass of collected airborne particles. The high flowrate used in this study increased aerosol dose but failed to achieve cell stability. For example,RNA in the ALI BEAS-2B cells in vitro was stable at 0.15 L/minute but decayed at high flowrates. The ESP device and the resulting model were applied to in vitro studies (i.e.,viability and IL-8 expression) of toluene SOA using ALI BEAS-2B cells with a flowrate of 0.15 L/minute,and no cellular RNA decay occurred. View Publication

BACKGROUND The Rhinovirus C (RV-C),first identified in 2006,produce high symptom burdens in children and asthmatics,however,their primary target host cell in the airways remains unknown. Our primary hypotheses were that RV-C target ciliated airway epithelial cells (AECs),and that cell specificity is determined by restricted and high expression of the only known RV-C cell-entry factor,cadherin related family member 3 (CDHR3). METHODS RV-C15 (C15) infection in differentiated human bronchial epithelial cell (HBEC) cultures was assessed using immunofluorescent and time-lapse epifluorescent imaging. Morphology of C15-infected differentiated AECs was assessed by immunohistochemistry. RESULTS C15 produced a scattered pattern of infection,and infected cells were shed from the epithelium. The percentage of cells infected with C15 varied from 1.4 to 14.7% depending on cell culture conditions. Infected cells had increased staining for markers of ciliated cells (acetylated-alpha-tubulin [aat],p < 0.001) but not markers of goblet cells (wheat germ agglutinin or Muc5AC,p = ns). CDHR3 expression was increased on ciliated epithelial cells,but not other epithelial cells (p < 0.01). C15 infection caused a 27.4% reduction of ciliated cells expressing CDHR3 (p < 0.01). During differentiation of AECs,CDHR3 expression progressively increased and correlated with both RV-C binding and replication. CONCLUSIONS The RV-C only replicate in ciliated AECs in vitro,leading to infected cell shedding. CDHR3 expression positively correlates with RV-C binding and replication,and is largely confined to ciliated AECs. Our data imply that factors regulating differentiation and CDHR3 production may be important determinants of RV-C illness severity. View Publication

Long-lived multipotent stem cells (ISCs) at the base of intestinal crypts adjust their phenotypes to accommodate normal maintenance and post-injury regeneration of the epithelium. Their long life,lineage plasticity,and proliferative potential underlie the necessity for tight homeostatic regulation of the ISC compartment. In that context,the guanylate cyclase C (GUCY2C) receptor and its paracrine ligands regulate intestinal epithelial homeostasis,including proliferation,lineage commitment,and DNA damage repair. However,a role for this axis in maintaining ISCs remains unknown. Transgenic mice enabling analysis of ISCs (Lgr5-GFP) in the context of GUCY2C elimination (Gucy2c -/- ) were combined with immunodetection techniques and pharmacological treatments to define the role of the GUCY2C signaling axis in supporting ISCs. ISCs were reduced inGucy2c -/- mice,associated with loss of active Lgr5+cells but a reciprocal increase in reserve Bmi1+cells. GUCY2C was expressed in crypt base Lgr5+cells in which it mediates canonical cyclic (c) GMP-dependent signaling. Endoplasmic reticulum (ER) stress,typically absent from ISCs,was elevated throughout the crypt base inGucy2c -/- mice. The chemical chaperone tauroursodeoxycholic acid resolved this ER stress and restored the balance of ISCs,an effect mimicked by the GUCY2C effector 8Br-cGMP. Reduced ISCs inGucy2c -/- mice was associated with greater epithelial injury and impaired regeneration following sub-lethal doses of irradiation. These observations suggest that GUCY2C provides homeostatic signals that modulate ER stress and cell vulnerability as part of the machinery contributing to the integrity of ISCs. View Publication

In recent years,the differentiation of bone marrow cells (BMCs) into myocytes has been extensively investigated,but the findings remain inconclusive. The purpose of this study was to determine the conditions necessary to induce myogenic differentiation in short-term cultures of adult BMCs,and to identify the BMC subpopulation responsible for this phenomenon. We report that high-density cultures of murine hematopoietic BMCs gave rise to spontaneous beating cell clusters in the presence of vascular endothelial and fibroblast growth factors. These clusters originated from c-kit(pos) cells. The formation of the clusters could be completely blocked by adding a c-kit/tyrosine kinase inhibitor,Gleevec (imatinib mesylate; Novartis International,Basel,Switzerland,http://www.novartis.com),to the culture. Cluster formation was also blunted in BMCs from c-kit-deficient (Kit(W)/Kit(W-v)) mice. Clustered cells expressed cardiomyocyte-specific transcription factor genes Gata-4 and Nkx2.5,sarcomeric proteins beta-MHC and MLC-2v,and ANF and connexin-43. Immunostaining revealed alpha-sarcomeric actinin expression in more than 90% of clustered cells. Under electron microscopy,the clustered cells exhibited a sarcomeric myofiber arrangement and z-bands. This study defines the microenvironment required to achieve a reproducible in vitro model of beating,myogenic cell clusters. This model could be used to examine the mechanisms responsible for the postnatal myogenic differentiation of BMCs. Our results identify c-kit(pos) bone marrow hematopoietic cells as the source of the myogenic clusters. View Publication