Chronic cigarette smoke exposure induces systemic hypoxia that drives intestinal dysfunction

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract (GIT). Cigarette smoke (CS) exposure and chronic obstructive pulmonary disease (COPD) are risk factors for CD, although the mechanisms involved are poorly understood. We employed a mouse model of CS-induced experimental COPD and clinical studies to examine these mechanisms. Concurrent with the development of pulmonary pathology and impaired gas exchange, CS-exposed mice developed CD-associated pathology in the colon and ileum, including gut mucosal tissue hypoxia, HIF-2 stabilization, inflammation, increased microvasculature, epithelial cell turnover, and decreased intestinal barrier function. Subsequent smoking cessation reduced GIT pathology, particularly in the ileum. Dimethyloxaloylglycine, a pan-prolyl hydroxylase inhibitor, ameliorated CS-induced GIT pathology independently of pulmonary pathology. Prior smoke exposure exacerbated intestinal pathology in 2,4,6-trinitrobenzenesulfonic acid-induced (TNBS-induced) colitis. Circulating vascular endothelial growth factor, a marker of systemic hypoxia, correlated with CS exposure and CD in mice and humans. Increased mucosal vascularisation was evident in ileum biopsies from CD patients who smoke compared with nonsmokers, supporting our preclinical data. We provide strong evidence that chronic CS exposure and, for the first time to our knowledge, associated impaired gas exchange cause systemic and intestinal ischemia, driving angiogenesis and GIT epithelial barrier dysfunction, resulting in increased risk and severity of CD

Dietary Supplementation with Soluble Plantain Non-Starch Polysaccharides Inhibits Intestinal Invasion of Salmonella Typhimurium in the Chicken

Soluble fibres (non-starch polysaccharides, NSP) from edible plants but particularly plantain banana (Musa spp.), have been shown in vitro and ex vivo to prevent various enteric pathogens from adhering to, or translocating across, the human intestinal epithelium, a property that we have termed contrabiotic. Here we report that dietary plantain fibre prevents invasion of the chicken intestinal mucosa by Salmonella. In vivo experiments were performed with chicks fed from hatch on a pellet diet containing soluble plantain NSP (0 to 200 mg/d) and orally infected with S.Typhimurium 4/74 at 8 d of age. Birds were sacrificed 3, 6 and 10 d post-infection. Bacteria were enumerated from liver, spleen and caecal contents. In vitro studies were performed using chicken caecal crypts and porcine intestinal epithelial cells infected with Salmonella enterica serovars following pre-treatment separately with soluble plantain NSP and acidic or neutral polysaccharide fractions of plantain NSP, each compared with saline vehicle. Bacterial adherence and invasion were assessed by gentamicin protection assay. In vivo dietary supplementation with plantain NSP 50 mg/d reduced invasion by S.Typhimurium, as reflected by viable bacterial counts from splenic tissue, by 98.9% (95% CI, 98.1–99.7; P<0.0001). In vitro studies confirmed that plantain NSP (5–10 mg/ml) inhibited adhesion of S.Typhimurium 4/74 to a porcine epithelial cell-line (73% mean inhibition (95% CI, 64–81); P<0.001) and to primary chick caecal crypts (82% mean inhibition (95% CI, 75–90); P<0.001). Adherence inhibition was shown to be mediated via an effect on the epithelial cells and Ussing chamber experiments with ex-vivo human ileal mucosa showed that this effect was associated with increased short circuit current but no change in electrical resistance. The inhibitory activity of plantain NSP lay mainly within the acidic/pectic (homogalacturonan-rich) component. Supplementation of chick feed with plantain NSP was well tolerated and shows promise as a simple approach for reducing invasive salmonellosis

A non-tight junction function of claudin-7—Interaction with integrin signaling in suppressing lung cancer cell proliferation and detachment

Background Claudins are a family of tight junction (TJ) membrane proteins involved in a broad spectrum of human diseases including cancer. Claudin-7 is a unique TJ membrane protein in that it has a strong basolateral membrane distribution in epithelial cells and in tissues. Therefore, this study aims to investigate the functional significance of this non-TJ localization of claudin-7 in human lung cancer cells. Methods Claudin-7 expression was suppressed or deleted by lentivirus shRNA or by targeted-gene deletion. Cell cycle analysis and antibody blocking methods were employed to assay cell proliferation and cell attachment, respectively. Electron microscopy and transepthelial electrical resistance measurement were performed to examine the TJ ultrastructure and barrier function. Co-immunolocalization and co-immunoprecipitation was used to study claudin-7 interaction with integrin β1. Tumor growth in vivo were analyzed using athymic nude mice. Results Claudin-7 co-localizes and forms a stable complex with integrin β1. Both suppressing claudin-7 expression by lentivirus shRNA in human lung cancer cells (KD cells) and deletion of claudin-7 in mouse lungs lead to the reduction in integrin β1 and phospho-FAK levels. Suppressing claudin-7 expression increases cell growth and cell cycle progression. More significantly, claudin-7 KD cells have severe defects in cell-matrix interactions and adhere poorly to culture plates with a remarkably reduced integrin β1 expression. When cultured on uncoated glass coverslips, claudin-7 KD cells grow on top of each other and form spheroids while the control cells adhere well and grow as a monolayer. Reintroducing claudin-7 reduces cell proliferation, upregulates integrin β1 expression and increases cell-matrix adhesion. Integrin β1 transfection partially rescues the cell attachment defect. When inoculated into nude mice, claudin-7 KD cells produced significantly larger tumors than control cells. Conclusion In this study, we identified a previously unrecognized function of claudin-7 in regulating cell proliferation and maintaining epithelial cell attachment through engaging integrin β1

Progenitor cells of the rod-free area centralis originate in the anterior dorsal optic vesicle

<p>Abstract</p> <p>Background</p> <p>Nervous system development is dependent on early regional specification to create functionally distinct tissues within an initially undifferentiated zone. Within the retina, photoreceptors are topographically organized with rod free area centrales faithfully generated at the centre of gaze. How does the developing eye regulate this placement? Conventional wisdom indicates that the distal tip of the growing optic vesicle (OV) gives rise to the area centralis/fovea. Ectopic expression and ablation studies do not fully support this view, creating a controversy as to the origin of this region. In this study, the lineage of cells in the chicken OV was traced using DiI. The location of labelled cells was mapped onto the retina in relation to the rod-free zone at embryonic (E) 7 and E17.5. The ability to regenerate a rod free area after OV ablation was determined in conjunction with lineage tracing.</p> <p>Results</p> <p>Anterior OV gave rise to cells in nasal retina and posterior OV became temporal retina. The OV distal tip gave rise to cells above the optic nerve head. A dorsal and anterior region of the OV correlated with cells in the developing rod free area centralis. Only ablations including the dorsal anterior region gave rise to a retina lacking a rod free zone. DiI application after ablation indicated that cells movements were greater along the anterior/posterior axis compared with the dorsal/ventral axis.</p> <p>Conclusion</p> <p>Our data support the idea that the chicken rod free area centralis originates from cells located near, but not at the distal tip of the developing OV. Therefore, the hypothesis that the area centralis is derived from cells at the distal tip of the OV is not supported; rather, a region anterior and dorsal to the distal tip gives rise to the rod free region. When compared with other studies of retinal development, our results are supported on molecular, morphological and functional levels. Our data will lead to a better understanding of the mechanisms underlying the topographic organization of the retina, the origin of the rod free zone, and the general issue of compartmentalization of neural tissue before any indication of morphological differentiation.</p

Claudin-7 Is Frequently Overexpressed in Ovarian Cancer and Promotes Invasion

Background: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC. Methodology/Principal Findings: A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration

The Promigratory Activity of the Matricellular Protein Galectin-3 Depends on the Activation of PI-3 Kinase

Expression of galectin-3 is associated with sarcoma progression, invasion and metastasis. Here we determined the role of extracellular galectin-3 on migration of sarcoma cells on laminin-111. Cell lines from methylcholanthrene-induced sarcomas from both wild type and galectin-3−/− mice were established. Despite the presence of similar levels of laminin-binding integrins on the cell surface, galectin-3−/− sarcoma cells were more adherent and less migratory than galectin-3+/+ sarcoma cells on laminin-111. When galectin-3 was transiently expressed in galectin-3−/− sarcoma cells, it inhibited cell adhesion and stimulated the migratory response to laminin in a carbohydrate-dependent manner. Extracellular galectin-3 led to the recruitment of SHP-2 phosphatase to focal adhesion plaques, followed by a decrease in the amount of phosphorylated FAK and phospho-paxillin in the lamellipodia of migrating cells. The promigratory activity of extracellular galectin-3 was inhibitable by wortmannin, implicating the activation of a PI-3 kinase dependent pathway in the galectin-3 triggered disruption of adhesion plaques, leading to sarcoma cell migration on laminin-111

Pneumocystis murina colonization in immunocompetent surfactant protein A deficient mice following environmental exposure

<p>Abstract</p> <p>Background</p> <p><it>Pneumocystis spp</it>. are opportunistic pathogens that cause pneumonia in immunocompromised humans and animals. <it>Pneumocystis </it>colonization has also been detected in immunocompetent hosts and may exacerbate other pulmonary diseases. Surfactant protein A (SP-A) is an innate host defense molecule and plays a role in the host response to <it>Pneumocystis</it>.</p> <p>Methods</p> <p>To analyze the role of SP-A in protecting the immunocompetent host from <it>Pneumocystis </it>colonization, the susceptibility of immunocompetent mice deficient in SP-A (KO) and wild-type (WT) mice to <it>P. murina </it>colonization was analyzed by reverse-transcriptase quantitative PCR (qPCR) and serum antibodies were measured by enzyme-linked immunosorbent assay (ELISA).</p> <p>Results</p> <p>Detection of <it>P. murina </it>specific serum antibodies in immunocompetent WT and KO mice indicated that the both strains of mice had been exposed to <it>P. murina </it>within the animal facility. However, P. <it>murina </it>mRNA was only detected by qPCR in the lungs of the KO mice. The incidence and level of the mRNA expression peaked at 8–10 weeks and declined to undetectable levels by 16–18 weeks. When the mice were immunosuppressed, <it>P. murina </it>cyst forms were also only detected in KO mice. <it>P. murina </it>mRNA was detected in <it>SCID </it>mice that had been exposed to KO mice, demonstrating that the immunocompetent KO mice are capable of transmitting the infection to immunodeficient mice. The pulmonary cellular response appeared to be responsible for the clearance of the colonization. More CD4+ and CD8+ T-cells were recovered from the lungs of immunocompetent KO mice than from WT mice, and the colonization in KO mice depleted CD4+ cells was not cleared.</p> <p>Conclusion</p> <p>These data support an important role for SP-A in protecting the immunocompetent host from <it>P. murina </it>colonization, and provide a model to study <it>Pneumocystis </it>colonization acquired via environmental exposure in humans. The results also illustrate the difficulties in keeping mice from exposure to <it>P. murina </it>even when housed under barrier conditions.</p

The DIAMIND study: postpartum SMS reminders to women who have had gestational diabetes mellitus to test for type 2 diabetes: a randomised controlled trial - study protocol

BACKGROUND Postpartum follow up of women who have been found to have gestational diabetes during pregnancy is essential because of the strong association of gestational diabetes with subsequent type 2 diabetes. Postal reminders have been shown to increase significantly attendance for oral glucose tolerance testing postpartum. It is possible that a short message service (text) reminder system may also be effective. This trial aims to assess whether a text message reminder system for women who have experienced gestational diabetes in their index pregnancy will increase attendance for oral glucose tolerance testing within six months after birth. METHODS/DESIGN Design: Single centre (Women’s and Children’s Hospital, South Australia), parallel group randomised controlled trial. Inclusion criteria: Women diagnosed with gestational diabetes in their index pregnancy (oral glucose tolerance test with fasting glucose ≥ 5.5 mmol/L and/or two hour glucose ≥ 7.8 mmol/L), with access to a mobile phone, whose capillary blood glucose profile measurements prior to postnatal discharge are all normal (fasting glucose < 6.0 mmol/L, postprandial glucoses < 8.0 mmol/L). Exclusion criteria: Pregestational diabetes mellitus, triplet/higher order multiple birth or stillbirth in the index pregnancy, requirement for interpreter. Trial entry and randomisation: Allocation to intervention will be undertaken using a telephone randomisation service (computer-generated random number sequence generation, with balanced variable blocks, and stratification by insulin requirement). Study groups: Women in the intervention group will receive a text reminder to attend for an oral glucose tolerance test at 6 weeks postpartum, with further reminders at 3 months and 6 months if they do not respond to indicate test completion. Women in the control group will receive a single text message reminder at 6 months postpartum. Blinding: Baseline data collection will be undertaken blinded. Blinding of participants and blinded collection of primary outcome data will not be possible for this study. Primary study outcome: Attendance for the oral glucose tolerance test within 6 months postpartum. Sample size: 276 subjects will be required to show an 18% absolute increase in the rate of attendance (α=0.05 two tailed, β=80%, 5% loss to follow up) from 37% to 55% in the intervention group. DISCUSSION Given the heightened risk of impaired glucose tolerance and type 2 diabetes in women who have had gestational diabetes, ensuring the highest possible rate of attendance for postpartum glucose tolerance testing, so that early diagnosis and intervention can occur, is important. A text message reminder system may prove to be an effective method for achieving improved attendance for such testing. This randomised controlled trial will assess whether such a system will increase rates of attendance for postpartum oral glucose tolerance testing in women who have experienced gestational diabetes. TRIAL REGISTRATION Australian New Zealand Clinical Trials Registry - ACTRN12612000621819Emer Heatley, Philippa Middleton, William Hague and Caroline Crowthe