A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in filtered, sterilized distilled water. We used different concentrations of ciprofloxacin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and ciprofloxacin. The mixture was incubated at different incubation times at 37 °C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solution or orange acridine. The filters were prepared and examined by epifluorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.
Introduction and Aims: Sarcoidosis is a multifactorial disease characterized by the formation and diffusion of granuloma in lungs and many other organs. Genetic and environmental factors have been involved in etiology of the disease. The existence of familial forms suggests that genetic predisposition plays a significant role and we recently identified a series of deleterious mutations in genes related to regulation of autophagy, around the Rac1 and mTor related pathways. Our aim was to replicate the genetic screening in a series of patients of the French GSF cohort in order to identify genetic markers putatively correlated with severe forms of sarcoidosis. Methods: A panel of 88 genes identified from previous familial exome analysis was applied to a series of 100 patients classified in : SEV-1: advanced pulmonary disease, SEV-2: extrapulmonary lesions and CTRL: 9benign9 and/or resolving disease. Results: A significant clustering of mutations in genes direcly related to autophagy (e.g. ATG), mTOR signaling pathways (e.g. DDIT4/L, BRSK2, Annexins) and Rac1 regulation (e.g. EPHA, KALRN, STEF, DOCK) occurs in SEV-1 and SEV-2 groups compared to CTRL suggesting that specific defects in these pathways may be related to progressive and/or multisystemic sarcoidosis. Perspectives: Identification of genetic biomarkers linked to severe forms of sarcoidosis is a challenge for the early management of patients and optimal therapeutic adaptation. Our study allows the definition of a first set of genes which could be implemented by further functional analysis and international cooperative studies.
Introduction: Lung fibrosis is associated with the reactivation of molecular pathways involved in lung development. Mesothelial cells are involved in the fibrotic lung process but their exact role is debated. Fibroblast Growth Factor-9 (FGF-9) is expressed by epithelial cells and visceral pleura during embryonic lung development. Mice homozygous for a targeted disruption of FGF-9 exhibit lung hypoplasia with reduced mesenchyme and early postnatal death. The aim of this study was to evaluate the role of FGF-9 expression by mesothelial cells in the adult lung. We used adenovirus-mediated (FGF-9) gene transfer in mesothelial cells in vivo. Material and Methods: AdFGF9 or a control adenovirus (AdCont) (108pfu/mL) was administered in 100 μl of 0.9% NaCl by pleural injection of C56/Bl6 male mice. Mice were studied at day 3, day 5, and day 14.We quantified FGF-9 in pleural fluid by ELISA. The expression of FGF-9, inflammation and pro fibrotic markers were assessed by qPCR, and immunochemistry. Results: Intrapleural injection of AdCont led to pleural inflammation and pleural remodeling beginning at day 5 and maximal at day 14, with pleural thickening, and accumulation of α-SMA+ cells and collagen. AdCont induced murine FGF-9 expression in the pleura. AdFGF-9 led to human FGF-9 overexpression in the pleural fluid and in the lung at day 5. AdFGF-9 completely inhibited the development of pleural remodeling, with a significant decrease of Collagen I, III, fibronectin, PAI-1, CTGF mRNA expression. Conclusion: Intrapleural injection of adenovirus induces pleural fibrosis. It is prevented by pleural overexpression of human FGF-9 suggesting that mesothelial FGF-9 has anti fibrotic properties.
