Wortmannin blocks Yersinia invasin-triggered internalization, but not interleukin-8 production by epithelial cells

In response to bacterial infection epithelial cells up-regulate expression and secretion of proinflammatory cytokines. Previous work from our laboratory showed that basolateral infection of polarized T84 cells with Yersinia enterocolitica induces interleukin-8 (IL-8) secretion in the absence of significant invasion. Here we studied Y. enterocolitica-induced IL-8 secretion by epithelial HeLa cells as a function of Yersinia invasion or adhesion. For this purpose we tried to separated induction of IL-8 secretion from invasion by treating HeLa cells with signal transduction inhibitors prior to infection. While staurosporin and genistein inhibited both Yersinia invasion and Yersinia-triggered IL-8 secretion, wortmannin, an inhibitor of the phosphatidylinositol-3-phosphate kinase (PI3-K), blocked invasion of Y. enterocolitica into HeLa cells but did not show any effect on IL-8 secretion. These results suggest that Yersinia adhesion might be sufficient to induce IL-8 secretion by epithelial cells. Further analysis demonstrated the requirement of the Yersinia invasion locus inv for adhesion-mediated induction of IL-8 secretion. Thus, HeLa cells infected with an E. coli strain expressing the Y. enterocolitica inv locus induced IL-8 secretion in the presence and absence of wortmannin. Reverse transcription-polymerase chain reaction analysis revealed that adhesion of inv-expressing Y. enterocolitica or E. coli results in the transcriptional activation of the IL-8 gene. These results suggest that Y. enterocolitica adhesion to host cells via Inv activates de novo synthesis and secretion of IL-8.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Detection of pneumonia associated pathogens using a prototype multiplexed pneumonia test in hospitalized patients with severe pneumonia

Severe pneumonia remains an important cause of morbidity and mortality. Polymerase chain reaction (PCR) has been shown to be more sensitive than current standard microbiological methods - particularly in patients with prior antibiotic treatment - and therefore, may improve the accuracy of microbiological diagnosis for hospitalized patients with pneumonia. Conventional detection techniques and multiplex PCR for 14 typical bacterial pneumonia-associated pathogens were performed on respiratory samples collected from adult hospitalized patients enrolled in a prospective multi-center study. Patients were enrolled from March until September 2012. A total of 739 fresh, native samples were eligible for analysis, of which 75 were sputa, 421 aspirates, and 234 bronchial lavages. 276 pathogens were detected by microbiology for which a valid PCR result was generated (positive or negative detection result by Curetis prototype system). Among these, 120 were identified by the prototype assay, 50 pathogens were not detected. Overall performance of the prototype for pathogen identification was 70.6% sensitivity (95% confidence interval (CI) lower bound: 63.3%, upper bound: 76.9%) and 95.2% specificity (95% CI lower bound: 94.6%, upper bound: 95.7%). Based on the study results, device cut-off settings were adjusted for future series production. The overall performance with the settings of the CE series production devices was 78.7% sensitivity (95% CI lower bound: 72.1%) and 96.6% specificity (95% CI lower bound: 96.1%). Time to result was 5.2 hours (median) for the prototype test and 43.5 h for standard-of-care. The Pneumonia Application provides a rapid and moderately sensitive assay for the detection of pneumonia-causing pathogens with minimal hands-on time.SCOPUS: ar.jinfo:eu-repo/semantics/publishe