Molecular Profile: A Newsletter of Molecular Diagnostics & Molecular Pathology, 2007-2013
AuthorUniversity of Maryland School of Medicine. Department of Pathology
University of Maryland Medical Center. Molecular Diagnostics Laboratory
MetadataShow full item record
Other TitlesNewsletter of molecular diagnostics & molecular pathology
Newsletter of molecular diagnostics and molecular pathology
DescriptionNo newsletter released in 2011.
KeywordUniversity of Maryland Medical Center. Molecular Diagnostics Laboratory--Newsletters
University of Maryland, Baltimore. Department of Pathology--Newsletters
Identifier to cite or link to this itemhttp://hdl.handle.net/10713/2248
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Development of a molecular diagnostic, an animal model and a transposon mutagenesis system for studying fish tuberculosisTalaat, Adel Mohammed; Reimschuessel, Renate (1998)World wide, 88 million people are predicted to be infected with Mycobacterium tuberculosis (M. tuberculosis) during the current decade, 1990-99, of which 30 million people are expected to die. The slow growth rate and clumping of mycobacterial cells has hindered the application of "conventional" genetic techniques to study the molecular pathogenesis of mycobacterial infections. To overcome the problems encountered in working with M. tuberculosis, M. marinum and the goldfish, Carcassius auratus were developed as a model system for studying mycobacterial pathogenesis. This model system was used to evaluate the virulence of the slowly growing mycobacteria, M. marinum compared to the rapidly growing, M. fortuitum and M. smegmatis. Depending on the dose of mycobacteria inoculated into animals, an acute or a chronic form of the disease develops. With M. marinum, injection of 10 to the 9 or 10 to the 8 cfu per fish induced an acute disease with all animals dying within 17 days postinfection. Inoculation of 10 to the 2 to 10 to the 7 cfu per fish induced a chronic disease with all fish surviving until the end of the experiment (56 days). In contrast, with M. fortuitum, only an inoculum of 10 to the 9 cfu per fish was able to induce an acute form of the disease. Surprisingly, our results showed that M. smegmatis, once considered a nonpathogenic strain of mycobacteria, is pathogenic to fish. Additionally, a genus specific PCR-based protocol was developed to amplify a 924 bp DNA fragment from the 16S rRNA present in all Mycobacteria, followed by restriction enzyme analysis (REA) to identify the species. To develop an efficient protocol for genetic transfer of plasmids to mycobacterial cells, several protocols were tested to and the optimal conditions for M. marinum electroporation. Electrocompetent cells prepared at room temperature from M. marinum cultures grown to late-exponential phase in the presence of ethionamide (1 mug/ml) gave the highest transposition efficiency. Signature-tagged mutagenesis protocol was applied to identify virulence factors in M. marinum. By screening M. marinum mutant library in goldfish, we were able to identify several M. marinum mutants unable to survive in the goldfish. Genetic analysis of such mutants is currently underway.
Human herpesvirus 8: Diagnostic and serologic investigations of a novel human pathogenEdelman, Daniel Charles; Constantine, Niel T. (2005)Human herpesvirus 8 (HHV-8), a lymphotropic gammaherpesvirus, is the etiologic agent of Kaposi's sarcoma (KS). Results from serologic assays for HHV-8 infection have variable concordance, primarily in low risk populations. The detection of HHV-8 infection could be improved by optimization of existing serologic tests, development of new assays and effective algorithms, better understanding of the immune response, and identification of new diagnostic antigens. To address this, a number of HHV-8 serologic tests were developed or modified and compared to existing methods. IgG, IgK and IgA isotype seroprevalences against the Orf 65 antigen were 93%, 87%, and 40% in KS samples, respectively---much higher than observed in USA blood donors (BDs: <3%). In comparison studies, the K8.1 IgG ELISA had the best overall diagnostic performance. For seropositive BDs, there was poor agreement among multiple tests. HHV-8 seroprevalence in non-KS samples was highest in Africa (9%--42%), followed by the Middle East (6%--20%), Asia and South America (0%--18%), the USA (0%--11%), and the Caribbean (1%--2%). Rates were highest in KS patients (74%--100%), high in African BDs (22%), lowest in non-African BDs (2%--7%), and significantly higher in HIV+ individuals (17%) as compared with HIV- (8%). Antibody reactivities using multiple assays were observed in patients with sarcoidosis (0%--33%: P = 0.007) and multiple myeloma (0%--21%: P = 0.017). A diagnostic algorithm was established exhibiting high sensitivity (99%) for KS samples using an Orf 65 ELISA followed by a lytic IgG IFA. A study of the kinetic humoral response to HHV-8 showed 4-fold changes in Orf 65 reactivity, and increasing IgM or IgA responses suggested viral reactivation. In addition, Western blot analysis showed temporal IgG and IgM reactivities against specific HHV-8 lytic proteins. Epitope expression proved effective in identifying a new HHV-8 antigen; antibodies to the Orf 64 tegument protein had a higher seroprevalence in KS sera (30%) than in BDs (2%). In summary, HHV-8 diagnostic tests were developed or modified to have improved test indices, prevalence studies showed the utility of the tests, antibody isotype reactivity against HHV-8 was better characterized, the kinetics of the immune response were better defined, and a new HHV-8 antigen was discovered through epitope expression.
Physiological and pathological role of zonulin in the regulation of intercellular tight junctionsWang, Wenle; Fasano, Alessio; Bucci, Enrico, M.D., Ph.D. (2001)The paracellular route is the dominant pathway through which passive solutes flow across both the endothelial and epithelial barriers, and its functional status is regulated, in part, at the level of intercellular tight junctions. Tight junctions readily adapt to a variety of developmental, physiological, and pathological circumstances. The physiological regulation remains undefined. Zonula occludens toxin (Zot), a protein elaborated by Vibrio cholerae, reversibly regulates tight junction permeability. Zot interacts with a specific surface receptor(s) with subsequent protein kinase C alpha-dependent polymerization of actin microfilaments strategically localized to regulate the paracellular pathway. Based on this observation, we postulated that Zot might mimic an endogenous modulator(s) of tight junctions and that Zot and its putative eukaryotic analogue could be structurally and immunologically related. Accordingly, the specific anti-Zot antibody and the Using chamber assay were used to screen for the human intestinal Zot analogue(s). Non-primate intestinal tissues were used as an indicator system to identify and purify this analogue. A single protein was picked up and named zonulin. Purified zonulin reduced the electrical resistance of small intestine in a reversible fashion similar to that of Zot. All characteristics of zonulin that have been tested are comparable with Zot's characteristics. We conclude that zonulin and Zot share the same mechanism in regulating tight junctions. To date, there is no clear explanation for the disturbed physiological regulation of the intestinal permeability secondary to proximal bacterial contamination. Zonulin can, however, protect the small intestine from bacterial colonization. When rabbit ileum was exposed to Salmonella typhimurium and E. coli 6-1, resistance decrement was induced. These changes in Rt correlated with increases in zonulin secretion into the luminal (but not the serosal) side of the mucosa. This result suggests that when there is bacterial colonization in the small intestine, the tissue will secrete zonulin that is either stored or newly synthesized. Pre-treatment with FZI/0, a zonulin receptor binding inhibitor that prevents the permeating effect of zonulin, completely blocked the E. coli 6-1-induced Rt changes in both jejunum and ileum and the paracellular passage of inulin without affecting zonulin luminal secretion. Taken together, our data demonstrate the some tissues contain zonulin which can regulate tight junctions by using the same mechanism as Zot. They share the same receptors and the same signal transduction pathway, and they share a conservative domain responsible for binding to receptors. Zonulin is induced when there are bacteria or other specific proteins in the small intestine. In addition, overexpression of zonulin has been found in celiac disease. (Abstract shortened by UMI.)