Browsing by Author "Brennan, Patrick J., advisor"
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Item Open Access Characterization of the mannosyltransferase RV3779, which forms polyprenyl phosphomannose for the biosynthesis of phosphatidylinositol mannoside and lipoarabinomannan, and elucidation of the polymerization stages of galactan biosynthesis in mycobacteria(Colorado State University. Libraries, 2008) Scherman, Hataichanok, author; Brennan, Patrick J., advisorThe cell wall complex of mycobacteria, dominated by highly unique structural polysaccharides and a rather impermeable layer of lipids, can give these bacteria the ability to resist the immune response and cause a prolonged and deadly illness. Various lipoglycans and glycolipids in the cell wall complex of mycobacteria, such as lipoarabinomannan (LAM), as well as the synthetic precursors of LAM; lipomannan, and the phosphatidyl myo-inositol mannosides, are essential for the normal growth and viability of mycobacteria, although the specifics of their synthesis are still not fully defined. Their synthesis are governed by a myriad of enzymes, including a class of enzymes called glycosyltransferases, which are one of the most diverse and important groups of enzymes in nature. A particular glycosyltransferase, Rv3779, is the primary focus of this study, and from sequence and bioinformatic analysis, we identified it as a putative mannosyltransferase belonging to the GT-C superfamily found to be involved in various aspects of synthesis of the higher forms of the phosphatidyl myo-inositol mannosides and the subsequent lipoglycans. Rv3779 is present in a prominent gene cluster involved in cell wall biosynthesis. From the experimental evidence gathered from construction and analysis of a Rv3779 knockout mutant of M. tuberculosis, the in vitro assay of the membrane fractions of an Rv3779-overexpressed strain of M. smegmatis, and analysis of the reaction products by thin layer chromatography and mass spectrometry, we have concluded that Rv3779 possesses polyprenyl phosphomannose synthase activity in a homologous manner to Ppm1. Polyprenyl phosphomannose is the key mannosyl donor that is utilized heavily by later enzymes in the extracytoplasmic hypermannosylation of the higher forms of these lipoglycans. The galactan chain serves as the covalent attachment point for mycolated arabinan and is a central structural polymer in the cell wall complex that is the second focus of this study. The exact stages of the buildup galactan are not fully elucidated, and involve unique polymerization steps. We have utilized various organic extraction and analytical techniques from a cell-free assay utilizing UDP-D-galactofuranose as a donor and the results of these studies suggest that the galactan chain is polymerized one galactofuranosyl residue at a time.Item Open Access Development and evaluation of new leprosy skin test antigens as diagnostic tools(Colorado State University. Libraries, 2012) Rivoire, Becky Louise, author; Brennan, Patrick J., advisor; Beaty, Barry, committee member; Gonzales-Juarrero, Mercedes, committee member; Peersen, Olve, committee memberAn early diagnostic test for leprosy that is adequately sensitive and specific to identify infected individuals before the onset of clinical symptoms continues to be one of the greatest needs in the field. Preclinical diagnosis would expedite the delivery of chemotherapy to patients, prevent disabilities, decrease stigma, intercept transmission, and measure the true incidence of disease. To address this pressing need, three new leprosy skin test antigens were investigated. MLSA-LAM [M. leprae soluble antigens devoid of lipoglycans, primarily lipoarabinomannan (LAM)], MLCwA (M. leprae cell wall associated antigens), and MLMA-LAM (M. leprae membrane antigens devoid of lipoglycans, primarily LAM). Two of these antigens, MLSA-LAM and MLCwA, were developed for manufacturing and testing for safety and efficacy in phase I and phase II human clinical trials. Skin test antigens were derived from M. leprae purified from experimentally infected armadillo tissues under current good manufacturing practice conditions. A skin test pilot plant was created at Colorado State University for this purpose. Quality control testing of skin test antigens included potency and stability testing in guinea pigs, safety testing in guinea pigs and mice, integrity testing by gel electrophoresis and immunoblotting, and purity testing for residual dextran, collagenase, detergent, and endotoxin. An investigational new drug (IND) application was submitted to the Food and Drug Administration (FDA) and clinical protocols with respective informed consent forms were generated. Training in good laboratory, manufacturing, and clinical practice (GLP, GMP, and GCP) was a prerequisite for these studies. The phase I clinical trial was conducted at a non-endemic region for leprosy with both antigens at 2.5, 1.0, and 0.1 µg dosages. A randomized double blind phase II clinical trial (stages A, B, and C) followed in an endemic region for leprosy with both antigens at 1.0, and 0.1 µg dosages. Antigens were tested in the phase I and phase II, stage A/B trials using the intradermal delayed type hypersensitivity (DTH) skin test in healthy subjects without known exposure to leprosy, while the phase II, stage C trial compared the DTH skin test to the IFN-γ test and the M. leprae specific phenolic glycolipid I antibody test in target populations, including: leprosy patients, household contacts of leprosy patients, and tuberculosis patients. Both skin test antigens, MLSA-LAM and MLCwA, were found to be safe at each dose tested in the phase I and II clinical trials. The phase II, stage A/B clinical trials showed the baseline in healthy endemic controls for both leprosy antigens at the low dose of 0.1 µg was negligible, while slightly elevated with the high dose of 1.0 µg. Efficacy findings from the phase II, stage C clinical trial showed that the antigens were immunologically potent; highly specific, but lacked sensitivity at the low dose. The response to PPD did not correlate with either leprosy antigen at either dose. The IFN-γ release test provided the best diagnostic accuracy at the high dose with both antigens. Household contacts with the highest risk of infection reacted in each test. MLSA-LAM and MLCwA are the first skin test antigens to show specificity for leprosy in the field. The interferon gamma release assay with MLSA-LAM at the high dose provides the best diagnostic accuracy for tuberculoid leprosy patients. The PGL-I antibody assay provides the best diagnostic accuracy for lepromatous leprosy patients. Optimization of the antigen dosage or use of these tests in parallel or combination could lead to enhanced sensitivity, resulting in a good early diagnostic test for leprosy. Results from these research studies provide proof that a product can be translated from the bench to the clinic in an academic setting.Item Open Access Identification and characterization of leprosy T cell antigens in the context of early diagnosis and CD1a restricted T cell activation(Colorado State University. Libraries, 2011) Kim, Hee Jin, author; Brennan, Patrick J., advisor; Schenkel, Alan R., committee member; Reist, Noreen E., committee member; Vissa, Varalakshmi D., committee memberTo view the abstract, please see the full text of the document.