CHARACTERIZATION OF THE SERO-REACTIVITY OF PROTEINS MAP1152 AND MAP1156 FROM \u3ci\u3eMYCOBACTERIUM AVIUM\u3c/i\u3e SUBSPECIES \u3ci\u3ePARATUBERCULOSIS\u3c/i\u3e

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease (JD) in ruminants. Development of genetic tools and completion of the MAP genome sequencing project expanded opportunities for antigen discovery. In this thesis, I review the current trends in diagnosis and disease control of JD and present the results of the studies on the seroreactivity of two proteins encoded for by the MAP1152-MAP1156 gene cluster. MAP1152 encodes for a PPE protein and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Maltose-binding protein (MBP) tagged recombinant MAP proteins were purified from Escherichia coli. Western immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of immunized mice and rabbits, and naturally infected cattle. MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme linked immunosorbent assay (ELISA) for the recombinant proteins was developed and used to test pre-classified positive and negative serum samples from naturally infected and non-infected cattle. Samples, with one exception, displayed no seroreactivity against MBP-LacZ (P \u3e 0.05), the negative control antigen. MAP1152 displayed seroreactivity against all positive sera, but no seroreactivity to the negative sera (P \u3c 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between non- infected and infected cattle (P \u3c 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, MAP infected cattle mount a humoral response to both MAP1152 and MAP1156. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immuno-pathogenesis of JD. Adviser: Raul G. Barlett

Immunogenicity and Reactivity of Novel Mycobacterium avium subsp. paratuberculosis PPE MAP1152 and Conserved MAP1156 Proteins with Sera from Experimentally and Naturally Infected Animals ▿ †

Mycobacterium avium subsp. paratuberculosis causes Johne's disease (JD) in ruminants. Development of genetic tools and completion of the M. avium subsp. paratuberculosis genome sequencing project have expanded the opportunities for antigen discovery. In this study, we determined the seroreactivities of two proteins encoded at the 5′ and 3′ regions of the MAP1152-MAP1156 gene cluster. MAP1152 encodes a PPE protein, and MAP1156 encodes a diacylglycerol acyltransferase involved in triglyceride metabolism and classified in the uncharacterized protein family UPF0089. Recombinant MAP proteins were overproduced and purified from Escherichia coli as maltose-binding protein (MBP) fusions. Immunoblotting analysis indicated that both MAP1152 and MAP1156 displayed reactivity against sera of mice and rabbits immunized with live M. avium subsp. paratuberculosis cells and against samples from naturally infected cattle. In immunoblot assays, MAP1156 yielded a stronger positive signal than MAP1152 against sera from cattle with JD. An enzyme-linked immunosorbent assay for the recombinant proteins was developed and used to test preclassified positive and negative serum samples from naturally infected and noninfected cattle. Samples, with one exception, displayed no seroreactivity against the MBP-LacZ fusion protein (P > 0.05), the negative-control antigen. MAP1152 displayed seroreactivity against all positive sera but no seroreactivity to the negative sera (P < 0.01). MAP1156 displayed stronger and more variable reactivity than MAP1152, but significant differences were observed between noninfected and infected cattle (P < 0.05). Otherwise, degrees of reactivity followed the same trend as the positive reference antigen. In conclusion, both proteins are immunogenic in mice and rabbits, and M. avium subsp. paratuberculosis-infected cattle mount a humoral response to both MAP1152 and MAP1156 cross-reactive epitopes. These findings have potential applications to diagnostics, vaccine production, and elucidation of the immunopathogenesis of JD

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS): the mechanism, present strategies and future perspectives of therapies

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), which manifests as non-cardiogenic pulmonary edema, respiratory distress and hypoxemia, could be resulted from various processes that directly or indirectly injure the lung. Extensive investigations in experimental models and humans with ALI/ARDS have revealed many molecular mechanisms that offer therapeutic opportunities for cell or gene therapy. Herein the present strategies and future perspectives of the treatment for ALI/ARDS, include the ventilatory, pharmacological, as well as cell therapies