Tuesday, April 23, 2024
4/23/2024
ABSTRACT Acute rheumatic fever (ARF) and rheumatic heart disease are the leading cause of preventable cardiovascular morbidity and mortality in children worldwide. Acute rheumatic fever is a multisystem inflammatory autoimmune disease that occurs in a subset of patients as a delayed complication of an improperly or untreated throat infection with a rheumatogenic strain of S. pyogenes, Group A Streptococcus, (GAS). While the exact triggers and mechanisms of ARF have not been proven, studies indicate that aberrant immune responses develop against host tissues approximately 3 weeks after GAS infection. The most severe pathology of ARF takes place in the heart, where infiltrating immune cells, cytokines and auto-antibodies cause destruction of the heart muscle and permanent damage to the heart valves. While there is no current diagnostic kit available, a method of detecting children who are more susceptible to ARF (3-6% of the total population) would have global health and economic benefits. As such, over the last century investigators have searched for genes related to ARF susceptibility. As ARF is an autoimmune complication of infection, most the research has focused on genetic markers related to different components of the immune response and effects of various immune mediators, but no direct correlations could be made across ARF patients from different ethnic groups and geographical locations. As an alternative, D8/17, a murine monoclonal IgM antibody capable of binding an unknown allogenic epitope on B- cells from ARF patients, was found to bind B lymphocytes in 90-95% of ARF patients from diverse ethnic groups and different geographical locations compared to only 11% in matched controls. While many studies have successfully tested cell culture supernatants of the D8/17 antibody as a marker for rheumatic fever susceptibility across several different population groups, no group to date has identified the specific target antigen that the D8/17 antibody binds to on the surface of B cell lymphocytes. We plan on use cutting edge techniques involving antibody sequencing and recombinant engineering, to elucidate the sequence of the D8/17 antibody variable regions. We then produce new recombinantly expressed mouse IgM and IgG1 versions of the D8/17 antibody. We hypothesize that the IgG1 class- switched antibody will be a better reagent to identify the D8/17 antigen and more amenable for use in diagnostic testing. We will also compare the activity of these newly engineered antibodies and utilize them in fluorescence activated cell sorting (FACS) analysis, immunoprecipitation and mass spectrometry experiments to more specifically derive the D8/17 antigen from our previously immortalized B-cell lines isolated from rheumatic fever patients and controls. By utilizing the class switched D8/17 antibody, we hope to discover novel susceptibility markers in patients predisposed to acute rheumatic fever (ARF), allowing us to derive better diagnostic reagents and gain further insight into the role of the associated genetic factors in pathogenesis. This will be achieved though proteomic and expression analysis of immortalized B lymphocyte lines from rheumatic fever patients to identify the D8/17 marker. We hope that our findings will lead to an ARF specific-diagnostic test for susceptibility to this illness and further elucidate the mechanisms behind this treatable and preventable disease.
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