Formalin-fixed, paraffin-embedded sections (4 to 6 6 m) were deparaffinized in xylene and rehydrated with graded ethanol. and CD68+ cells into valve cells, consistent with human being studies that suggest that RF/RHD are mediated by inflammatory CD4+ T cells and CD68+ macrophages. The current study provides additional information that supports the use of the rat autoimmune valvulitis model for investigating RF/RHD. There is a wealth of evidence to indicate the immunopathogenic mechanisms in rheumatic heart disease involve autoimmunity as a result of molecular mimicry between streptococcal and sponsor cells proteins (7, 16), although the precise mechanisms are not completely understood. Progress has been made in this part of study by analyzing cellular and humoral reactions of peripheral blood samples from rheumatic fever and RF9 rheumatic heart disease (RF/RHD) individuals (11, 15, 17, 19). However, study of T cells and connected cytokines involved in initiating tissue damage is required. Limited access to cells samples from RF/RHD individuals is definitely a major obstacle to these studies, and an acceptable animal model would facilitate further studies. An animal model, in which the immunopathological mechanisms or end result of disease resembles those that happen in humans, is definitely a logical adjunct to human being studies. For many years, attempts to establish a suitable animal model for RF/RHD experienced limited success, with none of the proposed models showing the same pathological changes as those seen in human being individuals (23). The rat autoimmune valvulitis (RAV) model, developed by Quinn and colleagues (30) whereby Lewis rats immunized with recombinant streptococcal M protein develop hallmark RHD lesions in heart valves, has shown promise as a suitable animal model of rheumatic carditis. A role for molecular mimicry in RF/RHD immunopathogenesis has also been supported by the study of Quinn (30) and by others using the RAV model (14). Peripheral blood T-cell lines from M-protein-immunized rats proliferated in response to cardiac myosin (30), and T cells from heart lesions of cardiac myosin-immunized rats also responded to peptides from your RF9 B-repeat RF9 region of M protein (14). RF9 The RAV model has also been used in our laboratory to induce valvulitis/carditis by immunizing Lewis rats with C-terminal M-protein peptides (26). In this study, B- and T-cell reactions in Lewis rats immunized with group A streptococcus (GAS) M5 protein or selected M5 peptides were examined to further validate the use of the RAV model as a suitable animal model for RF/RHD. Immunostaining of cellular infiltrates in valvular and myocardial cells revealed that heart damage observed in streptococcal M-protein-immunized rats is definitely mediated by CD4+ T cells and macrophages, in agreement with human being studies (19). MATERIALS AND METHODS Animals. Lewis rats (LEW/SsN; albino: a,h,c: RT1) were originally purchased from Animal Resources Centre (Canning Vale, Western Australia, Australia) and consequently bred at the Small ERBB Animal Breeding RF9 Facility at James Cook University or college, Townsville, Australia. The rats were fed having a pelleted protein-rich commercial diet and water ad libitum. Experimental procedures were conducted with authorization from James Cook University Animal Ethics Committee and in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (National Health and Medical Study Council). Depending on the experimental design, treatment organizations included from three to five rats, with appropriate control organizations. Antigens. M protein is definitely a major virulence element of group A streptococcus, and serotype 5 was chosen for this study due to its potential like a rheumatogenic strain (34). The extracellular website of GAS M5 protein (amino acid residues 1 to 450) was amplified by PCR from genomic DNA from GAS research strain M5T5/B/PS PHLS.