ABSTRACT
Chronic cardiac rejection is represented by cardiac allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) known to cause severe complications. These processes are accompanied by remarkable changes in the cardiac extra cellular matrix (cECM). The aim of our study was to analyse the cECM remodelling in chronic rejection and to elucidate a potential role of ED-A domain containing fibronectin (ED-A(+) Fn), alpha smooth muscle actin (ASMA) and B domain containing tenascin-C (B(+) Tn-C). A model of chronic rejection after heterotopic rat heart transplantation was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade, to real-time PCR based analysis of 84 genes of ECM and adhesion molecules and to immunofluorescence labelling procedures, including ED-A(+) Fn, ASMA and B(+) Tn-C antibodies. Histological analysis revealed different grades of chronic rejection. By gene expression analysis, a relevant up-regulation of the majority of ECM genes in association with chronic rejection could be shown. For 8 genes, there was a relevant up-regulation in allografts as well as in the corresponding recipient hearts. Association of ASMA positive cells with the grade of chronic rejection could be proven. In CAV and also in CIF there were extensive co-depositions of ED-A(+) Fn, ASMA and B(+) Tn-C. In conclusion, chronic cardiac allograft rejection is associated with a cECM remodelling. ASMA protein deposition in CAV, and CIF is a valuable marker to detect chronic rejection. Interactions of VSMCs and Fibro-/Myofibroblasts with ED-A(+) Fn and B(+) Tn-C might functionally contribute to the development of chronic cardiac rejection.
Subject(s)
Actins/metabolism , Extracellular Matrix/metabolism , Fibronectins/metabolism , Gene Expression , Graft Rejection/metabolism , Heart Transplantation , Tenascin/metabolism , Actins/genetics , Animals , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Extracellular Matrix/genetics , Extracellular Matrix/pathology , Fibronectins/genetics , Fibrosis/metabolism , Fibrosis/pathology , Gene Expression Profiling , Graft Rejection/genetics , Graft Rejection/pathology , Male , Myocardium/metabolism , Myocardium/pathology , Protein Isoforms , RNA, Messenger/metabolism , Rats , Rats, Inbred F344 , Rats, Inbred Lew , Tenascin/genetics , Transplantation, HeterotopicABSTRACT
Mice develop a marked age-related susceptibility to myocardial coxsackievirus B3 (CVB3) infections. The lesions observed in mice resemble closely those seen in the human disease. Experimental murine models of CVB3-induced myocarditis have shown that both, host and viral genetic factors, can influence susceptibility to the infection as well as the persistence and progression of the disease. Recently, we have shown that CD4 T cell-deficient MHC Class II knockout mice develop a strong fibrosis with virus persistence in the heart tissue and without production of neutralizing antibodies. To examine the role of CD4+ T cells and especially the role of the T helper 1 cell response for the outcome and pathogenesis of CVB3-induced myocarditis in more detail, 2 different mouse strains with identical genetic background (H-2b) were infected with CVB3-Mü/J (Nancy strain). Immunocompetent C57BL/6 mice and mice with targeted disruption of interleukin (IL-)4 gene (IL-4-/- mice) developed a severe acute myocarditis on day 7 post infection (p.i.). The CVB3-induced inflammation was cured until the 21st day p.i. in hearts of C57BL/6 mice. IL-4-/- mice with insufficient T helper-2 cell immune response developed a severe myocardial damage between day 7 and 21 p.i. with prolonged virus persistence in the heart tissue. Therefore, we suggest that despite an obvious normal T helper-1 cell cytokine pattern, IL-4-/- mice are more susceptible to long-term heart muscle injuries after infection with CVB3.
Subject(s)
Cardiomyopathy, Dilated/immunology , Coxsackievirus Infections/immunology , Enterovirus B, Human/immunology , Immunity, Cellular/immunology , Myocarditis/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , Cardiomyopathy, Dilated/diagnosis , Cardiomyopathy, Dilated/pathology , Coxsackievirus Infections/diagnosis , Disease Models, Animal , Humans , Interleukin-4/genetics , Interleukin-4/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Myocarditis/diagnosis , Myocarditis/pathology , Myocardium/immunology , Prognosis , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/pathologyABSTRACT
For the development of multilayer fluorescent immunoassays to determine directly a variety of therapeutic drugs high specific monoclonal antibodies against the anticonvulsants carbamazepine, phenobarbital, phenytoin and valproic acid have been prepared. For each antibody a standard curve could be established showing good linearity and a suitable sensitivity scope. To validate the eligibility of the desired antibodies a variety of drugs and metabolites sharing structural similarities to the individual drugs were tested for their cross-reactivity. It could be shown that only a few very closely related compounds exhibited substantial cross-reactivity. These monoclonal antibodies are therefore assumed to be suitable for a TDM-assay development.
