The applicants propose to study the effects of treatment with a nondepleting CD4 mAb (RIB-5/2) or rapamycin (RPM) + synthetic fibronectin (FN) peptides in sensitized rat recipients of cardiac allografts (Tx). RIB-5.2 mAb or RPM monotherapy abrogates rejection at 24 h and prolongs cardiac Tx survival to >60 days and ca. 50 days respectively. The approach to preventing Tx rejection with FN peptides is based upon observations that : (i) rejection triggers increased intra-Tx deposition of FN, (ii) adhesive FN- MNC associations are decisive for intra-Tx MNC recruitment, (iii) FN-MNC interactions are vital for cell activation/migration/adhesion, (iv) treatment with FN peptides prevents/reverses acute or chronic inflammatory conditions (e.g. rat model of erosive arthritis, TGFB1 knockout mice).
The specific aims are as follows: 1. To investigate intraTx expression /function of FNs, and FN-MNC interactions: Cardiac transplants from untreated and RIB-5.2 mAb or RPM treated hosts will be analyzed for FN expression at the mRNA (Northern blotting/RNase protection assay) and protein (immunohistochemistry) levels. The expression of total FN, and FN splicing variants (EIIIA, EIIIB, CS-1) will be correlated with FN receptors on infiltrating MNC. The FN producers will be identified by in situ hybridization. MNC positioning in relation to FNs will be studied by laser scanning confocal microscopy. The adhesive/migratory/differentiation properties of MNC will be tested in vitro on FN-coated substrata. The ability of anti-integrin mAb, FN peptides interacting with integrins or cell surface proteoglycans, and FN fusion proteins to modulate cell functions will be evaluated. The effects of treatment with FN peptides/fusion proteins on in vivo cell migration will be analyzed. 2. To assess the effects of adjunctive treatment with FN peptides on Tx survival and FN-MNC interactions. Individual or combined FN peptide preparations will be administered into RIB-b/2 mAb or RPM treated hosts bearing long-term (>30 day) cardiac Tx. The effects of treatment with FN peptides upon adhesive/migratory/differentiation properties of intra-Tx infiltrating MNC will be evaluated in vitro and in vivo as outlined in Aim 1. 3. To evaluate the effects of applied therapies upon host cell-mediated immunity: Cardiac Tx will be examined by immunohistology to identify infiltrating MNC by phenotypic/activation markers. The binding patters of naive or host LNL to HEV of naive or recipient lymph nodes and cardiac T will be studied: the specificity will be ascertained in mAb inhibition experiments. in vitro LMC, MLR, flow cytometry and in vivo adoptive transfer, allo-Ag challenge experiments will unravel putative feedbacks between immunomoulatory agents and cellular repertoire in host lymphoid organs. 4. To explore the cytokine networks: Intra-Tx expression of cytokines will be examined by RT-PCR and immunohistochemistry. The studies will focus on Th1 (IL-2, IFN- ) vs Th2 (IL-4, IL-10) cytokines, and IL-12; CTL specific granzyme B: FN enhancing growth factor (TGF-B1); neutrophil (MIP-2, KC) or macrophage (MCP-1) stimulatory chemokines. To clarify the relevance of gene products, Tx hosts will be challenged with rIL-2, rIFN- , rIL-12, or cytokine specific Abs. 5. To assess the effects of applied therapies on host humoral immunity: The utilization patterns and isotype switching of allo-Abs will be studied in the circulation (flow cytometry), and at the Tx site (immunochemistry, radial immunodiffusion). The functional significance of host humoral reactivity will be probed by testing sera samples in CDC, ADCC, and in passive transfer studies. The effects of sera on the alloreactive T cell functions will be evaluated in MLR.
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