| ObjectivesInduction of antigen specific tolerance in organ transplant recipients is the fundamental way for reaching long term survival of grafts. In recent studies, peptides derived from certain regions of human class I HLA molecules were known to have immunomodulatory effects. In particular, amino acid residues 75-84 of the HLA-B7 and HLA-B2702 molecules had demonstrated allele nonspecific immunosuppression in several animal transplant models. However, the effect of the peptides derived from the class I HLA molecules was not satisfactory. Therefore, to optimize novel peptides derived from class I HLA molecules can be a breakthrough in this field. Although these class I HLA derived peptides functioned, at least in part, by inhibiting CTL and NK cells, their mechanism of action was incompletely understood. The allele nonspecific effects of the 07.75-84 and the fact that both L- and D-isomers of peptide 2702.75-84 prolong graft survival suggested that the peptides' immunomodulatory activity was not based on direct interaction with the T cell receptor or on presentation by MHC molecules. Affinity chromatography with one of these immunomodulatory peptides had identified heme oxygenase-1 (HO-1) as a potential receptor for these agents.In this study, a novel rational design was approached to develop a new immunomodulatory peptides based on peptide 2702.75-84, RDP1258. And peptides were synthesized by using Fmoc chemistry, then the effects of RDP1258 to rat T lymphocyte and PBMC proliferation to ConA, MLR in vitro and in vivo were observed. This study establishes the rat renal allograft model, and observes its effects on the survival days of receptor rats, renal allograft's functions, and occurrence of the acute rejection. Also we evaluate the effects of RDP1258 on rat HO-1 activity in vitro and in vivo, and explore the mechanisms of its immunomodulatory effects.Methods1. Peptides were synthesized by Fmoc chemistry method with the peptide synthesizer ABI 431A and purified by reversed phase HPLC method. The molecular weight was validated by mass-spectrum. The sequential decoding of the peptides were 2702.75-84: RENLRIALRY, RDP1258: RNleNleNleRNleNleNleGY.2. Rat spleen lymphocytes and human peripheral blood mononuclear cells were isolated by Ficoll-paque density gradient centrifugation. Then rat T lymphocyte and PBMC proliferation to ConA, MLR were assayed by [3H]-thymidine uptake. The effects of the peptides on alloreactive cytotoxic activities of cytotoxic T lymphocytes and NK cells were observed by [3H]-thymidine release method. RDP1258, HLA-B2702.75-84 and control peptide were administrated respectively in every experiment.3. In vivo, the peptides were administered intraperitoneally at a dose of 2.5, 5, 10 mg/kg on days -1, and rat spleen lymphocytes were isolated. Then rat T lymphocytes to ConA, MLR were assayed by [3H]-thymidine uptake.4. F344 rats underwent unilateral nephrectomy and heterotopic transplantation with a LEW kidney. On day 5, the second native kidney was removed. Group 1 (n = 8) received no immunosuppression, group 2 (n = 8) received subtherapeutic CsA alone, group 3 (n = 8) received RDP1258 alone, group 4 (n = 8) received subtherapeutic CsA + RDP1258 peptide. RDP1258 peptide was administered intraperitoneally at a dose of 5 mg/kg on days -7, -1 and 0 and then twice weekly until day 30; CsA in all cases was administered subcutaneously at a dose of 5 mg/kg per day from days 0 to 5.5. The renal allograft survival days of receptor rats were observed. Serum creatinine was measured and Colour Doppler Flow Imaging was checked at every week after transplantation. The transplanted kidneys were harvested for light and electron microscopic examination at day 7. The immunologic tolerance status of rat renal allografts were detected by MLR of 60th day receptor rat spleen cells cultured with the donor spleen cells and the third party, proliferation to MLR were assayed by [3H]-thymidine uptake. 6. HO activity in Vitro: Rat spleen homogenates were prepared and used as the source of HO for acti...
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