Targeting of glycosaminoglycan-cytokine interactions as a novel therapeutic approach in allotransplantation.

BACKGROUND: Glycosaminoglycans (GAGs) are heteropolysaccharides present as integral components of the extracellular matrix (ECM), cell and basement membranes. GAGs play an important role in immune and inflammatory responses because of their ability to interact with cytokines and chemokines, promoting the localization of these molecules onto the ECM or cell membranes at specific anatomical sites. The main goal of these studies was to test the hypothesis that interference with the binding of cytokines/chemokines to GAGs will interfere with a graft rejection response. METHODS: MC-2, a cationic peptide derived from the sequence of the heparin-binding domain of mouse interferon gamma, was used as an inhibitor of the binding of cytokines/chemokines to GAGs. The effects of this peptide were studied in an allogeneic transplantation model involving vascularized rat skin flaps. RESULTS: The MC-2 peptide was found to inhibit binding of interferon-gamma, as well as that of the chemokines, interleukin-8, interferon gamma inducible protein-10, and regulated on activation normal T cell expressed and secreted (RANTES), to GAGs in vitro. Direct administration of MC-2 in an allogeneic skin flap transplantation model resulted in a significantly delayed time of rejection, from 5.4 +/- 0.5 days (control; n=6) to 12.6 +/- 1.6 days (treated animals; n=10). Histopathologic analysis of the skin biopsies was consistent with the delayed rejection process in those animals receiving the peptide, showing only mild signs of rejection up to day 11 (in contrast, all control animals had rejected their flaps by day 6). CONCLUSIONS: These results are consistent with the idea that GAG-cytokine interactions constitute valid therapeutic targets and suggest the potential applicability of such an approach in the prevention of graft rejection.

Composite tissue allotransplantation in chimeric hosts: part I. Prevention of graft-versus-host disease.

BACKGROUND: Mixed allogeneic chimerism (MAC) has been shown to induce tolerance to composite tissue allografts (CTA). However, transplantation of unmanipulated donor-specific limbs results in severe graft-versus-host disease (GVHD). This suggests that nontolerant mature donor-derived cells in the CTA may affect the stability of chimerism, potentially resulting in GVHD. The aim of this study was to develop an approach to study and prevent GVHD in a mixed chimeric-rat hind-limb transplantation model. METHODS: [ACI-->WF] chimeras received a limb from Wistar Furth (WF) (syngeneic), Fisher (third-party), or ACI (irradiated [1,050 cGy] or nonirradiated) rats. In vitro tolerance was assessed using mixed lymphocyte reactivity (MLR) assays at the time the animals were killed. RESULTS: [ACI-->WF] chimeras with greater than 85% chimerism exhibited rejection-free survival of donor-specific hind limbs. However, 100% of these animals developed lethal GVHD 22.4+/-2.8 days after limb transplantation. [ACI-->WF] chimeras that underwent transplantation with irradiated ACI or syngeneic WF limbs showed no signs of rejection or GVHD at 5 months. Nonchimeric and third-party controls rejected limbs within 10 days. CONCLUSIONS: Conditioning of the host WF rats with 950 cGy of irradiation (sublethal, myeloablative) led to high levels of MAC without GVHD. The mature T-cell content of nonirradiated donor (ACI) limbs was sufficient to induce lethal GVHD in 100% of tolerant mixed chimeric [ACI-->WF] hosts. Irradiation of donor limbs before transplantation resulted in long-term donor-specific tolerance and prevented GVHD. These data demonstrate that (1) established chimeras could be susceptible to GVHD caused by immunocompetent donor cells transferred with the hind limb, and (2) inactivating these cells with irradiation prevents GVHD and destabilization of chimerism, and permits rejection-free graft acceptance.

Low-dose immunosuppression in a rat hind-limb transplantation model.

Composite tissue allografts (CTAs) offer an alternative to conventional reconstructive methods. However, the toxicity of the drugs that are required to prevent rejection has prevented its widespread clinical application. The purpose of this study was to determine whether a low-dose, corticosteroid-free combination regimen of tacrolimus and mycophenolate mofetil (MMF) would prevent rejection in a rat hind-limb model, with minimal toxic side effects. Three groups were used in this study. In group I, Wistar Furth (WF) rats received a syngeneic WF hind-limb. In groups II and III, WF rats received an ACI hind-limb. The latter were treated with tacrolimus-MMF. Assessment for rejection, flow cytometry, and mixed lymphocyte reactions was performed. Biopsies were taken regularly and at the time of killing. Combination therapy with low-dose tacrolimus-MMF effectively prolonged CTA survival indefinitely, with minimal side effects. Toxicity associated with immunosuppressive drugs can be avoided in a low-dose combination corticosteroid-free regimen.