Allorecognition in T-cell receptor (beta-chain) transgenic mice.

Recent advances in knowledge of the structure of the T-cell receptor and of major histocompatibility complex (MHC) molecules have increased our understanding of the nature of their interaction in the immune response. Nevertheless, it remains unclear how the T-cell receptor recognizes foreign MHC molecules in the process of graft rejection. One approach to this problem is to characterize the alloreactivity of a given T-cell receptor. We have chosen to take this approach in vivo by examining patterns of rejection of vascularized heart allografts in transgenic mice carrying a rearranged T-cell receptor-beta-chain gene, in which essentially all alpha beta T cells bear the rearranged gene product. Heterotopic heart allografts were performed in transgene-positive and transgene-negative recipients. The data show that transgene-positive mice will reject fully allogeneic grafts of three different haplotypes after a modest delay, but will not reject grafts from F1 mice that bear H-2 antigens from these same haplotypes and from the recipient strain. Transgene-negative animals reject all grafts promptly. These results suggest that the restricted T-cell receptor repertoire expressed by transgene-positive recipients affects their ability to respond to an alloantigen as expressed on a vascularized graft and that this response is influenced by the presence of self-MHC molecules on the graft.

RO 23-6457 prolongs survival of vascularized allografts in rodents and primates.

The ability of RO 23-6457, a retinoid compound with marked in vitro immunosuppressive properties, to prolong vascularized allografts was examined in several in vivo transplantation models. In the murine heterotopic heart model, efficacy was shown in two H-2 incompatible strain combinations, with indefinite graft survival at some doses. In the rat heterotopic heart model, oral administration prolonged Wistar-Furth grafts in Lewis hosts an average of 1 week, with no long-term survivors at a variety of doses. Given subcutaneously, grafts were further prolonged, but the compound proved toxic. In a bilaterally nephrectomized renal transplant model in cynomolgus monkeys treated intravenously at a dose of 2 mg/kg/day, host survival was prolonged to 18, 32, 33, and 74 days, compared with 11, 11, 12, 13, and 26 days in untreated controls (P less than 0.05 by rank-sum testing). The three shorter surviving recipients died from anorexia and weight loss with normal renal function, while the longest survivor rejected its kidney when exhaustion of iv sites precluded further treatment. The toxic effects of the compound resemble the syndrome of hypervitaminosis A. RO 23-6457 will prolong graft survival as a single agent, justifying further preclinical testing and efforts to reduce toxicity.

Enhancement of immunosuppression by substitution of fish oil for olive oil as a vehicle for cyclosporine.

As previously reported, acute cyclosporine-induced nephrotoxicity is characterized by a decline in glomerular filtration rate and a selective intrarenal production of the vasoconstrictor thromboxane (TxA2), but not vasodilator prostaglandin E2 (PGE2), or prostacyclin (PGI2), cyclooxygenase metabolites. Fish oils (FO), that are rich in n-3 polyunsaturated fatty acids have a high affinity for cyclooxygenase but serve as poor substrate inhibit TxA2 synthesis. We have shown that when FO replaces olive oil (OO) as the vehicle for CsA, CsA-induced nephrotoxicity and increased TxA2 synthesis are obviated in rodent models. In this study, we demonstrate that the FO vehicle for CsA does not compromise CsA's immunosuppressive properties as deduced from studies of a delayed-type hypersensitivity (DTH) model in BALB/c mice and in a rat heart transplant model. In fact, concurrent FO administration with CsA actually enhances immunosuppression. A dose of CsA incapable of blunting DTH when injected in OO was suppressive when given in FO. Administration of as little as 0.05 ml of FO vehicle potentiated the suppressive action of CsA. In addition, nonconcurrent dietary supplementation of FO in animals receiving CsA caused an increase in the immunosuppressive action of CsA in DTH. FO alone reduced DTH as compared with OO, but was far less effective than CsA plus FO. Furthermore, doses of CsA (5 mg/kg/day or 1.5 mg/kg/day), which are subtherapeutic when administered with OO, prolonged engraftment of Lewis recipients of Lewis x Brown-Norway F1 hearts when CsA was solubilized with FO. These studies indicate that concurrent administration of CsA and FO potentiates the activity of CsA and thus increases its therapeutic index. Thus, CsA plus FO is potentially a safe, potent antirejection therapy worthy of clinical testing, especially insofar as FO prevents CsA-induced acute nephrotoxicity in the rodent.

Cardiac allograft survival in mice treated with IL-2-PE40.

IL-2-PE40 is a chimeric protein composed of human interleukin 2 (IL-2) genetically fused to the amino terminus of a modified form of Pseudomonas exotoxin lacking its cell recognition domain. IL-2-PE40, which is extremely cytotoxic to IL-2 receptor-positive cells, was examined for its ability to prevent graft rejection in mice in which activation of T cells is prominent. We demonstrate that intraperitoneally administered IL-2-PE40 specifically and significantly prolongs the survival of vascularized heart allografts in mice. The chimeric toxin, IL-2-PE40, offers an alternative approach to the treatment of autoimmune diseases and transplant rejection in humans.

Prolongation of cardiac allograft survival in murine recipients treated with a diphtheria toxin-related interleukin-2 fusion protein.

A diphtheria toxin-related IL-2 fusion gene has been constructed that encodes a 68KD recombinant toxin in which the diphtheria toxin receptor-binding domain has been replaced with amino acids 2-133 of IL-2. This chimeric IL-2 toxin is cytotoxic for cells expressing the high-affinity IL-2 receptor but not for cells lacking this receptor. The ability of this IL-2 toxin to prolong allograft survival was examined in a murine vascularized, heterotopic heart transplant model in the strain combination B10.BR into C57B1/10. When given at a dose of 1.0 micrograms/day for 10 days, the IL-2 toxin significantly prolonged allograft survival in all recipients. CRM-45, a fragment of diphtheria toxin missing the binding domain, was ineffective, confirming the specificity of the therapy. The results demonstrate that this IL-2 toxin, which targets activated T cells expressing the IL-2 receptor, will prolong allograft survival, offering a new option for immunosuppressive therapy.

Monitoring the bioenergetics of cardiac allograft rejection using in vivo P-31 nuclear magnetic resonance spectroscopy.

Monitoring human cardiac allograft rejection is currently accomplished by endomyocardial biopsy. Available noninvasive methods for identifying rejection have lacked the necessary sensitivity or specificity, or both, for routine clinical application. In vivo phosphorus-31 (P-31) nuclear magnetic resonance (NMR) spectroscopy has been used for monitoring phosphorus metabolism in both animal models and humans. In the present study this technique was employed as a noninvasive means to assess the bioenergetic processes that occur during cardiac allograft rejection in a rat model. Brown Norway rat hearts were transplanted subcutaneously into the anterior region of the neck of Lewis rat recipients (allografts). Control isografts employed Lewis donors and recipients. Phosphocreatine to inorganic phosphate (PCr/Pi), phosphocreatine to beta-adenosine triphosphate (PCr/ATP beta), beta-adenosine triphosphate to inorganic phosphate (ATP beta/Pi) ratios and pH of the transplanted hearts were monitored using surface coil P-31 NMR spectroscopy (at 4.7 tesla) daily for 7 days. To allow recovery from the compromise induced by the surgical procedure, the measurements obtained on day 2 were taken as a baseline. PCr/Pi was unchanged or increased in the isografts but decreased continually in allografts, with the difference becoming significant by day 4 when compared with levels in day 2 allografts (p less than 0.005) and by day 3 when compared with levels in the isograft group (p less than 0.05). PCr/ATP beta in isografts did not change throughout the study; however, allografts demonstrated a significant decrease as early as day 3 (p less than 0.01), although a significant difference between isografts and allografts did not become manifest until day 4 (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)

Myopericarditis and enhanced dystrophic cardiac calcification in murine cytomegalovirus infection.

In mice inoculated with murine cytomegalovirus (MCMV) an acute myopericarditis developed which varied from a focal lymphohistiocytic inflammation to intense inflammation with necrosis and cytomegalic inclusion-bearing cells. Sublethal doses caused focal transient nonspecific chronic inflammation, followed months later by an increased frequency and extent of dystrophic cardiac calcification. When such latently infected hearts were heterotopically transplanted into uninfected animals which were then immunosuppressed (IS), a fatal generalized CMV infection followed. Cytomegalic inclusion-bearing endothelial, fibroblastic, and myocardial cells were seen in the intense inflammation found in hearts taken from mice 4 days after lethal inoculation and transplanted into uninfected mice, which were then IS. These findings may be relevant to human cardiac transplantation because they show that MCMV regularly causes cardiac infection with both acute and chronic consequences; chronic injury may follow a morphologically nonspecific myopericarditis which might not be attributed to CMV infection.

The protective effects of hyperimmune anti-murine cytomegalovirus antiserum against lethal viral challenge: the case for passive-active immunization.

The administration of 0.2 ml of hyperimmune anti-mouse cytomegalovirus (CMV) antiserum intraperitoneally (ip) or intravenously provided complete protection against lethal challenge (10(5.8) PFU ip) with murine CMV. Antiserum protection was complete when the antiserum was administered as long as 24 hr after viral challenge. The administration of antiserum had little effect on the titers of virus in the organs of these animals. Ammonium sulfate-treated antiserum provided similar complete protection. Animals rechallenged with 10(6)-10(6.5) PFU of murine CMV 1 month after initial challenge, at a time when the administrated antiserum was no longer detectable, all survived. We conclude that hyperimmune antiserum can provide significant protection against otherwise lethal murine CMV infection, that the protecting material lies within the immunoglobulin fraction, and that long-term immunity results from the combined exposure to virus and antiserum. Such passive-active protection could be useful in protecting against human CMV infection.

The effect of anti-interleukin-2 receptor monoclonal antibody on allograft rejection.

During immune response to an allograft, activated T cells express a number of cell surface activation antigens, among them the membrane receptor for the lymphokine interleukin 2 (IL-2). As the IL-2 receptor is not present on resting T cells, it offers an attractive target for potentially specific immunosuppressive therapy. The rat monoclonal antibody M7/20, which binds to the murine IL-2 receptor, was studied for its effect on allograft survival in two H-2-incompatible strain combinations in inbred mice. Treatment with M7/20 for 10 days markedly prolonged survival of vascularized, heterotopic heart allografts in both strain combinations, with indefinite graft survival in 50% of recipients. The same treatment significantly prolonged skin allograft survival in one of the two combinations. The results support the important role of the IL-2 receptor in the mechanism of graft rejection and confirm its suitability as a target for immunosuppressive therapy in transplantation.

Administration of an anti-interleukin 2 receptor monoclonal antibody prolongs cardiac allograft survival in mice.

Administration of the monoclonal antibody M7/20, which binds to the murine interleukin-2 (IL) receptor, significantly prolongs cardiac allograft survival in two H-2-incompatible strain combinations of inbred mice. The results support the important role of the IL-2 receptor in the mechanism of graft rejection, and suggest its suitability as a target for immunosuppressive therapy.

Primary cytomegalovirus infection following cardiac transplantation in a murine model.

A murine cytomegalovirus (CMV) model was utilized to determine the source of primary CMV infection in cardiac transplantation. Hearts were taken from actively or latently infected BALB/C mice and then transplanted as primary, heterotopic isografts into CMV-negative BALB/C recipients. The transplantation of hearts from acutely infected donors into nonimmunosuppressed recipients resulted in asymptomatic primary infection as manifested by detectable virus in both donor and recipient hearts, liver, spleen, and salivary glands and by the development of anti-CMV antibody. When hearts from latently infected animals were transplanted into nonimmunosuppressed recipients, a transient primary infection occurred that was manifested by detectable virus in the spleen and salivary glands and the appearance of anti-CMV antibody. When recipient animals were immunosuppressed with cortisone acetate (125 mg/kg/day i.p.) and rabbit antimouse thymocyte globulin (0.2 ml i.p. twice weekly), after transplantation of hearts from acutely and latently infected mice, lethal primary CMV infection developed. High titers of virus were recovered in all organs tested in these animals, including both the donor and recipient hearts. We conclude that the heart is infected during the course of primary murine CMV infection, and that hearts from latently infected animals are a source of serious primary infection in immunosuppressed recipients. This experimental system should be a useful model relevant to human cardiac transplantation.