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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.

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.

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.

Previous reports have suggested that the immunosuppressive effects of lymphoid irradiation (LI) are related to changes in T cell peripheral blood levels. We prospectively studied 13 patients with Stages I and II Hodgkin's disease before, during, and after a course of Lt. Forty to 45Gy delivered to the mantle field in 412–5 weeks was followed by a 3–4 week break and 35–40Gy to the paraaortic field. Daily fraction size was 1.1–1.8Gy. Peripheral venous samples were drawn weekly. T cell subsets were identified by fluorescent staining of lymphocyte surface antigens using murine monoclonal antibodies OKT3, OKT4, and OKT8 (reactive with all T cells, helper/inducer cells (H/I), and cytotoxic/suppressor cells (C/S), respectively), and quantitated by flow cytometry. The absolute lymphocyte count prior to LI was 1400 ± 270/mm3. This fell to 597 ± 280/mm3 at the conclusion of LI and returned to 1490 ± 800/mm3 at 7–8 months post-LI. The percent total T cells and H/I were reduced from pre-LI values of 66 ± 8 % and 48 ± 10%, respectively, to 7–8 month post-LI values of 36 ± 16% and 19 ± 8%. The percent C/S cells was relatively unchanged by Lt: 20 ± 4% before treatment and 26 ±10 % at 7–8 months after Lt. Seven previously spleenectomized cynomolgus monkeys received 20Gy of LI in 20 equal daily fractions over a 4 week period through a single anterior field including the cervical, axillary, mediastinal, paraaortic iliac, and inguinal lymph node chains at a dose rate of 4.6–4.8cGy/minute. Renal allografts from unrelated monkey donors were placed following Lt. Graft function was monitored by urine output, serum BUN and creatinine, and renal biopsy. Low dose rate LI was well tolerated. The absolute lymphocyte count fell from a pre-LI level of 3080 ± 1227/mm3 to 534 ± 478/mm3 at 1 month post-LI. The percent H/I fell slightly during LI from a pre-treatment value of 28 ± 5% to 20 ± 10% at 1 month post-LI. There was a slight increase in percent C/S cells from a pre-LI value of 29 ± 13% to a 1 month post-LI value of 36 ± 19%. In 4 animals that received no LI (controls), survival post-transplant was 8–11 days prior to death from acute rejection. Five animals received transplants immediately following Lt. Four survived 11–56 days prior to death of presumed infection. One died at day 45 with rejection. Another group of two animals received transplants 312 weeks after Lt. One died of rejection at day 42. One remains well at day 394 post-transplant.

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.

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/ATPβ), beta-adenosine triphosphate to inorganic phosphate (ATPβ/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 iso-grafts but decreased continually in allografts, with the difference becoming significant by day 4 when compared with levels in day 2 allografts (p < 0.005) and by day 3 when compared with levels in the isograft group (p < 0.05). PCr/ATPβin isografts did not change throughout the study; however, allografts demonstrated a significant decrease as early as day 3 (p < 0.01), although a significant difference between isografts and allografts did not become manifest until day 4 (p < 0.005). The ATPβ/Pi differences between the two groups were similar to those for PCr/ATPβ, achieving a significant difference between groups on day 4 (p < 0.05). In the isograft group, a significant increase in ATPβ/Pi was present on day 3 (p < 0.05). The isograft group showed no change in intracellular pH; however, the allograft group demonstrated an initial alkaline shift followed by acidosis.This study, coupled with the development of new methods to apply P-31 spectroscopy clinically, suggests that the abnormal bioenergetics associated with cardiac allograft rejection may be a useful means for following cardiac transplant patients.

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, hetero topic 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.

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.

Experiences sur souris. Aucun pretraitement du donneur (soit cyclophosphamide a hautes doses soit irradiation) n'est efficace contre la transmission de cytomegalovirus. Le traitement devrait etre oriente sur le receveur

Cytomegalovirus (CMV) produces a latent infection in its host. The actively or latently infected transplanted kidney and heart are sources of debilitating CMV infection in transplant recipients [1], but the organs and cellular sites of viral latency and the mechanisms of CMV activation have not been fully defined. Latent murine cytomegalovirus (MCMV) has been activated in vitro by cocultivating spleen cells or peritoneal macrophages with allogeneic, syngeneic, or both mouse embryo cultures (MECs) [2-5]. MCMV has also been activated from explant cultures of spleens and prostate glands, cultured with or without MECs [6, 7]. Cultures of salivary glands, liver, kidney, and brain from latently infected mice, however, have failed to yield MCMV in vitro [8]. Latently infected, heterotopic cardiac isografts are the source of lethal MCMV infection in the immunosuppressed recipient [9]. Results presented below demonstrate the activation of latent MCMV from cardiac explants and dissociated cardiac cell cultures when cultured alone or with allogeneic MECs. The Smith strain of MCMV was maintained in this laboratory by serial passage of CD-1 Swiss mice (Charles River Breeding Laboratory, Wilmington, Mass) followed by salivary gland harvest as described elsewhere [9]. The stock virus had a titer of 2 x 10' pfu/ml. Noninfected salivary gland homogenates (NSG) were prepared in a similar manner after inoculation of weanling mice with saline. Virus stocks and NSG were stored at 70 C until use. Latent infection was established by ip inoculation of five-weekold female BALB/c mice (Cumberland View Farms, Clinton, Tenn) with 104 pfu of MCMV (0.1 ml of infected salivary gland homogenate). These mice were then maintained in our animal facility for four to six months, at which time their hearts were used for tissue culture. Control animals had been injected with an equal volume of NSG and maintained for four to six months in the same way. These animals showed no evidence of virus by salivary gland culture or by antibody testing at the time of death. Explants of cardiac tissue were prepared by aseptically removing hearts from latently infected or NSG-inoculated mice and placing them in cold, lactated Ringers solution with 507o dextrose. Hearts were transferred into petri dishes containing fresh Ringers solution and cut into 1x 2-mm pieces that were kept on ice. Secondary MECs (prepared as previously described [9] from pathogen-free CD-1 Swiss mice) seeded the previous day in six-well plates (Falcon Plastics, Oxnard, Calif) containing RPMI medium with