Decreased immunogenicity of human fetal pancreas allografts following hyperbaric oxygen culture.

Human fetal pancreas (HFP) is a potential source of transplantable islets for the treatment of type 1 insulin-dependent diabetes mellitus (IDDM). Pretransplant culture techniques such as long-term culture, high-oxygen culture, UVB irradiation, and low-temperature culture have previously been used to reduce the immunogenicity of tissue for transplantation. In this study, we use hyperbaric oxygen culture (HOC) to modify MHC Class I expression on HFP and to reduce the immunological response of human peripheral blood mononuclear cells (PBMC) to HFP using a sponge matrix allograft model. To study the interaction of naïve PBMC with HOC-treated or untreated HFP allografts, sponges embedded with HFP tissue were implanted into the peritoneal cavity of NOD-SCID mice and injected with 1 x 10(7) freshly isolated human PBMC at the time of transplant. By day 14, human CD45 cells represented less than 2% of the cells recovered from the sponges implanted with HOC-treated HFP. In contrast, human CD45(+) cells represented nearly 15% (P =.0018) of the cells isolated from sponges implanted with conventionally cultured HFP grafts. Approximately 75% of the human CD45(+) cells from conventionally cultured HFP allografts were producing IFNgamma as determined by intracellular cytokine analysis. These data suggest that HOC treatment of HFP abrogates the activation and proliferation of PBMC. Pretransplant HOC treatment of islets is a simple technique that could be used to reduce immunogenicity and increase allograft survival while decreasing the requirement for immunosuppressive drugs.

1,25-(OH(2))D(3) alters the transforming growth factor beta signaling pathway in renal tissue.

BACKGROUND: 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) plays an important role in regulating immune responses, in addition to its effects on bone metabolism. The cytokine transforming growth factor beta (TGF-beta) regulates diverse biological processes, including cellular proliferation and differentiation, immune modulation, and modulation of extracellular matrix deposition. 1,25-(OH)(2)D(3) interacts in vitro with Smad proteins, important regulators of TGF-beta signal transduction. We hypothesized that exogenous 1,25-(OH)(2)D(3) would alter levels of TGF-beta(1) and TGF-beta(1) signaling proteins in renal tissue. METHODS: C57BL6 mice and Lewis rats were placed on diets with or without 1,25-(OH)(2)D(3) for 14 days. Renal lysates were examined for TGF-beta(1), vitamin D receptor (VDR), and Smad3 protein levels using a cell proliferation assay and Western blot analysis. Coimmunoprecipitation was used to determine if any interaction between VDR and Smad3 proteins occurs in vivo. Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess messenger RNA (mRNA) levels for all of these molecules. RESULTS: Vitamin D supplementation decreased VDR and Smad3 protein levels. Coimmunoprecipitation of VDR and Smad3 revealed a Smad3-VDR interaction in vivo. Vitamin D-treated rats had a significant (P = 0.001) reduction in bioactive renal TGF-beta(1). RT-PCR demonstrated no difference in mRNA expression for either VDR or TGF-beta(1). CONCLUSION: Our results suggest that vitamin D has a significant effect in regulating levels of bioactive TGF-beta(1) and appears to affect aspects of the TGF-beta(1) signaling system. These effects, in combination with the immunomodulatory actions of vitamin D, may alter the evolution of chronic rejection in renal transplants.

Role of CD4+ regulatory T cells in hyperbaric oxygen-mediated immune nonresponsiveness.

We have previously shown that hyperbaric oxygen culture (HOC [95% O(2), 5% CO(2), 25 psi]) is an effective pretransplant tissue-modification technique that results in long-term allograft survival and the induction of systemic immune tolerance in a murine model. Here we address the immune modulatory effects of HOC-treatment of human immune responses using the in vitro mixed lymphocyte reaction (MLR). Pretreatment of allogeneic stimulator cells with HOC results in abrogation of cytotoxic T lymphocyte (CTL) activity, proliferative responses, and IFN gamma production in a 7-day MLR. These responses can be restored either by the addition of IFN gamma or IL-2 on day 0, or by blocking the activity of IL-4 and IL-10. The addition of IL-2 on day 4 does not restore allospecific CTL activity. The failure of HOC-treated cells to induce allospecific CTL is not due to the induction of anergy, demonstrated by the failure to restore responses after restimulation with allogeneic cells in the presence of IL-2. Removal of CD4(+) cells prior to restimulation, results in restoration of CTL activity in MLR cultures restimulated with HOC-treated allogeneic cells. These results suggest that HOC-induced immune nonresponsiveness is mediated by the development of CD4(+) regulatory cells in a Th2-type environment.

Enhancement of thyroid allograft survival following organ culture: II. Induction of recipient peripheral tolerance.

Hyperbaric oxygen culture (HOC) prolongs endocrine graft survival and decreases major histocompatibility complex (MHC) class I surface expression. If graft prolongation were the result of passenger cell inactivation and decreased class I expression, then simultaneous transplantation of both a nontreated and a HOC-treated graft should result in rejection of the nontreated graft and acceptance of the HOC-treated graft. Simultaneous transplant of a nontreated and a HOC-treated thyroid allograft beneath the kidney capsule of recipient mice resulted in prolonged survival of both grafts in three strain combinations differing at class I (K(K), D(d), D(b)). In vitro analysis of the recipient splenic population revealed the presence of primed donor-specific cytotoxic T cells. These results suggest that recipient tolerance was not because of anergy or clonal deletion. Splenectomy at the time of transplant, revealed that both graft prolongation and the induction of recipient tolerance were dependent on the spleen. Finally, analysis of graft infiltrating cells reveals the presence of CD8+ cells but no CD4+ cells in tolerant recipients, whereas graft infiltrating cells from rejecting recipients contained both populations. The results suggest that active peripheral tolerance can be generated following transplantation of a HOC-treated allograft and that tolerance results from redirection of the recipients immune response.

Update on preclinical and clinical experience with mycophenolate mofetil.

As initially predicted by in vitro and animal model experiments, MMF appears to demonstrate significant potential as a new immunosuppressive drug. Early clinical trials have shown that MMF can be used effectively to prevent allograft rejection, and to treat allograft rejection, in heart, liver, and kidney transplant patients. MMF may also help alleviate allograft arteriosclerosis associated with chronic allograft rejection. Three advanced, multicenter, international phase III trials have now shown that MMF at dosages of 2 g/d or 3 g/d improved immunosuppression in renal allografts when compared with either placebo or azathioprine. Our own experience in one of these trials demonstrated that ATGAM induction therapy, followed by a maintenance triple therapy of corticosteroids, cyclosporine, and MMF, is a safe, effective, and well-tolerated regimen for the prophylaxis of acute renal allograft rejection.

From mice to man: the preclinical history of mycophenolate mofetil.

In vitro studies demonstrating that mycophenolic acid (MPA) held promise as a powerful immunosuppressant led to a series of in vivo studies to further evaluate this interesting new agent. Experiments in mice showed that MPA had powerful lymphocyte-selective immunosuppressive effects. A prodrug of MPA, mycophenolate mofetil (MMF), was developed to improve bioavailability on oral administration. Numerous tests demonstrated that MMF, alone or in combination with other immunosuppressives, prolonged allograft survival of various organs in several species and may be useful for reversing ongoing rejection. MMF also was found to be useful in prolonging xenograft survival. These animal experiments indicated that MMF had the benefit of relatively low toxicity and a good safety profile and drove early clinical trials of MMF. These trials showed that MMF is relatively safe and well tolerated in man and is of potential use for the prevention and treatment of acute allograft rejection. This article describes the preclinical history of the development of MMF, concentrating on the animal experiments and phase I and II trials that preceded the large-scale clinical testing of this new immunosuppressant.

A comparison of the effects of hyperbaric oxygen culture on survival of murine and canine thyroid gland grafts.

Canine thyroid tissue (CTy) was subjected to hyperbaric oxygen culture (HOC) under conditions that affect immunoalteration in murine thyroid tissue (MTy). Survival of autografts and allografts implanted under the kidney capsule was determined after 21 days by 125I uptake and histology. Unlike MTy, autograft CTy subjected to normothermic HOC (95% O2, 5% CO2; 1.76 kg/cm2) for 48 h did not survive (0/8) whereas decrease of culture duration to 24 h resulted in autograft CTy survival (3/3). Under hypothermia (5 degrees C), HOC could be extended to 7 days with autograft CTy survival (3/3 after 4 days and 3/3 after 7 days). Allograft CTy after 24 h of normothermic HOC and 7 days of hypothermic HOC was rejected. Indicators of oxygen free radical injury were determined:catalase activity was comparable in MTy and CTy (means 14.82 and 6.3-10.8 mm/mg protein, respectively) but superoxide dismutase activity was low in CTy (means 0.01-0.29 and 4.75 U/mg protein, respectively). Malondialdehyde content after 48 h of normothermic HOC was higher in CTy than in MTy (means 2215 and 1275 nmol/g, respectively). The results show that CTy is injured by HOC under conditions tolerated by MTy, and that this difference is related to the greater sensitivity of CTy to oxygen free radical injury.

Prolonged survival of murine thyroid allografts after 7 days of hyperbaric organ culture in the UW preservation solution at hypothermia.

Organ culture of murine thyroid allografts in hyperbaric oxygen (95% O2 at 25 psi, 37 degrees C) for 48 hr, results in prolonged allograft survival. Endocrine tissues can be cultured at 37 degrees C--however, this method may not be applicable to vascularized organs at normothermia. The aim of this study was to apply hyperbaric oxygen culture (HOC) under organ preservation conditions (hypothermia, UW solution) that have been shown to be successful in clinical organ transplantation. B10BR/SGSNJ murine thyroid lobes were transplanted beneath the kidney capsule of C57BL/10J recipients. Thyroids were cultured in Eagle's MEM at 37 degrees C (controls) and at 5 degrees C, under hyperbaric conditions (95% O2:5% CO2, 25 psi). Alternatively, thyroids were cultured in UW solution (+/- allopurinol/GSH) at 5 degrees C, for up to 7 days. Graft survival was determined 21 days posttransplant by 125I uptake and by histology. In Eagle's MEM, HOC at 37 degrees C/48 hr and 5 degrees C/7 days, resulted in 93% and 20% allograft survivals, respectively. In UW solution (- allopurinol/glutathione [GSH]), HOC at 5 degrees C/7 days resulted in 83% allograft survival: immunoperoxidase staining showed a decrease of MHC class I alloantigen expression. Oxygen free radical scavenger (allopurinol/GSH) addition to the UW solution diminished this effect and suggested an oxygen free radical-mediated mechanism in immunoalteration. These results demonstrate that HOC for 7 days reduced the antigenicity and immunogenicity of murine thyroid grafts under conditions that simulate organ preservation. Hypothermic hyperbaric oxygen culture conditions require testing in a higher animal species and in vascularized grafts to determine if this method can be applied to whole-organ transplantation.

Enhancement of thyroid allograft survival following organ culture. Alteration of tissue immunogenicity.

Hyperbaric oxygen (95%, O2, 25 psi, 48-hr culture) resulted in prolonged thyroid allograft (B10.A) survival in both primary and sensitized recipients (B10.AQR). Recipients receiving noncultured thyroid allografts uniformly rejected the graft by 35 days, while 100% of cultured grafts survived. Noncultured thyroid grafts transplanted to skin-graft-primed recipients were rejected by 21 days. In contrast, 86% of cultured grafts transplanted to primed recipients were still functioning at 35 days. Donor spleen cells or peritoneal exudate cells transferred at the time of thyroid transplant were unable to stimulate cultured allograft rejection. Allografts histologically examined 35 days after transplant revealed, in some grafts, focal cellular infiltrates adjacent to normal, uninfiltrated tissue. To determine if tissue modification was the mechanism of prolonged allograft survival, hyperbaric-oxygen-cultured thyroids were examined for MHC class I expression. Immunoperoxidase staining with monoclonal antibody to MHC class I molecules showed that cultured thyroids were unstained in contrast to fresh thyroids that were uniformly stained. Cytotoxic T lymphocytes specific for H-2Kk administered 10 days following thyroid transplant were unable to eliminate cultured grafts (80% survival) but completely destroyed noncultured grafts. These results indicate that hyperbaric oxygen culture altered MHC class I expression such that it was no longer detectable by monoclonal antibody or cytotoxic T lymphocytes. Thus, the mechanism, explaining graft prolongation after hyperbaric culture in addition to passenger cell depletion, may be alteration of graft antigenicity such that the graft is no longer perceived as foreign.

[Effect of active interferonization and anti-thymocyte globulin on the growth of virus-induced hamster melanoma A-Mel-S]. / Der Einfluss einer aktiven Interferonisierung und der Einfluss von Anti-Thymozyten-Globulin auf das Wachstum des Virus induzierten Hamstermelanoms A-Mel-S

The present data show that biological and synthetic inducers of interferon are able to reduce the growth of a transplantable malignant melanoma in the Syrian hamster. The advantage of the biological inducer H-MP are that it is not toxic for humans and that a high titer of interferon can be maintained over a long period of time.