Role of the thymus in transplantation tolerance in miniature swine. III. Surgical manipulation of the thymus interferes with stable induction of tolerance to class I-mismatched renal allografts.

BACKGROUND: Previous studies have demonstrated that long-term tolerance of class I mismatched renal allografts in miniature swine is induced by a short course of cyclosporine (CyA), and that a total thymectomy 21 days before transplantation abrogates the induction of stable tolerance. We have now examined the effects of surgical manipulation of the thymus, with or without a reduction in the thymic volume, on the induction of tolerance. MATERIALS AND METHODS: Miniature swine receiving a transplant of a class I-mismatched renal allograft and 12 days of CyA underwent either (1) a partial thymectomy 21 days before kidney transplantation (day -21), (2) serial thymic biopsies (to evaluate the effect of surgical trauma and reduction in volume of the thymus) or serial incisions of the thymus thymus (to evaluate the effect of surgical trauma without changes in thymic volume), (3) a sham thymectomy on day -21, or serial sham thymic surgery on the same POD as the thymic biopsies and incisions (control animals). RESULTS: Control animals had a stable plasma creatinine, had donor-specific unresponsiveness in cell-mediated lympholysis (CML) assays, had absence of rejection in kidney biopsy specimens, and did not develop anti-donor class I immunoglobulin (Ig)G alloantibodies. Animals undergoing a partial thymectomy on day -21 or serial thymic biopsies showed severe renal dysfunction, histological evidence of rejection in kidney biopsy specimens and anti-donor reactivity in CML assays; all but one animal developed anti-donor class I IgG alloantibodies. Serial incisions of the thymus induced an increase in plasma creatinine and histological rejection in 1 of 3 animals and anti-donor cytotoxic T cells in vitro in all 3 animals. CONCLUSIONS: A partial thymectomy or serial thymic biopsies markedly interfere with the induction of tolerance to renal allografts. Serial thymic incisions also interfere with the induction of tolerance, but to a lesser degree. These studies may have implications for tolerance-inducing protocols that involve thymic manipulation.

Role of the thymus in transplantation tolerance in miniature swine: II. Effect of steroids and age on the induction of tolerance to class I mismatched renal allografts.

BACKGROUND: Recent studies in young (5-7 months) miniature swine have demonstrated that the thymus is involved in the rapid induction of stable tolerance to class I mismatched renal allografts after a 12-day course of Cyclosporine (CyA). Because both steroids and age are known to influence the structure and function of the thymus, we have now studied the effects of these two parameters on tolerance induction in this model. MATERIALS AND METHODS: In young swine, the administration of methylprednisolone (MP) during the standard tolerance-inducing regimen (a 12-day course of CyA) produced severe renal dysfunction and acute cellular rejection histologically. However, the renal allografts recovered and were accepted for >100 days with histological evidence of chronic rejection. To test the effect of age, two relatively old swine (55 and 71 months) received transplants of class I mismatched renal allografts and the standard 12-day course of CyA. One animal rejected the allograft acutely on postoperative day 22, and the second also rejected, but more slowly, with manifestations of chronic rejection. CONCLUSION: These findings suggest that both MP and old age interfere with the induction of stable tolerance in a fashion similar to the previously described effect of thymectomy. These results may have important implications for the mechanism of thymic-dependent tolerance, for the use of steroids in clinical protocols for the induction of allograft tolerance, and for the application of such protocols to adult patients.

Basic anatomical and physiological differences between species should be considered when choosing combinations for use in models of hepatic xenotransplantation: an investigation of the guinea pig-to-rat combination.

Published data on the guinea pig-to-rat hepatic xenotransplant model describe problems concerning poor graft reperfusion. To further investigate this phenomenon, orthotopic liver xenotransplantation between weight-matched guinea pigs and rats were performed using Kamada's technique. On reperfusion, all cases had portal venous inflow block with hypoperfusion of the hepatic parenchyma. Histological examination showed no evidence of hyperacute rejection, although deposits of IgG2a and C3 but not IgM were identified within the central area of the liver. To increase blood inflow, arterialized partial liver grafts were performed without changing the outcome. We hypothesize that the hypoperfusion may be related to anatomical and physiological differences between the species. Guinea pig portal vein branches were found to have muscular walls susceptible to spasm, and portal blood flow is four times greater in the guinea pig than in the rat because the guinea pig intestine is both longer (two times as long) and of greater diameter. The combination of reperfusion injury, early immunological events, and the rat's lower portal blood flow induces spasm of the intrahepatic portal system resulting in hypoperfusion. These findings demonstrate the importance of recognizing basic anatomical and physiological differences between species when selecting xenotransplantation models.

Role of the thymus in transplantation tolerance in miniature swine. I. Requirement of the thymus for rapid and stable induction of tolerance to class I-mismatched renal allografts.

The almost uniform failure in transplant patients of tolerance-inducing regimens that have been found to be effective in rodents, has made it necessary to examine large animal models before testing of new approaches clinically. Miniature swine have been shown to share many relevant immunologic parameters with humans, and because of their reproducible genetics, have proved extremely useful in providing such a large animal model. We have previously shown that indefinite systemic tolerance to renal allografts in miniature swine is induced in 100% of cases across a two-haplotype class I plus minor histocompatibility antigen disparity by a 12-d course of Cyclosporine A (CyA), in contrast to irreversible rejection observed uniformly without CyA treatment. In the present study, we have examined the role of the thymus during the induction of tolerance by performing a complete thymectomy 21 d before renal transplantation. This analysis demonstrated a striking difference between thymectomized and nonthymectomized animals. Thymectomized swine developed acute cellular rejection characterized by a T cell (CD25(+)) infiltrate, tubulitis, endothelialitis and glomerulitis, and anti-donor CTL reactivity in vitro. Nonthymectomized and sham thymectomized animals had a mild T cell infiltrate with few CD25(+) cells and no anti-donor CTL response in vitro. These results indicate that the thymus is required for rapid and stable induction of tolerance.

Pig to monkey bone marrow and kidney xenotransplantation.

BACKGROUND: The intensity of discordant xenograft cellular rejection makes it unlikely that safe doses of immunosuppressive drugs will alone be sufficient to permit long-term survival. We have therefore concentrated our efforts on establishing tolerance to xenogeneic organs through lymphohematopoietic chimerism and the elimination of preformed natural antibodies (nAbs). METHODS: Here we report the most recent series of 11 technically successful porcine to nonhuman primate transplantation procedures. In eight experimental animals induction therapy consisted of (1) 3 x 100 cGy nonlethal whole body irradiation (day -6 and day -5) to all animals, (2) horse anti-human thymocyte globulin (day -2, day -1, and day 0) to seven of the animals, (3) 700 cGy thymic irradiation (day -1) to five of the animals, and (4) pig bone marrow infused on day 0 (2-9 x 10(8)/cells/kg). On day 0, just before the renal xenograft, the recipient was splenectomized, and antipig nAbs were removed by means of perfusion of the monkey's blood through either a pig liver (n = 6) or a Gal-alpha (1,3)-Gal adsorption column (n = 5). There control animals did not receive this pretransplantation induction therapy but did undergo hemoperfusion and posttransplantation immunosuppression identical to the experimental animals. All 11 recipients were treated after transplantation with cyclosporin A and 15-deoxyspergualin. Recombinant pig-specific growth factors (interleukin-3 and stem cell factor) were given to six experimental animals from day 0 until the termination of the experiment. RESULTS: Analysis of recipients' sera by means of flow cytometry indicated the effective removal of immunoglobulin M and immunoglobulin G nAbs by either liver perfusion or column adsorption. In the eight experimental animals, nAb titers remained low until death (up to 15 days), but in the three control animals nAb titers increased substantially with time. The longest surviving recipient maintained excellent kidney function with creatinine levels at 0.8 to 1.3 mg/dl throughout its course. Death occurred at day 15 from complications caused by a urinary leak and pancytopenia. Histologic examination of the xenograft revealed only focal tubular necrosis and cytoplasmic vacuolization, with trace amounts of fibrin and C3 in peritubular capillaries. In this animal a fraction of the peripheral blood cells (3%) at day 7 were of pig origin as detected by pig-specific monoclonal antibodies. In addition, colony-forming assays performed on a bone marrow biopsy specimen taken at day 14 indicated that approximately 30% of the relatively few myeloid progenitors detected were of swine origin. CONCLUSIONS: We have demonstrated that our protocol is effective in the prevention of hyperacute rejection and in the maintenance of excellent function of the renal xenograft for up to 15 days. These results also indicate that at least short-term engraftment of the xenogeneic donor bone marrow cells is possible to achieve in this discordant large animal combination. Longer survivals will be required to assess the possible effect of this engraftment on induction of tolerance.

Molecular and cellular events implicated in local tolerance to kidney allografts in miniature swine.

Long-term tolerance to class I-mismatched renal allografts can be induced in miniature swine by treatment with a short course of cyclosporine (CsA). Kidney recipients treated with CsA and untreated control kidney recipients both demonstrated infiltration of the transplanted kidney by mononuclear cells, which reached a maximum between postoperative days 8 and 11. Recipients that did not receive the tolerizing regimen rejected their grafts between postoperative days 8 and 12 in this model. The kinetics of cytokine gene expression, including interleukin (IL)-1alpha, IL-1beta, IL-2, IL-6, IL-10, tumor necrosis factor, and interferon-gamma (IFN-gamma), within the grafted kidney of rejector and acceptor animals, were determined using Northern blot hybridization. A strong correlation between rejection and up-regulation of the IFN-gamma gene was observed, whereas animals with long-term tolerance showed low levels of IFN-gamma, but high levels of IL-10 gene transcription. None of the other cytokine genes demonstrated a reproducible pattern of expression that correlated with acceptance/rejection of allografts. Analysis of transcription patterns of cytokine genes in mononuclear cells purified from renal grafts confirmed the initial observations made on biopsies. The phenotype of graft-infiltrating cells (GIC) showed a dominance of CD8+ cells, with an average of 66% single-positive cells and 19% CD4/CD8 double-positive cells, compared with 30% and 14%, respectively, for peripheral cells. Predominance of CD8+ GIC was dictated neither by the MHC antigen disparity nor the rejector/acceptor status. These results, therefore, suggest that GIC represent a regulated combination of mononuclear cells producing local immune mediators that, in part, control the fate of allografts in this large animal model.

Effect of major histocompatibility complex matching on the development of tolerance to primarily vascularized renal allografts: a study in miniature swine.

Prevention of rejection and the induction of transplantation tolerance are two related but separable phenomena that must both be considered in the analysis of the response to a transplanted organ. It is frequently hard to separate these phenomena in assessing the outcome of clinical transplants, because patients are rarely studied in the absence of immunosuppressive agents. Use of our partially inbred miniature swine has permitted us to examine the effects of selective MHC matching on transplant survival, and the data indicate that matching has an effect on both phenomena. Prevention of early rejection with CyA was possible for all mismatches examined, although it was clearly more difficult with increasing degrees of mismatching. On the other hand, tolerance induction after cessation of the immunosuppressive agent was dependent on presence of at least one matched MHC locus between the donor and recipient, with complete class II matching appearing to be the most successful way of assuring long-term graft survival. It is also apparent from our data that although durable tolerance to primarily vascularized renal allografts could be induced across a variety of selective MHC disparities, all cases involving a class II mismatch (ie, selective class I matched or one-haplotype full MHC mismatched kidney allografts) underwent spontaneously reversible rejection crises during the early follow-up period. Such a clinical course might be unacceptable for human clinical trials, even though the transient renal dysfunction may reflect events involved in tolerance induction rather than true rejection (Gianello et al: Immunol Rev 133:19, 1993.). Indeed, we do not yet know whether or not further immunosuppressive treatment at the times of such crises may prevent rather than facilitate the induction of tolerance. On the other hand, in the case of selective two-haplotype class I mismatch the regimen utilized was capable of inducing tolerance to renal allografts in 100% of the recipients with minimal or no renal dysfunction throughout the follow-up period. Although the excellent results achieved with current antirejection agents has led to debate about the wisdom of HLA matching for cadaver transplants in terms of preventing rejection, our data would suggest that such matching might be of even greater importance for success of protocols in which attempts are made to induce transplantation tolerance. Because class II antigens are less polymorphic than are class I antigens, mismatching for class I antigens may be achievable for cadaver donor transplantation, and may provide the first situation in which these principles can be applied to clinical trials.

Long-term acceptance of primarily vascularized renal allografts in miniature swine. Systemic tolerance versus graft adaptation.

We have previously demonstrated that tolerance to two-haplotype class I-mismatched renal allografts can be induced uniformly by a short course of cyclosporine. We report here that following transplant nephrectomy, 8 such long-term acceptor animals all accepted a second renal transplant MHC matched to the original donor without additional immunosuppression. These results indicate that the mechanism of tolerance to primarily vascularized renal allografts involves modification of the host's immune system by the first transplant. To assess the possibility that "graft adaptation" is also involved in the maintenance of tolerance, we retransplanted class I-disparate kidneys from tolerant animals into naive recipients MHC matched to the original recipient. Three of 4 such transplants were rejected acutely, while one animal demonstrated a markedly prolonged survival, but also eventually rejected. These results, therefore, demonstrate that: (1) graft adaptation is not required in order to maintain tolerance; (2) graft acceptance involves induction of systemic tolerance; and (3) graft adaptation may participate in kidney graft prolongation but is not sufficient to transfer tolerance to a secondary host.

Induction of tolerance to renal allografts across single-haplotype MHC disparities in miniature swine.

We have previously demonstrated that a 12-day course of cyclosporine A (CsA) leads to the induction of tolerance to renal allografts in 100% of recipients selectively mismatched at class I for both haplotypes, and in 71% of recipients selectively mismatched at class II for both haplotypes, but in 0% of recipients mismatched for two haplotypes at both class I and class II. We have postulated that the mechanism by which tolerance is induced may therefore require matching for either class I or class II antigens. One might predict from this hypothesis that tolerance would also be induced in donor-recipient combinations sharing one full haplotype (e.g., AC-->AD), which mimics the clinically relevant transplant combination of parent to offspring. We have therefore investigated the effects of the CsA regimen on renal transplants in this combination. Without immunosuppression, such kidney allografts were uniformly rejected (n = 12; 10.6 +/- 2.4 days). In contrast, a course of CsA (10-13 mg/kg/day) during the first 12 postoperative days induced long-term acceptance of the allograft in 67% (4/6) of recipients. Some acceptor animals also showed specific unresponsiveness to donor antigens as measured by in vitro assays and by failure to develop anti-donor antibodies. Tolerance was confirmed in four of these animals by failure to reject a second transplant SLA-matched to the first kidney donor without additional immunosuppression. These results suggest the feasibility of inducing specific tolerance across a single-haplotype mismatch in the majority of the cases, which could have clinical implications for living-related transplants.

Tolerance to class I-disparate renal allografts in miniature swine. Maintenance of tolerance despite induction of specific antidonor CTL responses.

Miniature swine that become tolerant to renal allografts across an MHC class I barrier following a short course of cyclosporine are unresponsive to donor class I antigens in cell-mediated lymphocytotoxicity. However, skin grafts bearing donor class I plus third-party class II antigens are promptly rejected, and the animals then develop marked cell-mediated lymphocytoxic reactivity to donor class I antigens in vitro, but do not reject the kidney transplants. We show here that CTL generation is directed toward the same donor class I antigens as are expressed by the kidney donor, and is not the result of recognition in vitro of the tolerated class I antigen plus peptides of minor antigens shared between the skin graft donor and the stimulator/target cells. We also show that detection by CTLs of peptides expressed by skin but not by kidney is also not a sufficient explantation of the results, since the survival of skin grafts from the kidney donor is also prolonged, even after precursor CTL can be detected in vitro. The data are most consistent with suppression in vivo in tolerant animals of the helper pathways necessary for activation of precursor CTLs. Differences in patterns of cytokine expression by graft infiltrating cells may provide a mechanism for local suppression of help in this model. Finally, we have examined antibody production after sensitizing by skin grafts in long-term tolerant animals and have found that anti-donor class I antibodies are not produced, even though the same animals produce both anti-class II and anti-third-party class I antibodies.

Mechanism of cyclosporin-induced tolerance to primarily vascularized allografts in miniature swine. Effect of administration of exogenous IL-2.

Indefinite tolerance to kidney allografts across a two-haplotype class I disparity can be induced in miniature swine in 100% of cases by a short course of cyclosporin A (CyA). Without CyA, all recipients reject kidney allografts within 2 wk. These animals therefore provide a unique opportunity to study the mechanisms of induction and maintenance of tolerance in a large animal model. Previous studies of cellular and humoral immunity suggested that a T cell help deficit at the time of the first exposure of the host's immune system to alloantigens was involved in tolerance induction. We have now studied the effect of exogenous T cell help in the form of an IL-2 infusion during both the induction and maintenance phases of tolerance. Lymphoid infiltrates were seen in class I mismatched renal allografts by day 8 in all animals whether treated with CyA or not. Administration of i.v. IL-2 on postoperative days 8, 9, and 10 to animals receiving the full CyA tolerizing regimen led to acute rejection in four of four animals. These rejecting animals showed induction of IL-2R expression on graft infiltrating cells in the kidney, suggesting that the infiltrates present before IL-2 administration were capable of causing rejection once T cell help was provided. Treatment with IL-2 did not abrogate long-term tolerance. Thus, limitation of T cell help at the time of first exposure to Ag in this model appears to be required to prevent rejection during the time required for active tolerance to develop. Once established, this tolerance does not appear to require continuous limitation of T cell help to be maintained, suggesting loss, inactivation, or suppression of the cells capable of causing rejection.

[Aneurysms of the celiac trunk branches. Clinical study of 9 patients and review of the literature]. / Les anévrismes des branches du tronc coeliaque. Etude clinique de neuf patients et revue de la littérature.

During the period 1970-1983, nine patients presented with ten aneurysms of a branch of the coeliac trunk. An exact diagnosis, in these series, was based on a CT abdominal scanning with injection of contrast medium and on a selective visceral angiography. One patient with an inoperable pancreatic cancer was not submitted to surgery. Ligation of the alimentary artery of the aneurysm in the remaining 8 patients, had to be completed in three of them by a pancreatic resection. The non development of collateral hepatic circulation and the presence of segmental portal hypertension, necessitate an hepatic revascularisation (1 pat.) and splenectomy (2 pat.) in these series. Hospital mortality of all treated patients was nihil, the long term follow-up (5 years) excellent.

Pancreaticoduodenal resection. Surgical experience and evaluation of risk factors in 103 patients.

From an institutional review of 103 pancreaticoduodenal resections (PDRs) performed during the period 1968-1981, risk factors and selection criteria of this procedure were evaluated. A total of 43.7% of the patients were operated on for benign lesions, mainly right-sided chronic pancreatitis (35%); 56.3% of the interventions were performed for malignant disease, mainly carcinoma of the periampullary region. Despite the absence of any selection, the hospital mortality in 103 consecutive PDRs only reached 10.6% for the whole group (11/103 patients) and 8.3% for the elective group (8/96 patients). Mortality was significantly influenced by age barrier over 65 years (p less than 0.0001) and by urgent degree of surgery (p less than 0.03). All three patients with renal insufficiency had a fatal outcome after PDR. A total of 19.4% of the patients (20/103 patients) developed a surgical complication. The most important complication was pancreatic fistula (15/103 patients, 14.5%) responsible for all digestive-related fatal outcomes (six patients). Surgical treatment of pancreatic fistula (10 patients) is compromised by a high morbidity and a high mortality rate (50%). Postoperative morbidity as well as the incidence of the pancreatic fistula were significantly influenced by the age of the patients over 65 years (p less than 0.01 and less than 0.001, respectively), and by the serum bilirubin level over 6 mg/dl (p less than 0.002). The poor quality of the pancreatic tissue (p less than 0.03) and the urgent degree of the intervention (p less than 0.03) also raised the incidence of pancreatic leakage. Morbidity rate was more important in the malignant disease group (p less than 0.05). Corrected 5-year actuarial survival after PDR is excellent for ampullary cancer, moderate for chronic pancreatitis, and extremely poor for pancreatic and bile duct carcinoma. The decision to perform PDR should be taken after evaluation of the aforementioned risk factors: the emergency, age, serum bilirubin, quality of pancreatic tissue and renal insufficiency, underlying disease, and psycho-social status of the patient.