Heterotopic and orthotopic liver transplants in inbred rat strains

The functioning and survival of orthotopic liver allotransplants in toto were compared with heterotopic partial and whole allotransplants in inbred rats. In contrast to the rapid and aggressive rejection of the heterotopic allografts, the orthotopic whole-liver allografts survived for prolonged periods. These latter grafts were subjected to mild cellular attack 11 to 12 days following transplantation, but they survived without the massive cellular infiltration and hepatocytolysis seen in the heterotopic partial grafts. Biliary secretion by the orthotopic whole-liver allografts was functional throughout the survival period and was markedly increased during the final stages of survival, whereas the heterotopic liver grafts ceased to excrete normal bile by day 9, after which they were completely depigmented.     

A 12-day course of FK506 allows long-term acceptance of semi-identical liver allograft in inbred miniature swine

BACKGROUND: Spontaneous tolerance to liver allograft has been reported previously in outbred pig models, but the lack of genetic background did not allow to analyze the impact of the major histocompatibility complex (MHC) on tolerance induction. A model of semi-identical liver allograft was therefore developed in inbred miniature swine in order to mimic the clinical situation of living related liver transplant (parent into infant) and to study a protocol for inducing tolerance to liver allograft. METHODS: SLAdd (class Id/d, class IId/d) pigs received orthotopic liver allograft from heterozygous SLAcd (class Ic/d, class IIc/d) miniature swine. Eight animals did not receive immunosuppression. Fourteen SLAdd animals had a 12-day course of FK506 and were divided in two subgroups. In subgroup FK-1, six pigs received a daily intramuscular injection of FK506 at 0.1-0.4 mg/kg, in order to reach daily trough levels between 7 and 20 ng/ml; in subgroup FK-2, eight additional animals received two daily injections of FK506 at 0.05 mg/kg regardless of the daily trough levels. Graft survival, liver biological tests, histology, cellular and humoral immune responses, as well as detection of microchimerism were assessed in all groups. RESULTS: All untreated animals rejected their allograft and died within 28.1 +/- 9.5 days. These rejector animals developed a significant anti-donor cellular and humoral immune response. No peripheral or lymphoid tissue microchimerism was detected in this group. In contrast, long-term survival was obtained in five FK-treated animals (112, 154, 406, 413, and 440 days), whereas several pigs died with a normal allograft function from either overimmunosuppression or intercurrent causes. All FK-treated pigs developed a specific anti-donor unresponsiveness in both cell mediated lymphocytotoxicity and mixed lymphocyte reaction and did not develop anti-donor alloantibodies. The study of the anti-donor immune response by mixed lymphocyte reaction, during the first postoperative week, demonstrated a specific anti-donor unresponsiveness in the peripheral blood from the first posttransplant day. Although microchimerism was detectable in the peripheral blood for several postoperative weeks (maximum 10 weeks) in FK-treated animals, donor cells or DNA were not detected during the long-term follow-up in peripheral blood or lymphoid tissues. CONCLUSIONS: Spontaneous tolerance to semi-identical orthotopic liver allograft did not occur, whereas a 12-day course of FK506 allowed long-term graft acceptance. All FK-treated animals developed in vitro signs of specific immune unresponsiveness and transient peripheral microchimerism. The specific anti-donor cellular unresponsiveness occurred on the first postoperative day after surgery and was of long-term duration. The study of the early immunological events in this model could be of major importance regarding clinical living related liver transplantation.     

中国小型猪原位肝移植的围手术期处理

目的 总结中国小型猪非转流条件下原位肝移植的围手术期特点,建立成熟、稳定的移植模型.方法 通过预实验组8例次和实验组18例次非转流条件下中国小型版纳猪原位肝移植术,观察围手术期血流动力学及重要代谢指标的变化,改进围手术期处理措施,总结围手术期处理特点.结果 预实验组8例次均在术后当天死亡.实验组18例次无术中死亡,术后7天存活率为88.9%.平均手术时间(179.6±14.3)min,平均无肝期(27.3±3.4)min.无肝期血流动力学及生化代谢波动明显.术后当天麻醉清醒,术后3～5 h开始自行排出小便,术后第三天正常进食.结论 无肝期手术时间的缩短及血流动力学的相对稳定是非转流条件下猪原位肝移植成功的关键.     

Modeling chronic lung allograft rejection in miniature swine

BACKGROUND: The success of lung transplantation has been limited by the perplexing problem of chronic rejection. The development of a large-animal model for the systematic study of the mechanisms underlying chronic lung rejection has been problematic. We have developed a new preclinical model of chronic lung rejection using MHC-inbred miniature swine. METHODS: Using standard operative techniques, four orthotopic left lung allografts were performed using MHC-matched, minor-antigen-mismatched donors. Recipient animals received a 12-day course of postoperative cyclosporine. Grafts were followed with open biopsies and high-resolution computed tomography. Cellular immune responses were monitored by mixed lymphocyte reaction, cytometric analysis of graft-infiltrating lymphocytes, and skin grafting. RESULTS: All grafts survived > or = 5 months and developed manifestations of chronic rejection, including obliterative bronchiolitis, interstitial fibrosis, and occlusive vasculopathy. A mononuclear infiltrate was also present in all grafts by the fourth posttransplant month. High-resolution computed tomography demonstrated several cardinal radiographic findings known to correlate with chronic rejection. Cytometric analysis of graft-infiltrating lymphocytes showed a predominance of CD8+ cells. The development of alloreactivity in the host was confirmed by mixed lymphocyte reaction and skin grafting. CONCLUSIONS: We report a reproducible, whole-lung, large-animal model of chronic lung rejection. In this immunogenetically defined construct, we have observed a full spectrum of histopathologic lesions that reproduce with fidelity those lesions observed in human lung transplant recipients suffering from chronic rejection. We anticipate that this preclinical model will facilitate further study of the pathogenesis and therapy of chronic lung rejection.     

近交系大鼠DA到LEW肝移植急性排斥模型的特点

目的 探讨近交系大鼠DA到LEW原位肝移植模型的特点并判断排斥反应发生的强度.方法 采用Kamada二袖套法,利用封闭群大鼠SD和Wistar进行建模技能训练,在此基础上建立近交系大鼠DA到LEW肝移植模型,根据临床表现和Banff标准判断排斥反应发生的强度.结果 共施行DA到LEW大鼠肝移植模型15例,手术成功率86.7%,死亡原因为肝上下腔静脉漏血、肝下下腔静脉血栓、胆道梗阻.术后第3天开始出现排斥反应,7 d后逐渐达到高峰,除术后并发症致死外,其余均在12 d内死于Ⅲ级排斥反应.结论 DA到LEW为稳定、强烈的大鼠肝移植急排模型,是研究肝移植排斥反应及免疫耐受的理想动物模型.但近交系大鼠在组织结构上有其自身特点,给建模带来一定难度.     

Spontaneous long-term acceptance of RT-1-incompatible liver allografts in inbred rats. Analysis of the immune status

In several combinations of inbred rats, liver allografts are spontaneously tolerated, and after a few weeks liver tolerant rats are in a state of donor-specific transplantation tolerance. In vivo and in vitro experiments were conducted to analyze the immunological status of LEW or BN rats with spontaneously tolerated (LEW X BN) F1 liver allografts several months after transplantation. Acute rejection of secondary donor-specific heart allografts retransplanted from liver-tolerant rats to normal syngeneic hosts suggests that the state of tolerance in liver-tolerant rats is related to an active modification of the immune system of the rat and not to a reduced immunogenicity of the graft. No cytotoxic antibodies or cells were found in liver-tolerant rats. Reactivity in mixed lymphocyte culture was normal or slightly reduced. Arguments for the presence of splenic suppressor cells were found in LEW tolerant rats using a local graft-versus-host assay, but these could not be found in BN rats, or when attempting to transfer or to break the tolerance state. A nonspecific humoral blocking factor was found in vitro in liver-tolerant rats but transfer of serum from liver-tolerant rats to normal syngeneic hosts did not permit a significant prolongation of donor-specific heart allografts. These results suggest that more than one mechanism may be involved at the maintenance phase of liver allograft tolerance.     

小型猪非转流原位肝移植的围手术期处理

目的:探讨中国小型猪非转流条件下原位肝移植的围手术期特点，以建立成熟、稳定的移植模型。方法:通过预实验组8例次和实验组18例次非转流条件下中国小型版纳猪原位肝脏移植术，观察围手术期血流动力学及重要代谢指标的变化，改进围手术期处理措施，总结围手术期处理特点。结果:预实验组8例次均在术后当天死亡。实验组18例次无术中死亡，术后7d存活率为88.9%。实验组平均手术时间（179.6±14.3）min，平均无肝期（27.3±3.4）min。无肝期血流动力学及生化代谢波动明显。术后当天麻醉清醒，术后约3～5h开始自行排尿，术后第3天正常进食。结论:手术中无肝期时间的缩短及血流动力学的相对稳定是非转流条件下猪原位肝移植成功的关键。     

Chronic rejection of liver transplants revisited

We examined 27 hept-ectomy specimens to assess the frequency of foam cell endovasculitis and bile duct loss in chronic rejection. Arterial lesions, defined as total occlusion by subintimal foam cells and/or fibromuscular proliferation, were found mainly in hilar and septal arteries, whereas bile duct loss, defined as the absence of bile ducts in more than 50% of portal tracts, affected aminly small tracts. Both were found in 20 livers (74%). In two livers (7%) there was significant bile duct loss but no arterial lesions, whilst in five cases (19%) there were occlusive arterial lesions but no bile duct loss. Small arteries were involved in only 10% of the cases. These results indicate that in one-third of the cases arterial and bile duct lesions develop independently of each other, suggesting different pathogenetic pathways. In addition, liver biopsy may not be pathognomonic since small arteries are involved in only 10% of cases and bile duct loss may not be extensive. In such cases the diagnosis of chronic rejection should only be made in the presence of progressive clinical deterioration.     

Posttransplant lymphoproliferative disorder after liver transplantation in miniature swine.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a well-known, life-threatening complication of immunosuppressive therapy, occurring in both adult and pediatric transplant recipients. METHODS: To study the effect of major histocompatibility complex on tolerance induction to primarily vascularized liver allograft, a semi-identical miniature swine model was developed to mimic the clinical situation of parent-into-infant liver transplantation. Long-term acceptance of semi-identical liver allograft was obtained by a transient course of FK506. In a subgroup of six animals, three developed a lethal PTLD. These animals were studied by histology and immunohistochemistry and the anti-donor cellular immune response was assessed. In addition, the possible viral origin of the proliferative process was evoked. RESULTS: Histology and immunohistochemistry revealed an abnormal B-cell proliferation in many organs of swine suffering from PTLD. Evidence of human Epstein-Barr virus, cytomegalovirus, and adenovirus was not evidenced, but a porcine virus responsible for a respiratory and reproductive syndrome (PRRS) was identified in the lymphoid tissue of these animals. In mixed lymphocyte reaction, a significant antiself immune response confirmed an infection by a virus. CONCLUSIONS: This is the first report suggesting that PRRS virus might provoke PTLD in immunosuppressed miniature swine after orthotopic liver transplantation. Whether PTLD could be induced by injection of the PRRS virus in immunosuppressed animals, a pig model of PTLD might be developed and would represent an interesting preclinical model for testing anti-PTLD therapies.     

Early biological and immune response to semi-identical liver or kidney allograft in miniature swine

In inbred miniature swine, semi-identical liver allograft recipients survive up to 3 months without immunosuppression, whereas similarly mismatched kidney allografts are uniformly rejected within 2 weeks. The early biological and immunological events were assessed in this unique model. SLA(d/d) pigs (MGH, Harvard Medical School, Boston, MA, USA) received liver or kidney allograft from heterozygous SLA(c/d) miniature swine. Survival, graft function, histology, intragraft cytokines, peripheral lymphocyte and platelet count, plasma cortisol level and cellular/humoral anti-donor immune response were assessed. Kidney allografts were uniformly rejected within 2 weeks, whereas liver allografts survived for up to 87 days. After both liver and kidney transplantation, the peripheral lymphocyte count decreased during the first week concomitantly to a significant elevation of plasma cortisol level. Early decrease of peripheral platelet count was observed after liver but not renal transplantation. Up-regulation of transforming growth factor beta1 (TGF-beta1) and interferon-gamma (IFN-gamma) was observed during the first postoperative week in semi-identical liver allografts and IFN-gamma as well as IL-10 in kidney allografts. In liver recipients, labelled autologous lymphocytes accumulated in the liver graft and native spleen, whereas after renal allograft, lymphocytes accumulated in the native spleen and liver but never in the kidney allograft. Specific cellular anti-donor unresponsiveness was observed from the first post-transplant day in both liver and kidney recipients, while the humoral anti-donor response remained intact. In semi-identical liver allograft, recipient rejection is milder and slower than in similarly matched kidney allograft. The intragraft up-regulation of TGF-beta1 in semi-identical liver allograft might be one mediator to explain the modulation of rejection after liver transplant. The rapid, nonspecific accumulation of recipient lymphocytes in the liver allograft but not in kidney allograft might also play a role in the different survival time in this model.     

The prediction of acute cellular rejection in orthotopic liver transplantation.

The occurrence of acute cellular rejection after orthotopic liver transplantation is common. At present, no allowance is made in immunosuppressive regimens for parameters other than weight. We investigated parameters in 121 consecutive patients receiving their primary allograft to determine if there are pretransplantation factors predicting the occurrence of acute cellular rejection after transplantation. The case notes and dietetic notes of these patients were reviewed for age at transplantation, cause of liver disease, preoperative albumin and creatinine levels, lymphocyte count, anthropometric measurements, donor age, HLA DR mismatch, and cold ischemia time. Acute cellular rejection was more likely to occur in younger patients, patients with Child's class A disease, and those with normal midarm muscle circumference. Acute rejection was increased in transplant recipients from donors aged younger than 30 and older than 50 years. Acute cellular rejection was less likely to occur in patients who underwent transplantation for alcoholic liver disease. Chronic rejection was significantly increased in women and those patients who experienced recurrent acute rejection. On multivariate analysis, the only significant predictor was the decreased likelihood of acute rejection in patients with depleted midarm muscle circumference. In conclusion, it may be possible to individualize immunosuppressive regimens on the basis of pretransplantation characteristics.     

Late-onset acute rejection in orthotopic liver transplantation--associated risk factors and outcome.

A retrospective review of 375 consecutive orthotopic liver transplants was performed to determine the incidence and outcome of late rejection episodes ([LR] rejection occurring more than 6 months following transplant). A total of 31 episodes in 26 patients were identified. Eighteen of these episodes were associated with subtherapeutic levels of cyclosporine. Of these, 7 were due to noncompliance, 2 were due to biliary strictures, and 1 was due to malabsorption in a cystic fibrosis patient. All 31 episodes were treated initially with steroids, and 22 had a complete response, although one progressed to chronic rejection over a year later. Of the remaining 9, 1 received FK506 with a complete response, and 8 received OKT3. Of the 8 patients who received OKT3, 5 had a complete response, 1 received RS61443 following OKT3 and progressed to chronic rejection, and the remaining 2 received further steroids. Of these 2, 1 had a complete response following the steroids while the second was converted to FK506 with a complete response. Compared with 315 acute rejection episodes ([AR] occurring less than 6 months posttransplant), patients with late rejection episodes had an equivalent response to steroids (63.2% AR reversed vs. 71% LR reversed) but a lower response rate to OKT3 (91.5% AR reversed vs. 62.5% LR reversed). There was, therefore, a higher rate of persistent rejection (61% AR episodes vs. 15.4% LR episodes) but no increase in the incidence of chronic rejection (7% AR episodes vs. 7.7% LR episodes). We conclude that LR is a relatively common occurrence following liver transplant, which is most often associated with low cyclosporine levels. Many of these episodes are due to noncompliance, but biliary problems must also be investigated. The incidence of resistant rejection is higher in this group of patients but is not associated with a concurrent increase in chronic rejection.