Graft-versus-host disease in fully allogeneic small bowel transplantation in the rat

Small bowel and its mesentery contain considerable amounts of lymphoid tissue that can mediate graft-versus-host disease in small bowel transplant (SBT) recipients. Present studies determined the existence of GVHD in a fully allogeneic SBT model and examined the effect of donor pretreatment with ALS in eliminating GVHD. Adult male Lewis (Lew) rats received orthotopic small bowel transplants from untreated (LewxBN)F1 (LBNF1) donors (group 1) or Brown Norway (BN) donors that were untreated (group 2) or pretreated with ALS (days -2 and -1) (group 3). All recipients were treated with cyclosporine 15 mg/kg/day i.m. on days 0-6 postoperatively. Animals were weighed and examined daily for signs of rejection and GVHD. No animals in groups 1 or 3 showed any physical signs of GVHD, but all of those in group 2 had characteristic weight loss, diarrhea, and dermatitis between 4 and 6 weeks postoperatively, from which they all recovered. Histologic examination of skin and spleen at this time confirmed the presence of GVHD. The relative spleen weight [( spleen weight/body weight] x 100) of group 2 animals was also significantly greater than that of unoperated control Lew animals. Spleen cells obtained from group 2 animals at the time of subclinical GVHD, but not cells from group 1 or 3 animals, caused enlargement of popliteal lymph nodes when they were injected into the footpads of Lew rats. This study shows that GVHD can manifest itself in recipients of a fully allogeneic small bowel transplant even when rejection is prevented by effective immunosuppression with CsA. However, combined use of recipient treatment with CsA and pretreatment of donor animals with ALS eliminates all manifestations of GVHD.     

Stress responses in graft and native intestine after rat heterotopic small bowel transplantation

BACKGROUND: Cardiac transplantation has been shown to induce heat shock protein expression, and reactivity to these stress proteins has been implicated in acute and chronic allograft rejection. This study assessed Hsp60 and Hsp70 expression in graft and native small intestine after rat small bowel transplantation. METHODS: Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats, a subgroup of which received tacrolimus immunosuppression (1 mg x kg(-1) x day(-1)). Untransplanted and isografted (PVG-->PVG) animals served as controls. Paraffin sections of graft and native intestine on day 5 after transplantation were stained by immunohistochemistry, and heat shock protein expression was graded blindly by three observers. RESULTS: Villus epithelial cell expression of Hsp60, but not Hsp70, was increased in allografts. The induction of Hsp60 in the villus epithelium was not controlled by tacrolimus. Hsp60 and Hsp70 expression was induced in the lamina propria of isografts and allografts. This response was more pronounced in allografts and was significantly reduced, but not totally abrogated, by tacrolimus. Interestingly, heat shock protein expression was also induced in the native intestine lamina propria and epithelium of allograft recipients, suggesting the induction of stress responses at sites other than the transplanted organ. CONCLUSIONS: Small bowel transplantation induces a stress response in both the graft and native intestine. The early and prolonged expression of these proteins may influence the induction of anti-heat shock protein reactivity and have an adverse effect on graft outcome after small bowel transplantation.     

Persistence of allogeneic cells in graft and host tissues after small bowel transplantation.

Small bowel transplantation is associated with a significant risk of graft versus host disease owing to the large amount of organized lymphoid tissue within the graft. This study assessed whether graft lymphoid cells could persist in the long term following fully allogeneic small bowel transplantation when graft rejection was prevented by cyclosporin immunosuppression. Transplantation was carried out between PVG and DA strains of rat. Cyclosporin (15 mg/kg) was given daily from transplantation, and groups of animals were studied at 28 and 56 days after grafting. The proportions of donor- and recipient-derived cells in the graft and in the host gut and lymphoid tissues were assessed using immunohistochemical tissue staining and monoclonal antibodies specific for cells expressing class I antigens from the two strains of rat. Results demonstrated a persisting population of graft-derived T cells which were capable of migration to the host. Therefore, there may be a long-term risk of graft versus host disease after small bowel transplantation under cyclosporin immunosuppression.     

Involvement of endothelin in graft-versus-host disease after rat small bowel transplantation

Endothelin (ET-1) expression was evaluated by radioimmunoassay in both plasma and various tissue specimens serially obtained from LBN-F1 recipients of LEW heterotopic small bowel allografts. The recipients showed graft-versus-host disease (GVHD), which histologically became apparent on postoperative day (POD) 13. The ET-1 levels peaked on POD 9 in the kidney, lung, and host intestine at 51.0 ± 21.1, 90.9 ± 59.6, and 25.4 ± 11.8 pg/g wet, respectively, and peaked on POD 11 in the plasma at 7.7 ± 3.2 pg/ml; thereafter, they decreased to basal levels in both the plasma and tissue specimens on POD 13. An immunohistochemical study of these organs showed a corresponding increase in ET-1 staining in both the endothelial and epithelial cells on PODs 5 and 9, and a reduction in staining on POD 13. In conclusion, ET-1 was found to be increasingly released from the target cells of GVHD before any histological changes became apparent, thus suggesting the pathophysiological involvement of ET-1 in intestinal GVHD. Received: 11 June 1996 Received after revision: 3 September 1996 Accepted: 28 October 1996     

小肠移植术后巨细胞病毒感染二例的治疗体会

目的 总结小肠移植术后巨细胞病毒(CMV)感染的治疗经验.方法 1994年至2009年间完成15例小肠移植,分为3个阶段:1994-1995年为第1阶段(3例),2003-2006年为第2阶段(7例),2007年以后为第3阶段(5例).第1阶段术后未进行CMV感染的预防;第2阶段通过肠镜、病理检查和血清学检查(CMV IgM、CMV pp65和CMV DNA)进行CMV感染的诊断,术后静脉注射更昔洛韦2~3周,口服阿昔洛韦3个月以预防CMV感染;第3阶段在第2阶段的基础上,应用实时定量PCR技术检测CMV DNA,并制定计划性监测方案,术后静脉注射更昔洛韦2~3周,口服更昔洛韦3个月预防CMV感染,采用CMV感染的抢先治疗方案.结果 15例患者中有2例(13.3 %)术后发生CMV感染.其中第2阶段1例术后45 d发生移植肠CMV肠炎,术后64 d并发CMV肺炎,应用更昔洛韦和胸腺肽,并停用他克莫司,最终转为重度排斥反应后死亡;第3阶段1例术后第3个月发生CMV感染,经CMV抢先治疗后治愈.结论 小肠移植术后应进行CMV的预防性治疗,严密监测CMV血清学指标,适时进行抢先治疗.对于CMV侵袭性疾病在进行有效治疗的同时应注意排斥反应的发生.

Abstract：

Objective Cytomegalovirus (CMV) has remained the most significant pathogen that threatens the outcome of small bowel transplantation (SBTx). This paper To outline preliminary experience of prophylaxis and treatment of cytomegalovirus (CMV) in 15 cases subject to small bowel transplantation (SBTx) and also review current progress of diagnosis and treatment of CMV.Methods Fifteen cases of SBTx were divided into 3 eras: era Ⅰ (1994-1995)-3 SBTx treated with cyclosporine-based immunosuppression; era Ⅱ (2003-2006)-7 SBTx treated with tacrolimus-based immunosuppression; and era Ⅲ (2007-present)-5 SBTx treated with Alemtuzumab induction therapy and maintenance tacrolimus monotherapy. No antiviral prophylaxis after SBTx was applied during era Ⅰ; in era Ⅱ, ileoscopic and pathological diagnosis of CMV graft enteritis was defined, and plasma diagnosis tools including CMV-IgM, CMV pp65 and CMV DNA with PCR were introduced. 2-3 weeks intravenous ganciclovir prophylaxis of CMV was underway, followed by 3 months oral acyclovir; In era Ⅲ, more precise real-time PCR technique was used to detect CMV DNA copies, and the schedule of the CMV surveillance was set up, antiviral prophylaxis therapy was modified to 2-3 weeks intravenous ganciclovir and 3 months oral ganciclovir, and preemptive therapy to halt the progression of asymptomatic infection to clinical disease was also introduced.Results Two of 15 SBTx recipients suffered from CMV with the occurrence rate of 13.3%. One recipient in era Ⅱ suffered from CMV graft enteritis on postoperative day 45, and CMV pneumonia on postoperative day 64, he received intravenous ganciclovir and thymus peptide, paused tacrolimus maintenance, and finally he died from severe acute cellular rejection. 94 100 copies/ml of CMV DNA in periphery blood of a recipient in era Ⅲ was detected with real-time PCR at 3rd month after SBTx, and a preemptive therapy successfully halted the CMV infection.Conclusion Antiviral prophylaxis therapy and close surveillance of CMV infection after SBTx should be performed, and preemptive therapy can also halt the CMV infection. When CMV disease occurs, the recipient should receive effective antiviral therapy, and acute cellular rejection also should be closely monitored at same time.     

Improvement of inflammatory bowel disease after allogeneic stem-cell transplantation

Because causes of inflammatory bowel disease (IBD) remain obscure and a curative therapy is still lacking, the influence of stem-cell transplantation (SCT) on IBD is of major interest. We retrospectively analyzed the course of seven patients with Crohn's disease and four patients with idiopathic ulcerative colitis who underwent allogeneic SCT between July 1994 and August 2002 for acute and chronic myeloid leukemia and myelodysplastic syndrome. After a median follow-up of 34 months posttransplantation, 10 patients are alive. None of the patients showed IBD activity after SCT, except one patient with mild persistent symptoms of Crohn's disease early after transplant. Colonoscopy after complete discontinuation of prophylactic posttransplant immunosuppression revealed no pathologic findings. These observations imply that host immune dysregulation plays a central role in the perpetuation of IBD. It may be influenced by the implementation of a new allogeneic immune system resulting from the transplantation of hematopoietic stem cells.     

大鼠异位小肠移植淋巴管重建模型的建立

目的 建立适合于评估淋巴管重建对移植物存活，功能和对受者影响研究的小肠移植淋巴重建模型。方法 在小肠移植完成后，再将供者的胸导管与受者的乳糜池吻合，使得移植小肠的淋巴回流得以重建，结果 淋巴管重建的手术时间为16.2min，重建淋巴管的通畅率达82.5%。存活大鼠间重建淋巴管通畅率的差异无显著性；排斥反应对重建淋巴管通畅率的影响不显著。结论 在小肠移植完成后即进行移植小肠的淋巴管重建，淋巴管通畅率高，手术时间短，可作为进行其它相关研究的模型。     

Improved survival after allogeneic small intestinal transplantation in the rat using cyclosporine immunosuppression

We investigated the effect of cyclosporine on survival after intestinal transplantation between histoincompatible Brown Norway (AgB3/3) and Lewis (AgB1/1) rats. Intestinal grafts were primarily vascularized (by microsurgical techniques) and interposed isoperistaltically in the recipient's jejunum. All animals were weighed and observed daily. Survival of recipients given cyclosporine 20 mg/kg/d (112 +/- 92 days, n = 10) was significantly longer (P less than .02) than that of recipients given no drug (12 +/- 4 days, n = 8). Six out of ten cyclosporine-treated animals remained alive and well at the conclusion of the experiment. One of these was killed and showed no gross or microscopic evidence of rejection. The described experimental model involves a simple operative technique and minimal postoperative care and should permit systematic investigation of the detection and prevention of rejection.     

小肠移植后并发侵袭性真菌感染的治疗

目的 总结小肠移植术后侵袭性真菌感染(IFI)的治疗经验和教训.方法 将1994年至2009年6月间15例小肠移植患者分为3个阶段,1994-1995年的3例患者为第1阶段,2003-2006年的7例患者为第2阶段,2007年以后的5例患者为第3阶段.第1和第2阶段患者围手术期真菌感染的预防方案采用静脉注射氟康唑,IFI的治疗以静脉注射氟康唑为主,在病情危重时静脉注射两性霉素B或两性霉素B脂质体,首次用量为1～5 mg/d(或0.02～0.10 mg·kg~(-1)·d~(-1)),视患者耐受情况每日增加5 mg;第3阶段患者围手术期真菌感染的预防方案采用静脉注射两性霉素B脂质体,治疗IFI时,两性霉素B脂质体首次给药便达到目标治疗剂量,用量高达6 mg·kg~(-1)·d~(-1),并严密监测患者的生命体征,肝肾功能及电解质的变化,根据患者病情的变化和肾功能的状况调整剂量.结果 15例患者中有4例术后发生IFI,发生率为26.7%,其中第1、2和第3阶段患者中分别有1例、2例和1例发生IFI.第1和第2阶段3例发生IFI的患者经治疗无效死于严重IFI,第3阶段1例发生IFI的患者经两性霉素B脂质体治疗44 d后被成功救治.治疗期间,患者尿素氮和血清肌酐水平均显著升高,停药后逐渐下降至正常水平.3个阶段患者总体病死率为75%.结论 小肠移植术后IFI是极其凶险的并发症,病死率极高;两性霉素B脂质体能够成功救治IFI患者,在严密监测肾功能下,大剂量应用两性霉素B脂质体是安全的.     

Inhibition of host-versus-graft and graft-versus-host responses after small bowel transplantation in rats by rapamycin.

The effect of rapamycin (RAPA) on both host-versus-graft (HVG) and graft-versus-host (GVH) immune responses was examined in small bowel transplant models using strongly histoincompatible donor-recipient combinations. Normal Wistar Furth (WFu; RT-1u) recipients rejected Buffalo (BUF; RT-1b) small bowel allografts within a mean survival time (MST) of 10.5 +/- 0.5 days. Administration of RAPA (0.8 mg/kg) by continuous intravenous infusion for 14 days via an osmotic pump prolonged graft survival to 25.0 +/- 4.6 days (P = 0.01). In a second strain combination, the 12.5 +/- 2.2 day survival of Brown Norway (BN; RT-1n) small bowel allografts in Lewis (RT-1l) recipients was prolonged to 21.6 +/- 2.0 and 28.5 +/- 2.8 days by 14 days of i.v. RAPA at doses of 0.8 and 1.6 mg/kg, respectively. In this model RAPA is five times more effective than cyclosporine, which at 4.0 mg/kg prolongs BN small bowel allografts in Lewis recipients to 21.6 +/- 6.3. To isolate HVG and GVH immune responses, (BN x Lewis)F1 hybrid rats served as the graft donor or host, respectively. In the HVG model, (BN x Lewis)F1 small bowel allografts, which were rejected by normal Lewis recipients at 12.2 +/- 3.6 days, were prolonged to 40.8 +/- 5.8 days (P = 0.001) by RAPA (0.8 mg/kg x 14 days). In the GVH model, the ability of Lewis small bowel allografts to produce severe GVH disease in untreated (BN x Lewis)F1 recipients at 12.3 +/- 2.8 days was delayed to 21.3 +/- 5.2 days by 0.8 mg/kg RAPA (P = 0.025). Thus, RAPA protects small bowel allografts more effectively against HVG than GVH immune responses.     

Simplified techniques in rat heterotopic small bowel transplantation

AIM: Establish a simplified heterotopic small bowel transplantation (SBT) in the rat. METHODS: Ninety pairs of male Wistar rats were used as donors and recipients. The whole small intestine with a vascular pedicle composed of superior mesenteric artery (SMA) and portal vein (PV) was harvested as the graft. Revascularization was accomplished by end-to-side anastomosis between donor SMA and recipient infrarenal aorta and cuffed end-to-end anastomosis between donor PV and left renal vein of recipient. The distal end of graft was exteriorized to form an enterostoma. RESULTS: Average time of an operation was 130 minutes and the mean warm ischemia time of grafts was 30 minutes. The technical success rate of this model was 100% and 7-day survival was 95.6% (86/90). CONCLUSION: This simplified technique was effective and practical to improve the outcome of rat heterotopic SBT.     

Cyanoacrylate-assisted arterial anastomosis in rat small bowel transplantation

Purpose  

Microarterial anastomosis in rat small bowel transplantation (SBTx) remains technically difficult and time-consuming especially for beginners of microsurgery. In the exploration of the facilitated microarterial anastomosis strategies, we assessed the performance of n-butyl-cyanoacrylate-assisted suture technique (CAST) in comparison with simple continuous suture technique (SCST).