Intragraft heat shock protein-60 expression after small bowel transplantation in the mouse

The time course of heat shock protein 60 (hsp 60) expression after intestinal transplantation in syngeneic and allogeneic combination was correlated with the degree of rejection. Hsp 60 expression was assessed by immunostaining; rejection degree was established by histologic examination on posttransplantation days 1, 3, 6, and 8. No signs of rejection occurred in syngeneic grafts at any time. In the allogeneic setting, rejection was absent in all but 1 case on postoperative day 3. Three days later moderate rejection was evident based on focal crypt destruction and focal mucosal ulceration, whereas at postoperative day 8 extensive mucosal sloughing was the dominant feature, consistent with advanced rejection. Hsp 60 remained undetectable in the syngeneic setting at all times. In allografts, hsp 60 was initially expressed on posttransplant day 3, increasing synchronously with the progression of rejection at days 6 and 8. Hsp 60 expression was localized almost exclusively to the crypt area and the lower third of the villi. In conclusion, the rejection of murine allogeneic intestinal grafts is characterized by a progressive expression of hsp 60 in the epithelium.     

Intragraft gene expression profile during acute cellular rejection in clinical small bowel transplantation: a case report

BACKGROUND: Acute cellular rejection (ACR) is a common complication of small bowel transplantation (SBTx) and the major cause of graft loss. However, little is known regarding the genetic graft response to ACR in clinical transplants. In this study, we have determined a genetic expression profile of intestinal graft response to ACR after living related (LR) SBTx. RESULTS: By identifying the expression profiles of reported markers of rejection we were able to identify 57 genes that had significantly increased (more than twofold) expression in response to ACR. Known markers of rejection identified: MMP-9, MMP-2, VIP, IFNgamma, IL-2R, MADCAM-1, HSP-60, and HSP-70 all had greater than twofold increased expression after ACR diagnosed (week 3 to week 6). The newly identified genes were: IFI27, EPST11, APAF1, LAP3, STK6, and MDK. CONCLUSION: Newly identified up-regulated genes in response to ACR in small bowel graft are involved in the immune response, cell adhesion, neurogenesis, cell division and proliferation, DNA replication/repair, protein ubiquitin/proteolysis, and apoptosis. TNFalpha up-regulated early at week 2 biopsy may be an early genetic marker of ACR in SBTx.     

Intragraft distribution of lymphocytes expressing beta7 integrins after small bowel transplantation in mice

Lymphocytes with activated beta7 integrins (alphaEbeta7 and alpha4beta7) contribute to inflammatory reactions in the small bowel. Since the selective recruitment of lymphocytes to the lymphoid compartments of the small bowel is controlled by distinct adhesion molecule interactions, a compartment-dependent use of beta7 integrins may influence the rejection response within intestinal transplants. To further delineate the nature of beta7 integrin-mediated graft infiltration, we analysed their expression on T lymphocytes in the heterotopically transplanted small bowel of BALB/c and C57BL/6 mice. Lymphocytes isolated from the epithelium, lamina propria (LP), Peyer's patches (PP), and mesenteric lymph nodes (MLN) were analysed by three-color fluorescence flow cytometry using monoclonal antibodies (mAb) to integrin the subunits, lymphocyte markers, and MHC I of the donor and recipient strains. On postoperative day 5 (POD) after allogeneic small bowel transplantation (SBT), 43% of intraepithelial lymphocytes (IEL) and 63% of LP, 93% of MLN, and 93% of PP lymphocytes were of host origin. In the MLN and PP of allografts, a major infiltrating lymphocyte population consisted of CD8+ cells with increased expression of alpha4beta7 and decreased expression of L-selectin, an adhesion molecule profile characteristic of intestinal effector cell phenotypes. An increase in alpha4beta7 levels was also found on CD8+ host lymphocytes in the LP. The integrin profile of a number of host IEL suggests an ongoing transition from the phenotype of graft infiltrating lymphocytes with high levels of alpha4beta7 and low levels of alphaepsilonbeta7 to that of resident IEL with high levels of alphaepsilonbeta7 and low levels of alpha4beta7. The importance of beta7-mediated lymphocyte trafficking to the graft is attested by the significant reduction in the host lymphocyte population in the LP, PP, and epithelium following the administration of a beta7-blocking mAb to allograft recipients. In conclusion, while the infiltration patterns of lymphocytes may vary between the lymphoid compartments of intestinal allografts, host CD8+ lymphocytes with high levels of alpha4beta7 constitute a major effector cell population that affects the entire graft.     

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).     

简化的大鼠异位全小肠移植术

目的:建立简化的大鼠异位全小肠移植术技术,以提高手术成功率,为相关研究奠定基础.方法:供、受体均为雄性近交系Wistar大鼠,共180只,配对手术.采用供肠的肠系膜上动脉与受体肾下腹主动脉端侧吻合、门静脉与受体左肾静脉套管法端端吻合重建移植小肠血供,移植小肠远端腹壁造口.结果:手术耗时130min,移植小肠热缺血时间约30min.90只受全大鼠术扣即时存活率为100%,长期存活率(＞7d)为95.6%.结论:简化术式具有操作简便,移植小肠的热缺血时间短,手术成功率高等优点,有利于后续研究工作的开展.     

Apoptosis and CD8 and CD54 cell expression in rat small bowel transplantation

The importance of activated CD8 cells expressing IL-2R in small bowel and other organ rejection has been reported. Some authors even consider that a positive correlation might be demonstrated between the number of apoptotic enterocytes and the degree of graft rejection. In addition, moderate to intense activation of endothelial molecules in small bowel allograft in rats has been reported in chronic rejection. The aim of the present paper is to ascertain, in a heterotopic small bowel transplantation (HSBT) in rats, whether CD3, CD4, CD8, and CD54 cell expression in the allograft infiltrates shows some relationship with allograft enterocyte apoptosis when rejection is present. Wistar Furth male rats were allotted to two groups: group A was the control group without transplantation; group B received a heterotopic small bowel allograft from Fisher rats and an im dose of FK506 (0.25 mg/kg/day). A significant increase of CD8, CD54 cell receptor expression, and apoptosis in the group undergoing HSBT showed rejection. No significant differences have been observed in the variables under study between the control and HSBT without rejection groups or in CD3 and CD4 among the three groups. We observed a significant correlation between apoptosis and rejection, between CD8 and CD54 with apoptosis and with rejection, and between CD8 and CD54. This indicates that the activation of endothelial molecules and cells may play an important role in established HSBT chronic rejection. We consider that this study may contribute to the knowledge of small bowel allograft chronic rejection and its immunomodulation.     

改进技术的大鼠全小肠移植术

目的建立并发症少、成活率高的大鼠异位全小肠移植模型。方法应用显微外科技术对１９６只Ｗｉｓｔａｒ大鼠施行异位全小肠移植。术前缩短禁食时间,补充能量,术中静脉输液;减少对供肠的机械和缺血性损伤;重建供肠血管采用腹主动脉－腹主动脉吻合以及门静脉－左肾静脉套管法吻合。结果热缺血时间≤４０±５分钟,吻合口无血栓形成及狭窄,９８只接受小肠移植大鼠的存活率为８８．７８％（８７／９８）。结论改进技术后的大鼠全小肠移植术具有并发症少,存活率高的特点,并且稳定、实用     

2-Octylcyanoacrylate-assisted microvascular anastomosis in rat orthotopic small bowel transplantation

The purpose of this study was to investigate the potential advantages of 2-octylcyanoacrylate-assisted end-to-side arterial anastomosis over continuous suture-only anastomosis in the rat orthotopic small bowel transplantation (OSBT) model. OSBT was performed in 80 Sprague-Dawley rats, and they were divided into two groups. Group 1 was the study group (n = 40), using 2-octylcyanoacrylate-assisted end-to-side arterial anastomosis. Group 2 was the control group (n = 40), using a running 9-0 prolene suture only. The operation time and degree of leakage were observed. After sacrifice on postoperative days 90, histology and electron scanning microscope of arterial anastomosis were measured. The suture time and recovery time in group 1 were significantly shorter than that in group 2. First-degree leakage was observed mostly in group 1, and second-degree leakage was common in group 2. No difference was found between the two techniques considering the graft intestinal histological changes. Under electron scanning microscopy, the anastomoses of the two groups displayed no obvious difference. In conclusion, due to superior speed of operation and no toxicity to vessel wall, 2-octylcyanoacrylate-assisted end-to-side arterial anastomosis might be a quick and safe method that is much easier to apply in rat OSBT.     

Intragraft cytokine gene expression: implications for clinical transplantation

As our knowledge of the cytokine network in experimental transplant models grows, we need to understand how and to what extent cytokines mediate the various donor-directed immune events in clinical situations. This overview of clinical cytokine measurements shows that specific intragraft cytokine messenger RNA (mRNA) expression profiles can be associated with acute rejection, that they may reflect the efficacy of immunosuppression, and that they can identify patients at risk for the development of early chronic rejection. The literature also shows that acute rejection and immunological quiescence in humans are not restricted to the cytokine patterns defined in the type 1/type 2 paradigm. This apparent lack of association may be caused by the immunosuppression used in the clinic but may also be the result of the infinite diversity of donor and recipient factors, in which polymorphisms in cytokines and cytokine receptor genes may play a central role. Received: 15 July 1997 Received after revision: 5 January 1998 Accepted: 14 January 1998     

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     

Trophic effect of enteral rehabilitative therapy in rat small bowel transplantation

INTRODUCTION: We aimed to observe the trophic effects on graft structure and recipient metabolism of enteral rehabilitative therapy in rat small bowel transplantation (SBT). METHODS: Forty-eight recipients of rat allogeneic heterotopic SBT (Sprague-Dawley to Wistar rat) were divided into four groups randomly according to the presence of glutamine or growth hormone in the nutritional support regimen. We compared the histologic indices of graft mucosa and metabolic variables, including changes in body weight, nitrogen balance, urinary 3-methyl histidine excretion, and plasma albumin levels during 14 days after transplantation. RESULTS: Glutamine and growth hormone promoted recovery of graft structure and improved recipient protein metabolism, as evidenced by indices of the enteral rehabilitative therapy. CONCLUSIONS: Glutamine and growth hormone potentiated their effects in enteral rehabilitative therapy, which produced potent trophic effects on graft structure and recipient metabolism in rat SBT.     

Immunologic benefits of longer graft in rat allogenic small bowel transplantation

BACKGROUND: The effect of graft length on rejection reaction in small bowel transplantation (SBT), which is poorly understood, is tested using rat allogenic SBT models with a short course of tacrolimus. MATERIALS AND METHODS: Inbred Brown Norway rats (major histocompatibility complex: RT1) and Lewis rats (RT1) were used as donors and recipients, respectively. The intestinal tract of the recipient was partially or totally replaced by segmental (15 cm) or whole (70 cm) donor intestine, using two different SBT models. With tacrolimus treatment (0.64 mg/kg per day, 0-13 postoperative days, intramuscularly), recipients' body weights and their survival were evaluated. To compare the extent of peripheral chimerism, donor passenger leukocytes were followed using flow cytometry with a donor-specific monoclonal antibody, OX-27. For the periodical histologic analysis, heterotopic SBT and protocol biopsies of the graft were also performed with short or long intestinal grafts. RESULTS: In a classical Monchik and Russell orthotopic SBT model, whole SBT recipients survived more than 60 days. However, all of the allogenic segmental SBT recipients died within 14 days without histologic sign of graft rejection. In the modified orthotopic SBT model using a cuff technique without systemic clamping, all recipients with segmental allograft survived longer than 29 days. However, recipients with whole graft tended to survive longer than those with segmental graft. The suffering period, lasting from the onset of rejection to death, was significantly shorter in the segmental group than in the whole group. Flow cytometric analysis showed that recipients with whole intestinal grafts had significantly higher ratio of donor passenger leukocytes in peripheral blood. Histologic studies of the protocol biopsies showed that the shorter graft tended to be more severely rejected than the longer graft. CONCLUSIONS: We have demonstrated experimentally that long intestinal grafts have immunologic advantage over short grafts.