Migration of host and donor T cells in small bowel transplantation

Abstract In this study migration of host and donor CD4⁺ andCD8⁺ T cells in a fully allogeneic model was described and compared with the migration pattern in a graft-versus-host reaction (GVHR) model, where the T-cell traffic in the graft served as a physiological control. Heterotopic small bowel transplantations were performed in a rat model, with animals being sacrificed on postoperative days (POD) 2, 3, 4, 5, and 7. Graft and host mesenteric lymph nodes were harvested, homogenized, and stained with monoclonal antibodies against MHC class I, CD4 ⁺, and CD8 ⁺ antigens. The host and donor T cell migration patterns were studied using a double-staining flow cytometric technique. We found that during the development of rejection, the normal physiological circulation of graft and host T cells was disrupted. In the graft of the allogeneic model, a shift from host cell to graft cell dominance occurred on POD 3–4. This change in migration pattern coincided in the host with a 6 % peak in graft cell infiltration, which disappeared on POD 7. These patterns of T-cell migration may be further explored for diagnostic purposes.     

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     

Prevention of graft rejection and graft-versus-host reaction by a novel immunosuppressant, FTY 720, in rat small bowel transplantation

In the present study, we examined the immunosuppressive effect of a new drug, FTY 720, on small bowel transplantation (SBT) in rats. Grafts from (LEW × BN) F 1-to-LEW rats treated with FTY 720 at 0.5 mg/kg from day 0 to 14 post-SBT survived significantly longer than untreated grafts. In addition, the administration of FTY 720 combined with cyclosporin (CyA; 5 mg/kg per day) had a synergistic effect on allograft survival. The graft-versus-host reaction (GVHR) that occurred in the LEW-to-F 1 rats was markedly reduced after the administration of FTY 720. FTY 720 combined with a low dose of CyA completely abrogated GVHR without any adverse reaction. FTY 720 treatment resulted in a significant decrease in the number of lymphocytes in the peripheral blood and the spleen, but the number of peripheral neutrophils was unchanged. Thus, FTY 720 would appear to be an ideal drug to combine with CyA in order to control the immune reaction after SBT. Received: 19 February 1997 Received after revision: 23 May 1997 Accepted: 9 June 1997     

Migration of host and donor T cells in small bowel transplantation

In this study migration of host and donor CD4⁺ and CD8⁺ T cells in a fully allogeneic model was described and compared with the migration pattern in a graft-versushost reaction (GVHR) model, where the T-cell traffic in the graft served as a physiological control. Heterotopic small bowel transplantations were performed in a rat model, with animals being sacrificed on postoperative days (POD) 2, 3, 4, 5, and 7. Graft and host mesenteric lymph nodes were harvested, homogenized, and stained with monoclonal antibodies against MHC class 1, CD4⁺, and CD8⁺ antigens. The host and donor T cell migration patterns were studied using a doublestaining flow cytometric technique. We found that during the development of rejection, the normal physiological circulation of graft and host T cells was disrupted. In the graft of the allogeneic model, a shift from host cell to graft cell dominance occurred on POD 3–4. This change in migration pattern coincided in the host with a 6 % peak in graft cell infiltration, which disappeared on POD 7. These patterns of T-cell migration may be further explored for diagnostic purposes.     

Relevance of tumor necrosis factor to graft-versus-host disease after small bowel transplantation

The small bowel (SB), an organ replete with lymphocytes, may provoke graft-versus-host disease (GVHD) after transplantation (Tx). Since tumor necrosis factor (TNF) has been suspected of mediating the tissue lesions of GVHD, we sought to determine whether TNF could be detected in the serum of rats undergoing GVHD after SBTx or lymphocyte transfer. For this purpose, post-operative serum TNF activity was determined in Lewis x Brown after undergoing transplantation of an entire (group 1; n=8) or a segmental (group 2; n=4) Lew SB, or after i. p. injection with lethal doses (500×10⁶) of Lew lymphocytes (group 3; n=3). Control LBNF1 received i.p. small doses (50×10⁶) of Lew lymphocytes (group 4; n=4). Serum TNF activity was assessed using the WEHI bioassay. In rats with acute and lethal GVHD after entire SBTx (group 1) or injection with large doses of lymphocytes (group 3), TNF activity gradually increased and reached high levels by the time the rats were agonal. In segmental SBTx rats (group 2), GVHD was less severe than in entire SBTx rats. Similarly, the increase in TNF activity was less intense and only transient since it had returned to control levels by the time the rats had completely recovered from GVHD. In control rats primed with small doses of lymphocytes (group 4), GVHD did not occur and no increase in TNF activity was detected. We conclude that: (1) GVHD after SBTx or lymphocyte transfer is associated with the appearance of TNF in the serum and (2) the intensity and the reversibility of this phenomenon correlate with clinical severity and lethality of GVHD. These data strongly suggest that TNF is involved in the pathogenesis of GVHD.     

Host immune suppression after small bowel/liver transplantation in rats

Simultaneous liver grafting in the Lewis (RT1¹)-to-DA (RT1^a) rat strain combination protects small intestinal grafts from rejection. The present study examined host immune responses after combined small bowel/liver transplantation (SBL) in this model. Orthotopic liver transplantation and heterotopic small intestinal transplantation were performed simultaneously and compared with isolated small bowel allografts (SBA) and isolated small bowel isografts (SBI). All rats were sacrificed on postoperative day (POD) 7 or 14 for immunological and histological studies. The mean time to rejection of the SBA was 6.6±0.3 days. Incontrast, there was no clinical or histological evidence of intestinal rejection in SBL recipients during the 14 days of follow-up. The SBL recipients showed clinical and histological evidence of graft-versushost disease (GVHD). Lmphocyte proliferation and IL-2 production in response to donor antigens were suppressed after SBL transplantation compared with the SBA or the SBI controls (P<0.05). Cell-mediated cytotoxicity and lymphocytotoxic antibody production against donor cells were also significantly inhibited in the SBL recipients compared with the SBA control group (P<0.05). We conclude that SBL transplantation in the Lewis-toDA rat strain combination: (1) suppresses host alloimmune responses, (2) prevents early intestinal rejection, and (3) favors the development of GVHD.     

Review of various techniques of small bowel transplantation in pigs

Because of anatomical and physiological similarities to humans, porcine small bowel transplantation (SBTx) can be used as an appropriate experimental model in the field of surgical research. Various approaches to SBTx have been described in literature. The aim of this work is to present a review of different surgical techniques of SBTx which have been developed using the porcine model. Our analysis of Medline-cited studies dealing with different techniques of SBTx in porcine models was particularly focused on surgical aspects. With regard to graft procurement and enterectomy, the reported techniques vary widely. Arterial reconstruction is mainly conducted by performing the anastomosis between the superior mesenteric artery (SMA) of the donor and SMA or infrarenal aorta of the recipient. Alternatively, an aortic segment of the donor can be anastomosed to the infrarenal aorta of the recipient. Venous anastomosis is frequently performed between the superior mesenteric vein (SMV) of the donor and SMV or the inferior vena cava (IVC) of the recipient. Some studies also report venous anastomosis between the portal vein of the donor and the recipient. Bowel continuity is then restored by end-to-end or end-to-side anastomosis. Remarkable results were generated thanks to improved techniques which include proximal side-to-side ileo-ileal anastomosis with double-barrel ileostomy, or so-called "Paul-Mikulicz-Ileostomy". Most frequently used were jejunostomy and the "Bishop-Koop-Ileostomy"--where the proximal part of the bowel is anastomosed end-to-side to the distal part, which is then exteriorized as an ostomy. Based on the techniques presented in this review, one must select the most suitable surgical technique of porcine SBTx among those various models.     

Integrated intestinal capacity and nutritional status following small bowel transplantation

Successful small bowel transplantation requires normal functional capacity of the graft and unaltered metabolism of the host. Weight gain and wet weight of muscle groups and intra-abdominal fat pads were compared between transplanted, sham-operated, short bowel-operated, and normal rats that were fed either standard chow or fat-enriched (15 %) pellets. Weight gain and wet weight of muscle groups and fat pads for the control, transplanted, and sham-operated rats were identical, while short bowel animals showed reduced weight. Transplanted rats receiving fat-enriched food had lower wet weight of fat pads than control animals on the high-fat diet. We conclude that small bowel transplantation makes it possible to overcome the intestinal failure associated with short bowel syndrome, leading to overall normal weight gain and development of the recipient. However, altered fat metabolism, reflected in changed body composition, was observed in transplanted animals on the high-fat diet. Received: 19 November 1996 Received after revision: 10 April 1997 Accepted: 13 May 1997     

The pattern of rejection after combined stomach,small bowel,and pancreas transplantation in the rat

This study was designed to investigate whetherin combined stomach, small bowel, and pancreas transplantation allograft rejection occurs in the individual organs concomitantly and with the same intensity. Heterotopic enbloc transplantation of the stomach, small bowel, and pancreas was performed in a Lewis-to-Brown Norway rat combination. Group 1 animals received no immunosuppressive therapy while animals in group, 2 were treated with cyclosproin (10 mg/kg body weight, orally) daily. Grafts were histologically evaluated on the 5th (subgroups 1a and 2a) and 10th (subgroups 1b and 2b) postoperative days. The degree of rejection was defined as moderate, intermediate, or severe according to predefined criteria. The results indicate that the small bowel is more susceptible to rejection than either the stomach or the pancreas. Mucosal biopsies of the stomach are unlikely to provide a reliable guide to rejection in the small bowel.     

大鼠肝小肠联合移植模型的建立

本文报告一种封闭群大鼠进行的肝小肠联合移植模型。术中供体肝与小肠整块游离和灌注，分开切取。供肝原位、供肠异位移植于受体大鼠。主要血管用Ｋａｍａｄａ袖套法吻合，正式实验２１次，３天以上存活率为４３％。结果表明：减少手术时间和简化操作技术是提高成功率的关键因素和基本原则。     

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.     

Effects of donor pretreatment with antilymphocyte serum and cyclosporin on rejection and graft-versus-host disease after small bowel transplantation in immunosuppressed and nonimmunosuppressed rats

After fully allogeneic small bowel transplantation, both graft-versus-host disease (GVHD) and rejection may occur. Donor pretreatment may prevent GVHD, but this sometimes leads to accelerated graft rejection. To study a possible balance between GVHD and rejection, fully allogeneic total orthotopic small bowel transplantation was performed in rats using the WAG-to-BN donorhost combination. Untreated control grafts were rejected in 16.6±2.7 days (mean ±SEM), and 35% of the animals had mild, transient GVHD. Pretreatment of the donor with antilymphocyte serum on days-2 and-1 before grafting, either intravenously or intraperitoneally, completely eliminated the occurrence of clinical GVHD but led to significantly shortened survival times (12.3±0.8 and 10.3±0.9 days, respectively). Donor pretreatment with 50 mg/kg cyclosporin (CyA) on days-2 and-1 prolonged graft survival significantly to 22.1 days but had no significant effect on the incidence of GVHD. Administration of 25 mg/kg CyA on days 0, 1, 2, 4, and 6 after grafting prolonged survival to 38.3 days with no evidence of GVHD. Pretreatment of the donor with antilymphocyte serum (ALS), combined with the same postoperative, short-term CyA regimen, increased survival to more than 50 days, again with no evidence of GVHD. When CyA was used as both donor pretreatment and postoperative therapy, there was no survival advantage compared to the use of postoperative CyA alone. These results show that an in vivo balance between GVHD and rejection exists and that abrogation of GVHD leads to accelerated rejection. Immunosuppression of the recipient may overrule this accelerated rejection while preserving the beneficial effect of donor pretreatment: elimination of clinical GVHD.     

Experimental assessment of small intestinal submucosa as a small bowel graft in a rat model

Background/Purpose:

Small intestinal submucosa (SIS) is an extracellular matrix used in tissue engineering. The purpose of this study is to evaluate the feasibility of using SIS as a scafford for small bowel regeneration in a rat model.

Methods:

A 2-cm length tubular SIS graft from donor Sprague Dawley rats was interposed with bilateral anastomosis in the median tract of an isolated ileal loop of Lewis rats used to construct an ileostomy. The grafts were harvested and analyzed at each of the time-points ranging from 2 weeks to 24 weeks after operation using histology and immunohistochemistry.

Results:

Macroscopic examination found no adhesion in the surrounding area of neointestine by 24 weeks, and no stenosis was visible. The shrinkage of neointestine was indicated from 20% to 40%. Histologic and immunohistochemical evaluation showed that SIS grafts were colonized by numerous inflammation cells by 2 weeks. Neovascularization was evident, but the luminal surface was not epithelized. By 4 weeks, transitional mucosal epithelial layer began to line the luminal surface of the graft, and nearly 70% luminal surface of the graft had been covered by mucosal epithelium at 8 weeks. By 12 weeks, the luminal surface was covered completely by a mucosal layer with distinct bundles of smooth muscle cells in the neointestine. At 24 weeks, the neointestine wall showed 3 layers of mucosa, smooth muscle, and serosa.

Conclusions:

The preliminary study suggested that SIS allow rapid regeneration of mucosa and smooth muscle and might be a viable material for the creation of neointestine.     

Mucosal recipient-type mononuclear repopulation and low-grade chronic rejection occur simultaneously in indefinitely surviving recipients of small bowel allografts

Lewis rat recipients of long-term, surviving, orthotopic Brown-Norway rat intestinal allografts, initially treated with cyclosporin A (CyA) or FK 506, were evaluated for their functional capacity and morphology over 1 year after the immunosuppressive therapy had been discontinued. Functional parameters such as nitrogen and fat balances, maltose absorption, blood chemistry, hematologic studies, and the weight gained by the allografted animals did not differ from those of syngeneically grafted or agematched normal animals. Immunohistochemical studies showed that the lamina propria of the allografts was repopulated with recipient MHC class II+mononuclear cells and that a normal distribution of T helper, T suppressor/killer, and IgA+plasma cells had occurred. However, fibrous replacement of the mesenteric lymph nodes and Peyer's patches were detected in all, and an inflammatory obliterative arteriolopathy developed in the mesenteric vasculature of half of the allografted animals. No such findings were observed in recipients of syngeneic grafts. These results demonstrate that the limited use of potent immunosuppressive agents immediately after transplantation averts rejection and is followed by recipient-type mucosal lymphocytic repopulation. Simultaneously, a clinically not recognizable chronic rejection evolves. This suggests that the timely diagnosis of chronic rejection may not be possible with the use of standard tests of gut function and random mucosal biopsies alone.This study was presented in part at the 32nd Annual Meeting of the Society for Surgery of the Alimentary Tract, 21–22 May 1991, New Orleans, Louisiana     

Altered distribution of MHC class II antigens on enterocytes during acute small bowel allograft rejection in rats

Class II major histocompatibility complex (MHC) antigen induction was investigated on enterocytes of heterotopic rat small bowel allografts in the Lewis-Brown Norway strain combination and on isografts in the Lewis-Lewis strain combination. I a antigens were detected with monoclonal antibodies using an immunoperoxidase technique. Generally, MHC class II antigens were not exhibited in the isografted group, with the exception of two long-term isografts that presented the same pattern as normal small bowel. In these cases, I a was expressed in a patchy distribution predominantly in the villi, and only very few enterocytes stained positive in Lieberkühn's crypts. Allografted rats showed a typical pattern of I a expression on the enterocytes during the rejection course. The initial expression was confined to the crypts, indicating a very early stage of rejection when compared to histological findings. More advanced stages of rejection were accompanied by increasing I a biosynthesis in the crypts and I a expression by the epithelium lining the villi. Cyclosporin (CyA) was not able to fully inhibit MHC class II antigen expression; however, the appearance of I a was delayed.     

Low infectious complications in segmental living related small bowel transplantation in adults

Clinical small bowel transplantation (SBTx) has been associated with a high rate of infectious complications. Laparotomy, preservation injury, abnormal motility, lymphatic disruption, aberrant systemic venous drainage, rejection and antibiotic therapy could all be implicated in the etiology of these complications. In addition to the underlying disease, total parental nutrition could determine infections and liver impairment. Recently, standardized techniques for segmental living related SBTx (LR-SBTx) have been developed. This technique allows reduction of some of these factors, thus resulting in a reduced incidence of infections. We report the infectious complications observed in 3 patients with short bowel syndrome treated with LR-SBTx at our institution. A segment of 180-200 cm of ileum was transplanted with a neglectably short cold ischemia time (CIT). The donor bowel was decontaminated. Oral tacrolimus, prednisone and IV induction with ATG were used for immunosuppression. Blood, stool, urine, sputum and peritoneal fluids were collected and cultured as a routine surveillance. All recipients are alive with a current follow-up time up to 21 months. No bacterial infections were observed during the post-transplant period. One patient developed cytomegalovirus (CMV) enteritis 4 months after LR-SBTx and was treated successfully with IV ganciclovir. In our limited experience, LR-SBTx is associated with a low infection rate. This could be due to optimal graft decontamination, short CIT and to the reduced incidence of rejection and lower immunosuppression used in this immunologically well-matched combination.     

Allograft biopsy findings in patients with small bowel transplantation

In this study, we sought to determine the incidence of post‐transplant complications including acute cellular rejection (ACR), infection, and post‐transplant lymphoproliferative disease (PTLD) in mucosal allograft biopsies in patients with small bowel transplant at our institution. We retrospectively reviewed pathology reports from 5675 small bowel allograft biopsies from 99 patients and analyzed the following: indications for biopsy, frequency and grade of ACR, the presence of infectious agents, results of workup for potential PTLD, results of C4d immunohistochemistry (IHC), features of chronic mucosal injury, and findings in concurrent native bowel biopsies. Findings from 42 allograft resection specimens were also correlated with prior biopsy findings. Indeterminate, mild, moderate, and severe ACR were seen in 276 (4.9%), 409 (7.2%), 100 (1.8%), and 207 (3.6%) of biopsies, respectively. Although ACR may show histologic overlap with mycophenolate mofetil toxicity, we found the analysis of concurrent native bowel biopsies to be helpful in this distinction. Adenovirus was the most common infectious agent seen (11%), and we routinely performed adenovirus IHC on biopsies. Eighteen patients (18%) developed PTLD, 83% of which were EBV associated, but only 28% of PTLD cases were diagnosed on mucosal allograft biopsies. C4d IHC did not correlate with the presence of donor‐specific antibodies in limited cases.     

小淋巴结转移与结直肠癌分期和预后的关系

淋巴结转移是影响结直肠癌预后的重要因素。目前临床上对结直肠癌标本淋巴结检查没有统一的规范，对直径〈5mm的小淋巴结的检出和转移状况不重视。而传统的淋巴结检查方法，对小淋巴结漏检的可能性极大。多个研究表明：小淋巴结中不仅存在转移，且其占阳性淋巴结总数的50％以上。小淋巴结的检出精确了结直肠癌患者的病理分期，有利于术后正确辅助治疗方案的选择。临床上应改进结直肠癌标本淋巴结检查方法，提高小淋巴结的检出率。     

远端局部进展期胃癌肠系膜上静脉旁淋巴结(No.14v)清扫的临床意义

远端局部进展期胃癌肠系膜上静脉旁淋巴结(No.14v)转移常见。基于其解剖学特点,幽门下组淋巴结转移是影响No.14v组淋巴结转移的重要因素。生存分析显示No.14v组淋巴结转移病人的预后与没有No.14v组淋巴结转移的ⅢC组病人相当,因此No.14v应该视为局部(第二站)淋巴结,不应该视为远处转移(M1)。No.14v组淋巴结的治疗价值与第一站(No.5、No.7)和第二站(No.8a、No.9、No.12a)相当。因此,对于伴有No.6组淋巴结转移的远端进展期胃癌病例,常规清扫No. 14v组淋巴结可以显著提高病人的远期生存。     

The safety of donor in living donor small bowel transplantation – an analysis of four cases

Abstract:  Intestinal transplantation using living-related donors could potentially reduce the severity of rejection responses against this highly immunogenic organ by better tissue matching and shorter cold ischemia duration, compensating for the shortage of donor grafts. The purpose of this study is to assess the safety of donors receiving living donor small bowel transplantation (LDSBT) in our hospital by reviewing the risk of operations and absorbing capability recovering retrospectively based on the parameters, such as body weight loss, blood loss, blood transfusion, operation time, duration of hospitalization, complications, and d -xylose tolerance test. After a follow-up period (mean 72 months, range: 48–96 months) of four cases of donors performed LDSBT in Xijing Hospital of digestive diseases dated from May 1999 to September 2003, no complication occurred. Therefore, pre-operation angiography, meticulous management of operation and accurate post-operation monitoring were particularly necessary to guarantee the safety of donors.