Allogeneic Small Bowel Transplantation Research Articles

Graft infusion of adipose-derived mesenchymal stromal cells to prevent rejection in experimental intestinal transplantation: A feasibility study.

Mesenchymal stromal cells (MSC) have been proposed as a promising complement to standard immunosuppression in solid organ transplantation because of their immunomodulatory properties. The present work addresses the role of adipose-derived MSC (Ad-MSC) in an experimental model of acute rejection in small bowel transplantation (SBT). Heterotopic allogeneic SBT was performed. A single dose of 1.5x106 Ad-MSC was intra-arterially delivered just before graft reperfusion. Animals were divided into CONTROL (CTRL), CONTROL+Ad-MSC (CTRL_MSC), tacrolimus (TAC), and TAC+Ad-MSC (TAC_MSC) groups. Each Ad-MSC groups was subdivided in autologous and allogeneic third-party groups. Rejection rate and severity were similar in MSC-treated and untreated animals. CTRL_MSC animals showed a decrease in macrophages, T-cell (CD4, CD8, and Foxp3 subsets) and B-cell counts in the graft compared with CTRL, this decrease was attenuated in TAC_MSC animals. Pro- and anti-inflammatory cytokines and some chemokines and growth factors increased in CTRL_MSC animals, especially in the allogeneic group, whereas milder changes were seen in the TAC groups. Ad-MSC did not prevent rejection when administered just before reperfusion. However, they showed immunomodulatory effects that could be relevant for a longer-term outcome. Interference between tacrolimus and the MSC effects should be addressed in further studies.

P2A.07: Mucosal T-cell kinetic in the early phases of rat model of allogenic small bowel transplantationMucosal T-cell kinetic in the early phases of rat model of allogenic small bowel transplantation

Introduction: Intestinal transplantation (IT) faces many challenges, among them, the necessity to early detect and treat rejection processes. Improving our understanding on the dynamics of allogenic response will be useful to bring candidate targets to these aims. The objective of our study was to determine the kinetics of mucosal T-cells population in the early phase of small bowel rejection using a Heterotopic IT rat model. We made emphasis in phenotypical, activation and exhaustion markers for the early detection of the rejection phenomena. Methods: Allogeneic heterotopic small bowel IT (n=5) was performed following standard procedure. Brown Norway animals were used as donors and Lewis as recipients. Rats did not receive any immune suppressor treatment. Rejection was monitored by clinical scoring and hematoxylin-eosin staining of intestinal grafts (Gf). T-cells from the peripheral blood (PB) and small bowel mucosa were assessed by flow cytometry. CD3, CD4, CD8, CD25, CD45, CD45RC, PD-1, FoxP3 and anti-Lewis MHC I expression was analyzed. Gf samples were taken at day 0, 3 and 7 post-operative (POD). Results: No clinical signs of rejection or significant histological changes were observed at day 3. At this time, in PB 7.9 % ± 0,7 of T cells were from the donor, meanwhile in the Gf 42,7% ± 4,6 of T-cells were recipient cells. We observed a significant increase of CD3+CD4+CD25+, CD3+CD4+CD45RC+ and CD3+CD8+CD45RC+ % in comparison with basal levels (PB:p<0,05;Gf:p<0,01). CD3+CD4+CD25+FoxP3+ cells were also increased in the Gf. At day 7 animals present significant weight loss and histological features compatible with mild to moderate cellular rejection. Differently from day 3 POD, in PB we could not detect donor T-cells. In the Gf 70% ± 7 of T-cells were from the recipient. CD3+CD4+CD45RC+ and CD3+CD8+CD45RC+ % were significantly higher than at day 3 POD (PB p<0,001;Gf:p<0,01). We detected increased levels of CD3+CD4+PD-1+ and CD3+CD8+PD-1+ cells in the Gf in comparison to basal levels(p<0,01). No differences in CD25 expression was observed between day 3 and 7. Conclusion: T-cell replacement in the Gf mucosa is a very fast process and CD25 expression in both PB and the Gf is an early event that precede the histological changes and should be analyzed as a potential biomarker. Considering that T-cells expressing CD45RC have been described as key players in the Gf rejection processes, our results also suggest they could be a relevant target to treat/prevent ACR.

Adipose-derived mesenchymal stem cells suppress of acute rejection in small bowel transplantation.

Background/Aims:Adipose-derived mesenchymal stem cells (ADSCs) possess immunosuppressive activity and hold promise in autologous cell-based therapies. The aim of this study was to determine whether autologous ADSCs can improve outcomes in the rat small bowel transplantation (SBT) model.Materials and Methods:Allogeneic SBT followed by implantation of autologous ADSCs through the penile vein was conducted in Brown-Norway (BN) donor rats with Lewis (LEW) recipient rats infused with phosphate buffered solution as the control. Allograft and recipient peripheral blood were obtained. We assessed histopathology, apoptosis, cytokines, and regulatory T cells (Tregs). One-way analysis of variance was applied to assess the significance of the data.Results:It was found that ADSCs significantly reduced acute rejection and improved the allograft's survival rate. In addition, there were significantly fewer apoptotic cells in allograft mucosae in the ADSC group in comparison with the control group. Furthermore, levels of interleukin (IL)-10 and transforming growth factor-β1 were significantly elevated, whereas those of IL-2 and IL-17 levels were significantly reduced in the ADSC group when compared to the control group. Moreover, flow cytometry analysis revealed that there were significantly more peripheral Tregs after the infusion of ADSCs.Conclusions:These results demonstrate that implanted autologous ADSCs improve allogeneic small bowel allograft outcomes by attenuating the acute rejection and reducing inflammatory responses.

Reduction of acute rejection by bone marrow mesenchymal stem cells during rat small bowel transplantation.

BackgroundBone marrow mesenchymal stem cells (BMMSCs) have shown immunosuppressive activity in transplantation. This study was designed to determine whether BMMSCs could improve outcomes of small bowel transplantation in rats.MethodsHeterotopic small bowel transplantation was performed from Brown Norway to Lewis rats, followed by infusion of BMMSCs through the superficial dorsal veins of the penis. Controls included rats infused with normal saline (allogeneic control), isogeneically transplanted rats (BN-BN) and nontransplanted animals. The animals were sacrificed after 1, 5, 7 or 10 days. Small bowel histology and apoptosis, cytokine concentrations in serum and intestinal grafts, and numbers of T regulatory (Treg) cells were assessed at each time point.ResultsAcute cellular rejection occurred soon after transplantation and became aggravated over time in the allogeneic control rats, with increase in apoptosis, inflammatory response, and T helper (Th)1/Th2 and Th17/Treg-related cytokines. BMMSCs significantly attenuated acute cellular rejection, reduced apoptosis and suppressed the concentrations of interleukin (IL)-2, IL-6, IL-17, IL-23, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ while upregulating IL-10 and transforming growth factor (TGF)-β expression and increasing Treg levels.ConclusionBMMSCs improve the outcomes of allogeneic small bowel transplantation by attenuating the inflammatory response and acute cellular rejection. Treatment with BMMSCs may overcome acute cellular rejection in small bowel transplantation.

Nursing of one case undergoing allogeneic small bowel transplantation

Objective To summarize the main nursing point and experience of allogeneic small bowel transplantation.Methods One case underwent allogeneic small bowel transplantation in our hospital on the first of December 2010,the nursing measures were summarized.Results The patient passed the operation well-off,and survived the infection phase and repulsion phase in the transplantation ward.Conclusions Valid preoperative evaluation,taking active measures to prevent intraoperative hypothermia and infection,using self-made vacuum pads to prevent pressure sores during operation,are the guarantees for successful operation. Key words: Small intestine transplantation; Allogeneic; Nursing care

Effects of immunosuppressive therapy after experimental small bowel transplantation in rats

Significant improvements of graft and patient survival have been achieved over the past 20years in the field of intestinal transplantation. Tacrolimus monotherapy with corticosteroids, or in combination with sirolimus is the most commonly used immunosuppressive regimen. Early (24h) after experimental allogenic small bowel transplantation in rats, sirolimus reduces the cellular and molecular inflammatory response with subsequent graft dysmotility more efficiently than tacrolimus, with contrary effects at 7days after transplantation. This study evaluates three immunosuppressive strategies in the post-acute phase after intestinal transplantation – tacrolimus or sirolimus monotherapy and the combination therapy of tacrolimus with infliximab. After orthotopic intestinal transplantation between Brown Norway and Lewis rats, animals received 14days of immunosuppressive treatment. Histology, infiltration of neutrophils, monocytes and macrophages, cytokine and mediator mRNA expression (real time RT-PCR) and smooth muscle function in a standard organ bath were assessed at 14days after transplantation in all treatment groups and isogenic controls. Allogenic transplanted rats without immunosuppressive therapy and non-transplanted animals served as further control. Tacrolimus prevented acute rejection and graft dysmotility more effectively (p≥0.05) than sirolimus. Reduced mRNA expression levels of CD4, IFN-γ, IL-6, IL-10, iNOS, NFκB, TNF-α and MCP-1 (p≤0.05) were observed in tacrolimus treated animals compared to sirolimus. Additional infliximab application did not influence the cellular and molecular inflammatory response in the post-acute phase after transplantation.In conclusion, the severe cellular and molecular inflammatory response in allogenic transplanted grafts without immunosuppressive therapy is ameliorated by all three immunosuppressive regimens, but tacrolimus was found to be more efficient than sirolimus at 14days after transplantation. Our findings do not rule out the usage of sirolimus as single immunosuppressive therapy, but indicate and confirm the potency and effectivity of tacrolimus as basis immunosuppressant in the field of intestinal transplantation.

Cyanoacrylate-assisted arterial anastomosis in rat small bowel transplantation

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). Allogeneic small bowel transplantation was performed in a rat combination of F344 to Lewis. CAST and SCST were used for anastomosis of graft superior mesenteric artery to recipient infrarenal aorta. Success rate of anastomosis, anastomosis time, anastomotic bleeding, and stenosis as well as tissue reactivity of suture materials and their influence on allograft rejection were compared between the two techniques. CAST significantly reduced anastomosis time and anastomotic bleeding, although its success rate was similar to SCST. Moreover, CAST created regularly shaped anastomotic orifice and smooth luminal surface along the anastomotic line, and provided higher blood flow of graft superior mesenteric artery. The use of n-butyl-cyanoacrylate caused moderate to mild tissue reaction that was comparable to nylon suture; and it did not significantly influence allograft rejection. CAST is a simple, effective, and safe surgical technique for microarterial anastomosis in rat SBTx. It deserves the interest in exploration of facilitated microarterial anastomosis strategies in animal experiments as well as in clinical settings.

Application of a Self-Made Swivel Intravenous Transfusion Device in Constructing Allogenic Small Bowel Transplantation Rejection Model in Rats

Background Lacking a long-term continuous intravenous infusion device for experimental animals is the biggest obstacle to constructing an allogenic small bowel transplantation rejection model. Objective To establish a rat heterotopic small bowel transplantation model with the use of a swivel device for intravenous transfusion. Methods The randomized block design of controlled observations included 90 inbred male F344/NCrl BR rats as donors and 90 LEW/Crl rats as recipients. On the basis of body weight, the rats were divided into three groups each with 30 pairs of rats. Group 1, the control group, underwent small bowel transplantation. Group 2, the transfusion group, received small bowel transplantation and continuous infusion of parenteral nutrient solution. Group 3, the tacrolimus (FK506) treatment group, received small bowel transplantation, continuous infusion of parenteral nutrient solution, and intravenous injection of FK506. The general status and survival time of transplanted rats were observed for 5 weeks. Results The operative success rate was 100%. The survival rate was 83.3% (25/30) in the control group; and 96.7% (58/60) in groups 2 and 3 ( P = .039). The mean survival time of the FK506 group was 22.1 ± 13.4 days, which was significantly higher than that in the transfusion group (10.4 ± 2.9 days, P = .023). Conclusion The simple, practical swivel intravenous infusion device provided treatment for more than 30 days, a useful tool for heterotopic segmental small bowel transplantation.

Short-Term Anti-CD4 Plus Anti-TNF-α Receptor Treatment in Allogeneic Small Bowel Transplantation Results in Long-Term Survival

Despite improved immunosuppression, intestinal transplantation is still complicated by severe rejection episodes. To further improve immunosuppressive concepts, we evaluated an anti-CD4 antibody and an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody for their immunosuppressive efficacy in the standard rat model of intestinal transplantation. Intestinal transplantation was performed in the DA to Lewis combination, and recipients were treated perioperatively with either the anti-CD4 antibody RIB5/2 (day -1, 0, postoperative days 1, 2, 4, 7, 10, 14, 17, and 21), the anti-TNF antibody etanercept (60 min before reperfusion, postoperative days 3, 6, and 9) or a combination of both. Survival, histology and expression of immunologic mediator genes on days 3 and 4 after transplantation were investigated. Treatment with anti-CD4 antibody alone (19.71+/-5.94) and the antibody combination (171.58+/-122.76) prolonged survival. The chemokine MIP-1alpha was significantly decreased in both anti-CD4 antibody treatment groups, possibly indicating an additional effect of the TNF-alpha blockade on the immune modulation by RIB5/2. Our study demonstrated long-term graft survival in short-term treatment with a combination of an anti-CD4 antibody and a TNF-alpha antibody in more than 50% of the recipients of intestinal grafts. Such a combined approach could also be useful in clinical small bowel transplantation.

STAT-1 decoy oligonucleotide improves microcirculation and reduces acute rejection in allogeneic rat small bowel transplants

During acute rejection leukocyte-endothelial cell interaction fuelled by costimulatory molecules such as the CD40/CD154 receptor/ligand dyad disrupts microcirculation of the small bowel. Downregulating endothelial CD40 expression by employing a decoy oligonucleotide (dODN) neutralizing the transcription factor signal transducer and activator of transcription-1 (STAT-1) may protect the graft. Therefore allogenic small bowel transplantation was performed in the Brown Norway to Lewis rat model. Graft vessels were pretreated with STAT-1 dODN, mutant control ODN (20 microM) or vehicle (n=8). CD40 antisense ODN and scrambled control ODN-treated transplants served as target control (n=3 each). Intravital microscopy, histology, immunohistochemistry and Western blot analyses were performed 7 days later. Functional capillary density, red blood cell velocity and perfusion index in STAT-1 dODN and CD40 antisense ODN-treated transplants were improved whereas stasis index was reduced. Leukocyte-endothelial cell interaction showed no difference. Histological parameters of rejection, infiltrating CD3-positive cells and apoptotic bodies were also reduced in STAT-1 dODN and CD40 antisense ODN-treated transplants 7 days post-transplantation. CD40 protein abundance was reduced to less than 10% of control in STAT-1 dODN-treated grafts. STAT-1 dODN blockade of CD40 expression improves mucosal perfusion, reduces graft rejection, T-cell infiltration and apoptosis in rat small bowel allografts during acute rejection.

Features of chronic allograft rejection on rat small intestine transplantation

The aim of this study was to develop a model of chronic rejection of the entire small intestine transplantation and to analyze the features of chronic rejection. Allogenic small bowel transplantation was performed in a rat combination of Lewis to F344. Intestines were procured at the 60th and the 90th day after operation. We compared the semiquantitative score of histological parameters. The immunological components involved in the chronic rejection process were evaluated by immunohistochemical staining and the cytokine levels in grafts. The significant characteristics of the allograft on histology were changes of villous architecture, interstitial fibrosis, leukocyte infiltration, and obliterative arteriopathy. Allografts on the 60th day post-transplantation had more score in inflammatory events, while the grafts on the 90th day after operation had more values in ischemia/fibrotic events. The number of infiltrating CD4, CD8 and macrophage cells in allografts progressively decreased over time. The level of intragraft cytokines such as IL-6, TNF-alpha and IL-10 in the 90th day after transplantation also decreased compared with that in the 60th day. These data suggested that in the early stage (POD 60), there were more active and intense inflammatory events; later (POD 90) allografts manifested less inflammation and more arterial obliteration and fibrosis.

Small bowel transplantation tolerance achieved by costimulatory blockade leading to mixed chimerism

We evaluated mixed chimerism with costimulatory blockade for the achievement murine allogeneic small bowel transplantation (SBTx) tolerance. B6 mice received various combinations of anti-CD8 (day -2) and anti-CD154 mAbs with or without 3Gy total body irradiation (TBI) (day -1), and 20 x 10(6) fully MHC-mismatched B10.A bone marrow cells (BMC, day -1). Heterotopic SBTx was performed on day 0. Chimerism in peripheral blood was followed by flow cytometric (FCM) analysis and the frequency of TCR Vbeta usage was determined by FCM to assess deletion of donor-reactive T cells. All animals without any treatment (n=6) showed acute rejection within 18 days after transplantation. Mice treated with anti-CD8 and anti-CD154 mAbs alone rejected their grafts within 100 days after transplantation (n=10). Mice treated with anti-CD8 and anti-CD154 mAbs, TBI, and BMT achieved long-term multilineage mixed chimerism and accepted small bowel allografts permanently (>350 days) without any evidence of graft-versus-host disease(n=11). There was specific deletion of donor-reactive cells and skin was accepted as allografts from B10.A donors, but 3rd party B10.BR skin was rejected. Donor-specific tolerance was achieved by inducing mixed chimerism with costimulatory blockade in murine SBTx recipients. This approach which provides a reliable method to induce SBTx tolerance, has potential clinical applications.

Neuroendocrine-Immune Modulation May Be Useful for Allograft-Specific Immunosuppression in Small Bowel Transplantation

Purpose The authors have previously demonstrated that the neuropeptide bombesin (BBS) prevented allograft mucosal atrophy under tacrolimus (TRL) immunosuppression for rats small bowel transplantation (SBT). The present study investigated whether BBS had immunosuppressive effects on small bowel allografts. Methods Allogeneic SBT was performed heterotopically in rats ( n = 12) that received daily administration of 0.1 mg/kg/d TRL from postoperative day 0 to day 14. Rats divided into two groups of six rats each were administered BBS or normal saline as a control. Biopsy of the allograft was performed from the stomal site on postoperative days 6, 10, and 14. The state of the graft mucosal villi was evaluated by H & E staining and TUNEL immunohistochemistry. Results By postoperative day 14, extensive mucosal destruction accompanied by heavy transmural cellular infiltration had developed in the control group. Lymphocytes and plasma cells infiltrated the lamina propria of the allograft without the distorting villous architecture in the BBS group. The TUNEL index of graft mucosa in the control group was 1.26% ± 0.37% (mean ± SD) and that in the BBS group, 0.59% ± 0.20%, respectively (p < .001). Conclusion This study demonstrated an immunosuppressive effect of bombesin on transplanted allografts, which might dramatically reduce the dose of TRL required for postoperative immunosuppression.

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.

Immunologic Benefits of Longer Graft in Rat Allogenic Small Bowel Transplantation

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. 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. 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. We have demonstrated experimentally that long intestinal grafts have immunologic advantage over short grafts.

Intragraft distribution of lymphocytes expressing β7 integrins after small bowel transplantation in mice

Lymphocytes with activated β7 integrins (αEβ7 and α4β7) 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 β7 integrins may influence the rejection response within intestinal transplants. To further delineate the nature of β7 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 α4β7 and decreased expression of L-selectin, an adhesion molecule profile characteristic of intestinal effector cell phenotypes. An increase in α4β7 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 α4β7 and low levels of αεβ7 to that of resident IEL with high levels of αεβ7 and low levels of α4β7. The importance of β7-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 β7-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 α4β7 constitute a major effector cell population that affects the entire graft.

MIXED CHIMERISM WITH COSTIMULATORY BLOCKADE PROLONGS ALLOGENEIC GRAFT SURVIVAL IN MURINE SMALL BOWEL TRANSPLANTATION.

P1032 Aims: Mixed chimerism is known as one of the most powerful method to induce donor specific tolerance. Here we evaluate the potential advantage of mixed chimerism with costimulatory blockade in murine allogeneic small bowel transplantation. Methods: C57BL/6 received various combination of anti-CD8 (2.43; 1.4mg, day -2) and anti-CD154 (MR1; 2mg, day -1)mAbs with/without 3Gy total body irradiation (TBI, day -1), and 20×106 fully MHC-mismatched B10.A bone marrow cells (BMC, day -1). Heterotopic small bowel transplantation was performed on day 0 to assess induction of donor specific tolerance. Chimerism in peripheral blood was followed by FCM analysis and the frequency of TCR Vβ usage was determined by FCM to assess deletion of donor-reactive T cells. Stoma was daily observed, and acute rejection was evaluated with a microscope on POD14. Results: All animals without any treatment (n=6) showed acute rejection within 18 days after transplantation, and microscope examination presented the findings of acute rejection such as flatttend villous epithelium with the infiltration of inflammatory cells. Mice receiving anti-CD8 and anti-CD154mAbs were not induced mixed chimerism, and 4 of 8 mice rejected small bowel allograft by 79 days. All animals treated with anti-CD8mAb, anti-CD154, TBI, and BMC was induced long term multi-lineage mixed chimerism and accepted small bowel allografts (>60 days). Microscope examination presented the no findings of rejection. These chimeric animals showed specific deletion of donor reactive CD4+ peripheral blood lymphocytes (Vβ5 and Vβ11 cells). Conclusions: Prolongation of allogeneic small bowel graft survival was achieved using mixed chimerism with costimulatory blockade. Mixed chimerism with costimulatory blockade is the reliable regimen toward to the small bowel transplantation.

Immune responses of graft mesenteric lymph node in small bowel transplantation

Background The high proportions of lymphoid tissues are thought to be one of the underlying factors inducing severe allograft rejection following small bowel transplantation. Mesenteric lymph nodes (MLN) contained in the intestinal graft are not only a source of donor-derived professional antigen-presenting cells, but also offer a field for immune interaction between donor and host cells. We investigated immune responses in graft MLNs with or without FK506 to develop a novel strategy to control small bowel allograft rejection. Materials and methods Heterotopic small bowel transplantations were performed from Brown Norway donors to Lewis recipients. Changes in population of lymphocytes, expressions of costimulatory molecules, apoptosis, and cytokine profiles in graft MLNs were evaluated. Results The increase in apoptotic cells and cytokine responses relating to rejection in the graft MLNs developed prior to those in graft jejunum. While donor lymphocytes in graft MLNs were rapidly replaced to host-derived lymphocytes independent of FK treatment, increase in CD8 + T cells in host population was seen only in recipients without FK506 treatment. The expressions of B7 molecules on donor cells in graft MLNs were significantly lower in the recipients with FK treatment. Conclusions Immune responses in graft MLNs have significant impact on the outcome of the small bowel allograft. Apoptosis of graft MLN cells was well correlated with and ahead of progression of acute rejection. Modulation of costimulatory molecules on donor-derived MLN cells in the allograft and specific suppression of host CD8 + T cells are possible ways to control severe rejection after allogeneic small bowel transplantation.

Small bowel allografts maintained by administration of Bombesin while under immunosuppression

Background/Purpose: The aim of this study was to determine if Bombesin (BBS) could help maintain the mucosal villus state in small bowel allografts without inducing acute rejection under immunosuppression. Methods: Allogeneic small bowel transplantation was performed heterotopically in rats (n = 12). All rats received daily administration of FK506 from postoperative day 0 to day 28. On postoperative day 14, rats were divided into 2 groups of 6 rats each, and administered BBS or normal saline as a control. After 2 weeks of treatment, the rats were killed, and the graft mucosal villus state was evaluated by H&E staining, and crypt cell proliferation analysis was performed using immunohistochemistry with proliferative cell nuclear antigen (PCNA). Results: Villi were thin, and villus blunting was marked in the control group. The BBS group showed that the villi of the grafts were well maintained, and the volume of the lamina propria mucosa was adequately preserved. The PCNA labeling index of crypt cells in the control group was 40.06 ± 3.36 (mean ± SD) and that in the BBS group was 61.02 ± 4.27. There was a significant difference (P <.001) between the 2 groups. Conclusions: BBS maintained allograft epithelial cells and the volume of the lamina propria intestinal mucosa, stimulating proliferation of crypt cells under immunosuppression without inducing acute rejection. J Pediatr Surg 38:83-87. Copyright 2003, Elsevier Science (USA). All rights reserved.

Expression of Mucosal Addressin Cell Adhesion Molecule-1 (MAdCAM-1) During Small-Bowel Graft Rejection in Rats

Background: Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a critical endothelial adhesion molecule for lymphocyte trafficking to gut-associated lymphoid tissues (GALT) under physiological conditions. It is expressed on special postcapillary venules, that is the high endothelial venules (HEV) in GALT. In this study, we investigated changes in MAdCAM-1 expression during small-bowel graft rejection. Methods: Orthotopic small-bowel transplantation (SBT) with portacaval drainage was performed from brown Norway (BN) rats to Lewis (LEW) rats. Isografted (LEW → LEW) and untransplanted animals served as controls. Animals were killed on days 3, 4, 5, 6 and 7 after SBT. Cryostat sections (6 μm thick) were prepared from normal small-bowel tissues and small-bowel grafts, including Peyer patches (PPs). Indirect immunoperoxidase staining was performed using monoclonal antibodies (mAbs) against rat MAdCAM-1 (OST12). Results: In the PPs of controls, MAdCAM-1 antibodies specifically stained the endothelial cells of HEV, which were predominantly located in the interfollicular areas. In the allografts on day 4 after SBT, the MAdCAM-1 expression was weaker on the HEV in the PPs than in the controls. In the lamina propria of controls, a faint expression of MAdCAM-1 on vessels was observed. On day 4 after allogeneic SBT, the MAdCAM-1 was more strongly expressed on the endothelial cells of the vessels at the base of the villi. As rejection developed, the MAdCAM-1 expression on the vessels progressed toward the villus tip. Conclusions: The change of MAdCAM-1 expression may be involved in the development of small-bowel graft rejection. The vessels at the base of villi, which is associated with lymphocyte recruitment, may become a site of intense immune reactivity in the early phase of small-bowel allograft rejection.