Small Bowel Allograft Research Articles

A clinicopathologic study of isolated intestinal allografts with preformed lymphocytotoxic antibodies

Humoral rejection of human small bowel allografts has not been well documented. The aim of this study was to determine the significance of preformed IgG lymphocytotoxic antibodies, as determined by the modified Amos crossmatch technique, in 6 of 28 (21.4%) adult isolated intestinal allograft recipients treated with tacrolimus basal immunosuppression. The crossmatch with dithiothreitol (DTT) was strongly positive in 5 grafts and weakly positive in the remaining one. The strongly positive crossmatch grafts (n = 5) developed severe mucosal injuries immediately after reperfusion. Within the first 10 days after transplantation, 3 of the 5 strongly positive crossmatch grafts developed clinically and endoscopically documented severe mucosal congestion, cyanotic discoloration, and focal hemorrhage within the allograft. Simultaneous mucosal biopsy specimens demonstrated severe congestion, neutrophilic margination, and fibrin-platelet thrombi within the lamina propria microvasculature, along with focal hemorrhage, but with no histological neutrophilic or necrotizing arteritis, and the immunofluorescent findings were unremarkable. The other 2 strongly positive crossmatch allograft recipients developed macroscopic congestion of mucosa with microscopic foci of congestion and hemorrhage. In contrast, the recipient with a weakly positive crossmatch, as well as the 22 crossmatch negative recipients, exhibited none of these characteristic clinical, endoscopic, or microscopic findings ( P < .05). With prompt augmentation of the immunosuppression therapy, the 3 grafts with the characteristic clinicopathologic syndrome were successfully reversed with the monoclonal antilymphocyte preparation OKT3. Thus there was no statistical difference ( P > .05) in early graft survival and the frequency of acute rejection episodes between the crossmatch-positive and the crossmatch-negative groups within the first 6 months after transplantation. We conclude that preformed IgG lymphocytotoxic antibodies in isolated intestinal recipients can be associated with a characteristic clinicopathologic syndrome during the early postoperative course, similar to that observed in experimental animal models and in other solid organ transplantation. If recognized and treated aggressively, early graft failure can be avoided. The long-term significance of these preformed antibodies has yet to be determined.

REJECTION-MEDIATED INCREASE IN SMALL BOWEL (SB) EPITHELIAL TIGHT JUNCTION PERMEABILITY REQUIRES GRAFT EPITHELIAL NF-KAPPAB ACTIVATION

O389 Introduction: Sepsis is the major cause of morbidity and mortality in SB transplant recipients and enteric organisms are often responsible. Increased SB epithelial permeability has been shown to correlate with small bowel (SB) allograft rejection in humans and in animal models of SB transplantation. This compromise in SB allograft epithelial barrier function may exacerbate rejection and contribute to sepsis. The mechanism of rejection-mediated SB epithelial barrier dysfunction has not been defined. Tight junction proteins play a major role in regulating permeability in the intact SB epithelium. We hypothesize that alterations in graft epithelial tight junction function results in epithelial barrier dysfunction through NF-kappaB dependent mechanisms. Methods: SB tissues from C57BL6 → C57BL6 SB isografts and C57BL6 → BALB/c SB allografts were examined during early allograft rejection (POD 5). Mucosa permeability to different sized molecular probes (Alexa 350 hydrazide, FITC-Dextran 3000, and Texas Red-Dextran 10,000) were measured by Ussing Chamber. Tight junction-associated protein expression was examined by fluorescent staining with antibodies to occludin, claudin, ZO-1, and p-MLC. To determine the role of NF-kappaB activation in regulating tight junction protein expression following SB transplantation, SBs from double transgenic mice selectively expressing a mutated form of IkappaBalpha (mIkappaBalpha, a super-repressor of NF-kappaB) gene in their intestinal epithelial cells (mIkappaBalpha) were transplanted to BALB/c recipients. Results: 1) Ussing chamber studies identified a significant increase in mucosal to serosal flux of larger probes (i.e. Dextrans 3000 and 10,000) in the segments of the SB allograft compared to SB isograft (p<0.05) on POD 5. Flux of the smaller probe (Alexa 350 hydrazide) was not different from the isograft. 2) In SB isografts there were no differences in the epithelial distribution of these proteins compared to normal non-transplanted C57BL6 SB tissues. Conversely, the epithelial distribution of ZO-1, occludin, and p-MLC was altered in SB allografts compared to isografts and normal SBs. Specifically, while in the isograft and normal SB epithelium there was a smooth, linear, sub-membrane distribution of ZO-1, in SB allografts the distribution was beaded and irregular especially when viewed in tangential sections. More dramatic differences were seen with the epithelial distribution of occludin and p-MLC. While the epithelia of isografts and normal SBs had normal expression of occludin and p-MLC at the tight junctions, the SB allograft epithelia had a redistribution of these components from the tight junctions to intracytoplasmic inclusions. 3) Normal epithelial distribution of ZO-1, occludin, and p-MLC was seen in mIkappaBalpha allografts, suggesting a role of NF-kappaB activation in regulating tight junction functions during SB allograft rejection. Conclusions: These results indicate that early SB allograft rejection is associated with a defect in graft epithelial barrier function, which may be attributed to the changes in epithelial tight junction associated proteins. Furthermore, this rejection-mediated epithelial tight junction dysfunction is NF-kappaB–dependent.

EARLY SMALL BOWEL (SB) ALLOGRAFT REJECTION CAUSES A FUNCTIONAL DEFECT IN INTESTINAL EPITHELIAL SUCRASE ACTIVITY.

P1216 Introduction: Intestinal sucrase is a major epithelial brush border membrane hydrolase expressed by the SB epithelium. Sucrase activity has been used as a marker to differentiate mature villus cells from less mature crypt cells. A loss of sucrase activity has been associated with SB inflammation and reflects compromised epithelial function and/or a decrease in the ratio of villus to crypt cells, both features of early SB allograft rejection. Sucrase may therefore be useful as an early marker of acute SB allograft rejection and as a marker of graft dysfunction associated with chronic rejection. This study is to determine if changes in sucrase activity correlate with SB allograft rejection and to evaluate possible mechanisms underlying these changes. Methods: SB transplants using BALB/c donors and BALB/c recipients (isografts), or BALB/c donors and C57BL/6 recipients (allograft) were performed. Sucrase activity in graft epithelial proteins isolates on PODs 3, 5 and 7 were measured using standard methods. Sections of SB grafts were examined to establish the typical progression of histopathologic and morphometric changes. To evaluate the role of TNF/TNFR signaling pathway and Nuclear factor (NF)-kappaB activation in regulating sucrase activity during SB allograft rejection, SB grafts from B6 TNF receptor (R)-1 and R2 double knock out (TNFR−/−) mice or B6 mIkappaBalpha transgenic mice (with a SB epithelial specific defect in NF-kappaB activation) were transplanted to BALB/c recipients. Results: Our results revealed that posttransplant histopathologic changes including villus shortening, goblet cell loss and crypt hyperplasia progressed consistently in SB allograft between POD3 and POD7. Morphometric analysis showed a 40 percent decrease in the villus to crypt ratio by POD 5 in SB allograft, compared to isografts (P<0.01). Consistently, epithelial sucrase activity was significantly decreased in the SB allograft with 55% reduction on POD 5 (p<0.05,) and 85% on POD7 (p<0.01), compared to isografts. Interestingly, sucrase activity in both TNFR−/− SB allograft and mIkappaBalpha SB allografts were comparable to isografts. Conclusions: Small bowel (SB) allograft rejection is associated with an epithelial functional defect in intestinal epithelial sucrase activity, which suggests there is a defect of epithelial cell maturation or regeneration. Blocking TNF/TNFR signaling pathways or inhibiting epithelial NF-kappaB activation prevents this loss of sucrase activity, indicating an important connection between TNF and NF-kappaB in epithelial cell function during SB allograft rejection.

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.

BLOCKING NF-KAPPAB ACTIVATION IN RECIPIENT T CELLS PROLONGS SMALL BOWEL ALLOGRAFT SURVIVAL IN A MOUSE MODEL OF ORTHOTOPIC SB TRANSPLANTATION

P1027 Background: Previous studies reveal that blocking T-cell specific NF-kappaB activation permits long term survival of cardiac allografts but does not prevents skin allograft rejection, suggesting a differential mechanism for T-cell priming in response to different organs or tissues. It has also been known that NF-kappaB activation is critical in inflammatory bowel diseases. However, its role in small bowel allograft rejection has not been evaluated. Methods: To study the potential role of NF-kappaB activation in SB allograft rejection, nuclear extracts were isolated from mucosal scrapings of C57BL/6 (H-2b) isografts or C57BL/6 to BALB/c (H-2d) SB allografts on POD 7. Activation of NF-kappaB complex (p65) was quantified with an ELISA-based kit. To further determine if NF-kappaB activation in recipient T cells is required for SB allograft rejection, we have developed a mouse model of orthotopic small bowel transplantation in which host survival is entirely dependent upon graft function. IkappaBalpha transgenic mice (B6 background) with defective NF-kappaB translocation in T cells due to the presence of a NF-kappaB super repressor (a mutant IkappaBalpha transgene) were transplanted with BALB/c SB allografts with no additional immunosupression. Wild type B6 recipients of BALB/c SB allograft served as controls. Graft tissues were procured at POD 5 or when the recipient was moribund due to rejection (endpoint of rejection) for histology, immunopathology, as well as for function using an assay for sucrase activity. Results: We found that nuclear levels of NF-kappaB p65 subunit in SB isografts is 9.8+/− 1.6 ng/ml (mean+/− SD), while it was significantly increased in SB allografts (28.4+/−7.7 ng/ml, p<0.01). These results suggest that SB allograft rejection is associated with a significant upregulation of NF-kappaB. Our data on the survival of the orthotopic small bowel transplants showed that all B6 isografts (n=3) survival more than 100 days, while allografts (n=4) developed dehydration, diarrhea, weight loss and eventually died of graft failure by POD 9 with typical histologic features of SB allograft rejection. In addition, there was 50% decrease in epithelial sucrase activity as measured in SB allografts on POD 5 relative to the isografts. In contrast, BALB/c SB allografts transplanted into IkappaBalpha transgenic mice survived and continued to demonstrate weight gain at 4 weeks posttransplant. These grafts examined on POD 9 had no histologic evidence of rejection and demonstrated normal sucrase activity. Immunofluorescent staining showed significantly less CD4+ T cells and macrophages in the SB allografts transplanted into transgenic recipients compared to the WT SB allografts. Interestingly, CD8+ T cell infiltrates appeared similar in both SB allografts. Conclusions: Inhibition of NF-kappaB activation in recipient T cells prevents SB allograft rejection and preserves graft function, thereby prolonging survival in SB allografts and allograft recipients. These data suggest T-cell specific NF-kappaB activation is essential for SB allograft rejection and targeting this pathway may prove to be an effective strategy to prevent SB allograft rejection.

P0250* SAFETY AND EFFICACY OF SIROLIMUS IN CHILDREN FOLLOWING CHRONIC REJECTION AND/OR NEPHROTOXICITY POST INTESTINAL TRANSPLANT (ITX) AND LIVER TRANSPLANTATION (LTX).

Introduction: Chronic rejection after transplantation and drug induced nephrotoxicity continue to be major clinical problems. Sirolimus is a potent immunosuppressant acting on the mTOR receptor with no adverse effects on renal function. AIM To evaluate the safety and efficacy of Sirolimus in intestinal transplantation (ITx) and liver (LTx) transplant recipients in children. Methods: Retrospective review from Jan ’01 to Dec ’02. 14 children (9M): renal toxicity (n=5);recurrent rejection/acute steroid resistant rejection after ITx and/or LTx (n=9) were included. Results: Median age at transplant (Tx): 34 months. Median time post Tx at starting Sirolimus: 41.8 months. Sirolimus treatment followed up for 100–500 days. Patients receiving Sirolimus due to nephrotoxicity showed an improvement in cGFR from 52 to 73 ml/ml/1.73m2. Liver function tests improved in those with recurrent rejection and small bowel allograft function. Table 1 summarises data on efficacy and safety of Sirolimus. Adverse events: Infection: pneumonia (7 episodes), adenovirus (1), Influenza (1), EBV viraemia (4) and staphylococcus (2). Two children developed mucosal lesions, buccal mucosal ulcer (1) and candidial oesophageal lesions (1). 3 patients developed high triglycerides not requiring treatment. 5 children experienced transient thrombocytopaenia. Sirolimus temporarily withheld for EBV viraemia (1) and discontinued(1)for protracted neutropenia. Two patients with intestinal graft died after 8m and 15m exposure to Sirolimus (bowel perforation and fungal endocardititis).Table 1Conclusion: Sirolimus as a concomitant immunosuppressant is effective in acute steroid resistant and chronic rejection for ITx/LTx recipients. We recommend caution in the long term use of Sirolimus in ITx recipients

IP-10-induced recruitment of CXCR3 + host T cells is required for small bowel allograft rejection

Background & Aims : Chemokines mediate cell trafficking in inflammatory states such as allograft rejection. However, their role in small-bowel allograft rejection has not been defined. The aim of this study was to examine the roles of type 1 helper T-cell chemokines in small-bowel allograft rejection. Methods : Mucosal histology, chemokine messenger RNA (real-time polymerase chain reaction), and cell isolates were examined in small-bowel allografts and isografts. Interferon-γ-inducible protein-10/ CXC chemokine receptor (CXCR) 3 interactions were specifically evaluated by using allografts from interferon-γ-inducible protein-10 −/− donors and adoptive transfer of CXCR3 −/− T cells into recombination activating gene (RAG)-1 −/− recipients of small-bowel allografts. Results : Type 1 helper T-cell cytokine (interferon-γ) and chemokine (interferon-γ-inducible protein-10, monokine induced by interferon-γ, macrophage-inflammatory protein-1α, and regulated on activation, normal T cells expressed and secreted) messenger RNA up-regulation was detected (real-time polymerase chain reaction) by postoperative day 3 in small-bowel allografts. Interferon-γ-inducible protein-10 +/+ small-bowel allograft rejection was associated with a dramatic (>7-fold) increase in CXCR3 + host T cells in the graft lamina propria. With interferon-γ-inducible protein-10 −/− small-bowel allografts, CXCR3 + host T-cell infiltration of the graft lamina propria was markedly decreased and rejection was significantly delayed. Whereas adoptive transfer of wild-type B6 (CXCR3 +/+) T cells into B6 (RAG-1 −/−) recipients induced rapid rejection of CB6F1 small-bowel allografts, rejection was significantly delayed (29.2 ± 8.7 days vs. 16.5 ± 3.1 days; P < 0.01) in B6 (RAG-1 −/−) mice reconstituted with T cells from B6 (CXCR3 −/−) mice. Conclusions : Recruitment of CXCR3 + host T cells by donor derived interferon-γ-inducible protein-10 may precipitate small-bowel allograft rejection. These data highlight the importance of type 1 helper T cell-related chemokines in promoting cell-mediated rejection responses in small-bowel allografts and suggest that interferon-γ-inducible protein-10 is an attractive therapeutic target for humanized monoclonal antibody strategies.

Histological criteria for the identification of acute cellular rejection in human small bowel allografts: results of the pathology workshop at the VIII International Small Bowel Transplant Symposium

Acute cellular rejection remains a serious and frequent complication during the posttransplant course of small bowel allograft recipients. Currently, small bowel biopsies are the optimal method to identify this form of rejection. The morphological criteria for this diagnosis have been known for some time; however, no consensus study has classified these changes. To address issues in bowel transplant pathology, several pathologists experienced in this particular subdiscipline participated in a Pathology Workshop preceding the VIIIth International Small Bowel Transplant Symposium in Miami, Florida. Among the results of this workshop was the development a standardized grading scheme for acute cellular rejection in small bowel transplants.

Semiquantative analysis of expression of mucosal addressin cell adhesion molecule-1 during small bowel graft rejection in rats

Aim Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated lymphoid tissues (GALT). We performed a semiquantative analysis of MAdCAM-1 expression during small bowel graft rejection. Methods Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Animals were sacrificed on days 3, 4, 5, 6, and 7 after SBT. Cryostat sections were prepared from grafts, including Peyer's patches (PPs). Indirect immunoperoxidase staining was performed using mAbs against MAdCAM-1. The degree of vascular endothelial staining on high endothelial venules (HEV) in the PPs was graded from 1 (low levels) to 5 (high levels), and in the vessels of the lamina propria from 1 (faint), 2 (low at the base of villi), 3 (low to the middle of villi), 4 (high to the middle of villi), to 5 (high to villus tip). Results MAdCAM-1 expression on HEVs in PPs was down-regulated during rejection. In contrast its expression on endothelial cells of vessels in the lamina propria was up-regulated during rejection. Conclusion Alteration in MAdCAM-1 expression may be associated with the development of SB graft rejection. The vessels at the base of villi, which are associated with lymphocyte recruitment, may become sites of intense immune reactivity during the early phase of small bowel allograft rejection.

Lymphoplasmacytic hyperplasia (possibly pre-PTLD) has varied expression and appearance in intestinal transplant recipients receiving Campath immunosuppression

Posttransplant lymphoproliferative disorders (PTLD) are a frequent complication in bowel transplant recipients. Histological changes in PTLD range from expansile lymphoplasmacytic (LP) hyperplasia to frank lymphoma. Small bowel allograft biopsies obtained in the first 250 days posttransplant were retrospectively graded after patients had received induction immunosuppression with either anti-CD52 (Campath) or anti-CD25 (Zenapax) monoclonal antibodies. The biopsies were analyzed with respect to the onset intensity of lymphoplasmacytic infiltrates and presence of in situ EBV hybridization (EBER) positivity. We observed that lymphoplasmacytic infiltrates were a frequent change in all bowel transplant patients over the examined period. Campath-treated patients developed earlier LP infiltrates of mild to moderate intensity between day 1 and 100 posttransplant, thereafter decreasing to mild. No EBER positivity was detected in this group. Zenapax-treated patients presented with LP infiltrates later of mild to moderate intensity through day 100 posttransplant. However, more persistent and intense LP infiltrates was observed after day 101 in this group, including a case of lymphoma and two cases of EBER positivity. We conclude that Campath immunosuppression results in an earlier appearance of LP lesions that are generally less intense than those evident with Zenapax. We attribute these findings to the more profound immunodeficiency and cell targeting following Campath treatment.

Apoptosis of crypt cells and lymphocytes in gut-associated lymphoid tissues during small intestinal graft rejection in rats

Introduction We investigated the extent of apoptosis in crypt cells and Peyer's patches (PPs) during small bowel allograft rejection in rats to examine whether the Fas/FasL pathway participates in apoptosis within grafts during rejection. Materials and methods Orthotopic small bowel transplantation with portocaval drainage was performed from Brown Norway to Lewis (LEW) rats. Isografted (LEW → LEW) and nontransplanted animals served as the controls. Animals were sacrificed on days 3, 5, on 7 after SBT (each n = 5). An in situ end-labeling (ISEL) technique was used to detect apoptotic cells. Indirect immunoperoxidase staining was also performed using monoclonal antibodies against rat Fas or Fas-L. Results The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7. Similarly, in the PPs of the allografts, the number of ISEL-positive mononuclear cells increased significantly on days 3, 5, and 7. On day 7 the number of Fas- and FasL-positive enterocytes were increased significantly in the allografts compared with the nontransplanted controls. Similarly, in the PPs, Fas- and FasL-positive mononuclear cells also increased significantly on day 7 in the allograft. Conclusion Although an increase, number of apoptotic enterocytes and lymphocytes were observed in the early phase, activation of Fas/FasL system occurred during the late phase of small bowel graft rejection. These findings suggest that both rejection-associated and sepsis-induced forms of apoptosis may be associated with small bowel graft rejection.

Campath-1H immunosuppressive therapy reduces incidence and intensity of acute rejection in intestinal and multivisceral transplantation

Campath-1H, an anti-CD52 antibody, is being used at our institution as immunosuppression in multivisceral and intestinal transplantation. We reviewed the pathologic findings of 1696 small bowel allograft biopsies obtained in the first 250 days posttransplant from 78 patients who underwent isolated intestinal or multivisceral transplantation and received induction immunosuppression with Campath (n = 30) or Zenapax (n = 57). We found an overall reduced incidence of acute cellular rejection (ACR) in patients receiving Campath (19.1%) compared with those on Zenapax (32.8%). The majority of Campath patients showed no rejection or was indeterminate for rejection over the period of measurement. The frequencies of mild and moderate ACR were approximately twice and three times more common, respectively, in Zenapax-treated patients. The mean grade of ACR in Campath patients compared with Zenapax patients was significantly lower ( P < .01) during the first 6 weeks posttransplant. Thereafter, the grade of rejection in both patient groups showed fluctuation, with Zenapax patients sometimes having lower values (eg, at 2 to 4 months) than Campath patients. Patient and graft survival was not significantly different between the two groups. These data suggest that the incidence of ACR is significantly reduced with Campath during the first 2 months posttransplant, when compared with Zenapax. However, the incidence and intensity of ACR following this initial time period shows vacillation with both types of immunosuppression.

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.

Cyclosporin A differentially inhibits multiple steps in VEGF induced angiogenesis in human microvascular endothelial cells through altered intracellular signaling

The immunosuppressive agent cyclosporin A (CsA), a calcineurin inhibitor which blocks T cell activation has provided the pharmacologic foundation for organ transplantation. CsA exerts additional effects on non-immune cell populations and may adversely effect microvascular endothelial cells, contributing to chronic rejection, a long-term clinical complication and significant cause of mortality in solid-organ transplants, including patients with small bowel allografts. Growth of new blood vessels, or angiogenesis, is a critical homeostatic mechanism in organs and tissues, and regulates vascular populations in response to physiologic requirements. We hypothesized that CsA would inhibit the angiogenic capacity of human gut microvessels. Primary cultures of human intestinal microvascular endothelial cells (HIMEC) were used to evaluate CsA's effect on four in vitro measures of angiogenesis, including endothelial stress fiber assembly, migration, proliferation and tube formation, in response to the endothelial growth factor VEGF. We characterized the effect of CsA on intracellular signaling mechanisms following VEGF stimulation. CsA affected all VEGF induced angiogenic events assessed in HIMEC. CsA differentially inhibited signaling pathways which mediated distinct steps of the angiogenic process. CsA blocked VEGF induced nuclear translocation of the transcription factor NFAT, activation of p44/42 MAPK, and partially inhibited JNK and p38 MAPK. CsA differentially affected signaling cascades in a dose dependent fashion and completely blocked expression of COX-2, which was integrally linked to HIMEC angiogenesis. These data suggest that CsA inhibits the ability of microvascular endothelial cells to undergo angiogenesis, impairing vascular homeostatic mechanisms and contributing to the vasculopathy associated with chronic rejection.

Effect of local CTLA4Ig gene transfection on acute rejection of small bowel allografts in rats.

To evaluate the local expression of CTLA4Ig gene in small bowels and its effect on preventing acute rejection of the small bowel allografts. Groups of Wistar rats underwent heterotopic small bowel transplantation from SD rats. The recipients were randomly divided into experimental group (allografts were transfected with CTLA4Ig gene) and control group (non CTLA4Ig gene transfected). In the experimental group, the donor small bowels were perfused ex vivo with CTLA4Ig cDNA packaged with lipofectin vector via intra-superior mesenteric artery before transplantation, and the CTLA4Ig expression in the small bowel grafts post-transplantation was assessed by immunohistology. On d 3, 7 and 10 post-transplantation, the allografts in each group were harvested for the examination of histology and detection of apoptosis. Small bowel allografts treated with CTLA4Ig cDNA showed abundant CTLA4Ig expression after transplantation. Acute rejection of grade I on d 7 and grade II on d 10 after transplantation was noticed in the control allografts, and a dramatically increased number of apoptotic enterocytes in parallel to the progressive rejection could be recognized. In contrast, the allografts treated with CTLA4Ig cDNA showed nonspecific histological changes and only a few apoptotic enterocytes were found after transplantation. Local CTLA4Ig gene transfection of small bowel allograft is feasible, and the local CTLA4Ig expression in the allograft can prevent acute rejection after transplantation.

Sclerosing mesenteritis in small bowel transplantation: possible manifestation of acute vascular rejection

Background Acute rejection of human small bowel allografts is characterized by clinical symptoms combined with characteristic morphologic alterations. The typical geographic distribution of acute rejection in the bowel is involvement of the intestinal parenchyma, which can be transmural, particularly when the rejection is more severe. However, little is known concerning the potential for donor-derived soft tissue adjacent to the bowel to become involved by the host alloimmune response. Methods We describe a male patient who, several weeks after combined small bowel and liver transplantation, demonstrated sclerosing mesenteritis with vasculitis and acute rejection of the bowel. Results The vascular lesions in the mesentery demonstrated increased IgG deposition and the patient developed an alloantibody to the donor. Conclusions The changes described herein may represent a novel presentation of acute vascular rejection.

Histamine-degrading enzymes as cellular markers of acute small bowel allograft rejection

Abstract Intestinal histaminedegrading enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HNMT) activities are relatively constant per individual and bowel segment, and they reflect the functional integrity of the intestinal mucosa. It was, therefore, hypothesised that a decrease in these enzymes could be indicative of acute rejection of an intestinal allograft. Enzymatic activities of DAO and HNMT were determined in mucosal biopsies of isogeneic (Lewis-to-Lewis, n=48) and allogeneic (Brown Norway-to-Lewis, n=48) heterotopic small bowel transplants in a rat model at various time periods. Allograft recipients were not given any immunosuppression. While no changes in enzyme activities were observed in isografts up to day 8 following transplantation, significantly reduced activities of both enzymes were found in all allografts 6–8 days after transplantation. Activities of both DAO and HNMT exhibited a strong negative correlation with the histological rejection score (P<0.01). We can conclude that DAO and HNMT activities in gut mucosa are reliable quantitative markers of acute intestinal allograft rejection in the rat that support histopathological analysis.

Preclinical results of sirolimus treatment in transplant models

Sirolimus (SRL; rapamycin) is a macrolide antibiotic, which modest anticandidal and tumoricidal activities were superseded by its immunosuppressive potential to block allograft rejection. The most intriguing biological characteristic of SRL emerged after demonstration of its potent synergism with cyclosporine (CsA). Naïve T cells, residing in the G 0 phase of the cell cycle, become activated by three signals. Signal 1 (T cell antigen receptor/alloantigen) and Signal 2 (CD28/B7) progress T cell to the early G 1 phase inducing production of interleukin-2 (IL-2) and other T cell growth factors (TGFs). Signal 3 (cytokine/cytokine receptor) initiate cell division and differentiation in the late G 1/S phase. Whereas CsA binding to calcineurin blocks Signal 1/2, SRL binding to mammalian target of rapamycin (mTOR) blocks Signal 3. Our preclinical studies have established the in vivo principles of the effects exhibited by SRL alone on allograft survival, synergism between SRL and CsA as well as two drugs pharmacokinetic and pharmacodynamic interactions. In our experimental model, a 14-day i.v. continuous infusion of SRL by osmotic pump into rat recipients extended the survivals of heart allografts in a dose-dependent fashion. In comparison to untreated controls (MST of 6.3 ± 0.5 days), 0.08 mg/kg SRL extended MST to 34.4 ± 12.1 days, and 0.8 mg/kg to 74.1 ± 20.2 days, with 6/18 allografts surviving for more than 100 days. Since almost identical results were produced by 10-fold higher SRL doses delivered by oral gavage, we estimated its bioavailability at 10%. Similarly, SRL prolonged the survivals of kidney, pancreas, and small bowel allografts in rats. At the same time large animal models cautioned about potential toxicities, namely intestinal vasculitis. The synergistic interactions of CsA and SRL may be explained by sequential effects in the early G 0/G 1 versus late G1/S phases of cell cycle progression, respectively. The in vivo interaction of SRL with other immunosuppressive drugs was evaluated by the median effect analysis and the combination index (CI) values (CI = 1 shows additive, CI < 1, synergistic, and CI > 1, antagonistic, interactions). Oral SRL proved to be synergistic in both CsA-resistant mouse (CI = 0.4–1.5) and CsA-sensitive rat (CI = 0.3–0.6) models. The pharmacokinetic interactions of SRL and/or CsA were evaluated in rats for i.v. and oral formulations. Although low CsA and SRL i.v. doses did not affect each other levels, potent interaction was observed after oral gavage: CsA increased SRL levels by 2–11 folds; and, SRL increased CsA levels by 2–3-folds. Our results suggested that both pharmacodynamic and pharmacokinetic interactions contribute to the synergism between SRL and CsA. We also estimated the impact of CsA/SRL interaction on renal dysfunction, myelosuppression, and hyperlipidemia. Salt-depleted rats treated with SRL (0.4–6.4 mg/kg) and/or CsA (2.5–20 mg/kg) were examined for glomerular filtration rates (GFR), lipid levels, and bone marrow cellularity. CsA-induced kidney function deficiency was exacerbated by SRL. This exacerbation of renal dysfunction correlated with increased CsA levels in kidneys when combined with SRL. Furthermore, CsA potentiated SRL-mediated toxicities, namely myelosuppression and increased cholesterol. In conclusion, SRL therapy is synergistic with CsA but both drug levels should be carefully monitored to avoid toxic effects.

Histamine-degrading enzymes as cellular markers of acute small bowel allograft rejection.

Intestinal histamine-degrading enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HNMT) activities are relatively constant per individual and bowel segment, and they reflect the functional integrity of the intestinal mucosa. It was, therefore, hypothesised that a decrease in these enzymes could be indicative of acute rejection of an intestinal allograft. Enzymatic activities of DAO and HNMT were determined in mucosal biopsies of isogeneic (Lewis-to-Lewis, n=48) and allogeneic (Brown Norway-to-Lewis, n=48) heterotopic small bowel transplants in a rat model at various time periods. Allograft recipients were not given any immunosuppression. While no changes in enzyme activities were observed in isografts up to day 8 following transplantation, significantly reduced activities of both enzymes were found in all allografts 6-8 days after transplantation. Activities of both DAO and HNMT exhibited a strong negative correlation with the histological rejection score ( P<0.01). We can conclude that DAO and HNMT activities in gut mucosa are reliable quantitative markers of acute intestinal allograft rejection in the rat that support histopathological analysis.

A schema for histologic grading of small intestine allograft acute rejection.

Histologic evaluation of small bowel allograft biopsies is important for the diagnosis of acute rejection. However, a standard histologic schema to grade the severity of intestinal acute rejection is not currently available. The primary goal of this study was to develop a histologic grading system for the diagnosis of small bowel allograft acute rejection. We evaluated 3268 small bowel allograft biopsies obtained from adult patients who underwent small bowel transplantation at the University of Pittsburgh Medical Center between 1990 and 1999. A histologic grading system was proposed and validated by retrospective correlation with clinical outcomes. Among the 3268 biopsies, 180 acute rejection episodes were diagnosed (88 indeterminate, 74 mild, 14 moderate, and 4 severe). All four histologically diagnosed, severe acute rejection episodes resulted in graft failure before resolution, despite aggressive immunosuppressive therapy. Four of the 14 moderate acute rejection episodes were associated with unfavorable clinical outcomes. In contrast, the 74 mild and 88 indeterminate acute rejection episodes were not associated with unfavorable clinical outcomes. Statistical analysis for trend revealed that grades indicating more severe acute rejection episodes were associated with a greater probability of unfavorable outcomes (P<0.01). In addition, there was good overall agreement among different pathologists regarding the diagnosis of acute rejection using the proposed schema, suggesting that this system is practical. This study provides a reliable predictive schema for assessment of the severity of human small bowel acute rejection.