Small Bowel Allograft Rejection Research Articles

Crypt apoptotic body counts in normal ileal biopsies overlap with graft-versus-host disease and acute cellular rejection of small bowel allografts

Crypt apoptosis in intestinal epithelium is an important diagnostic feature of graft-versus-host disease (GVHD) and acute cellular rejection (ACR) of intestinal transplants (ITx). In ITx pathology, 2 or fewer apoptotic bodies in 10 consecutive crypts are considered normal, whereas 6 or more is consistent with mild ACR. The presence of 3 to 5 apoptotic bodies is problematic and is often classified as indeterminate for ACR. The minimum diagnostic threshold for GVHD is controversial but also depends on the apoptotic body count (ABC). We investigated how many crypt apoptotic bodies could be identified in histologically normal ileal biopsies from healthy subjects (native intestines, no bone marrow transplant) who underwent screening colonoscopy and had ileal biopsy to confirm complete colonoscopy. We recorded the number of biopsy pieces per specimen and the maximum ABC in 10 consecutive crypts. Twenty-six of 40 patients (65%) had an ABC of 3 or more in 10 crypts, thus only 35% were "normal." Four (10%) had an ABC of ≥6 (positive for ACR). Twenty-two (55%) had 3-5 (indefinite for ACR). Depending on the criteria, up to 60% of the cases could be diagnosed as positive for GVHD.

Attenuation of rat chronic small bowel allograft rejection by n‐3 polyunsaturated fatty acids is associated with reduced expression of graft IL‐15

Interleukin-15 was found to play key roles in various immunological processes including chronic rejection after renal and cardiac transplantation. n-3 polyunsaturated fatty acids (n-3 PUFA) have shown beneficial effects to chronic allograft rejection. The objective of this study is to search the possible mechanism of this inhibitory effect in chronic small bowel allograft rejection. Animals were divided into three groups: isograft (CsA+corn oil-supplemented diet); allograft (CsA+corn oil-supplemented diet); and allograft (CsA+fish oil-supplemented diet). Donor intestines from F344 rats were transplanted orthotopically into Lewis rat recipients. CsA was administered at 5mg/kg/day for 2wk post-operatively. Post-transplant weight was recorded. Histopathological changes and graft IL-15 expression were measured on POD 90. Chronic small bowel allograft rejection developed on POD 90. n-3 PUFA significantly decreased the score of chronic rejection and increased the post-operative weight gain rate. This attenuation is associated with reduced graft IL-15 expression. n-3 PUFA contributed to improved pathological and clinical outcome during chronic small bowel allograft rejection, and this improvement was associated with reduced graft IL-15 expression.

Cell Stress Response in Rat Chronic Small Bowel Allograft Rejection

BackgroundWe sought to assess the expression of five cell stress molecules: Heat shock protein (HSP)-60, HSP-70, interferon (IFN)-α, rat retinoic acid early induible-1 like transcript (RRLT), and vasoactive intestinal peptide (VIP) during rat chronic small bowel allograft rejection. MethodsA rat model of small bowel allograft rejection was induced on postoperative day 90 using F344 rat as donor and Lewis rat as recipient. ResultsWe demonstrated increased expression of lamina propria HSP-60 (mean rank grade 8.3 and 3.2, respectively), graft RRLT (relative mRNA amount 5.1 ± 1.8 and 3.0 ± 1.3, respectively) and IFN-α (relative mRNA amount 108.3 ± 49.2 and 59.2 ± 22.0, respectively) in allograft group compared with the isograft group. ConclusionThese data suggest the presence of cell stress during chronic small bowel allograft rejection.

Role of Recipient Derived Myd88 in Small Bowel Allograft Rejection

Background: Accumulating evidence suggests that Myd88, a common adaptor protein for signal transduction of most TLRs, plays an important role in ischemia/reperfusion injury and skin and heart allograft rejection. However, little is known concerning its role in small bowel (SB) transplantation. The purpose for this study was to determine whether MyD88 deficiency in recipients alleviates SB allograft rejection. Methods: Small bowels from B6 were transplanted into either wild type (WT) or Myd88−/− BALB/c mice (allografts) heterotopically. The recipients were followed up on POD7 and POD11. Frequencies of CD4+, CD8+, CD11b+, Gr-1+, IFN-g producing T cells, IL-17 producing cells, as well as CD4+Foxp3+ cells in spleen and SB allografts were measured by flow cytometry analysis on POD7 and POD11. Morphological changes of small bowel grafts were examined by histology and phenotypes of cellular infiltrates including CD4, CD8 were identified by immunohistochemical staining on POD7 and POD11. Results: B6 to BALB/c allografts developed severe rejection manifested by loss of epithelial integrity along with diffuse infiltration by CD11b+, CD8+ and Gr-1+ cells. In contrast, Myd88−/− allograft has minimal morphological changes with a marked decrease in the number of CD11b+, CD8+ and Gr1+ cell infiltrates on both POD7 and POD11. Moreover, data from phonotypical analysis of SB infiltrating cells reveal that compared to WT allografts on both POD7 and POD11, Myd88−/− SB allografts had decreased frequencies of CD8+ cells, IFN-g producing cells and IL-17 producing cells, whereas numbers of CD4+Foxp3+ T cells were markedly increased in both spleens and SB infiltrating cells of Myd88−/− allografts. Conclusion: Our results suggest that MyD88 dependent TLR signaling plays an important role in T cell differentiation by shifting the balance between effector T cells and Tregs, thereby influencing cellular infiltration and small bowel allograft rejection.

Elevated Myeloid: Plasmacytoid Dendritic Cell Ratio Associates With Early Acute Cellular Rejection in Pediatric Small Bowel Transplantation

Acute cellular rejection affects more than 60% of children after small bowel transplantation (SBTx). Dendritic cells (DCs) are potent antigen-presenting cells, modulate immune responses to gut microbes, and may serve as markers of rejection-prone small bowel transplantation (SBTx). Myeloid CD11c DC (MDC), which may have inflammatory functions, and plasmacytoid CD123 DC (PDC), which may have tolerogenic potential, were measured by flow cytometric analysis, longitudinally (pretransplant, and at days 1 to 60, 61 to 200 posttransplant) in 23 children after SBTx. All children received cadaveric allografts with rabbit anti-human thymocyte globulin induction and steroid-free tacrolimus maintenance therapy. Rejectors were those children (n=16), who experienced biopsy-proven acute cellular rejection within 60 days of SBTx. Of 69 maximum possible observations, 62 were available for analysis. Among rejectors, a significantly higher MDC:PDC ratio (P=0.004) was associated with numerically higher MDC counts and significantly lower PDC frequencies (P=0.017) during the 1- to 60-day time period, compared with nonrejectors. Logistic regression analysis, leave-one-out cross-validation, and receiver operating characteristic analysis revealed that MDC:PDC ratio more than or equal to 1.52 was associated with rejector status with sensitivity/specificity of 86/67% during the 1- to 60-day risk period for early SBTx rejection. Repeated measures analysis showed a significantly higher MDC:PDC ratio (P=0.043, F-test) among rejectors, compared with nonrejectors in cumulative data for pre-SBTx and 1- to 60-day time points. No correlation was seen between DC subsets and tacrolimus blood concentration at any time point. We conclude that an elevated MDC:PDC ratio associates with early small bowel allograft rejection and may, therefore, identify at-risk recipients in the clinic.

Significance of Serum Bile Acids in Small Bowel Allograft Rejection in Pigs

To examine changes in individual bile acids in serum in a pig model of allogenic small bowel transplantation. Seventeen pigs were divided into two groups: group 1 (n=10), controls; group 2 (n=7), allotransplantation, nonimmunosuppressed recipients. Both groups received a segmental intestine. Intestinal specimens for histologic studies were obtained at the end of cold flushing (T0) and on postoperative day 8 (T1). Total and individual bile acid concentrations were measured in serum samples taken at T0 and T1. All animals survived until the end of the study. In group 1, intestinal histology revealed no significant changes between T0 and T1 specimens. In contrast, in group 2, graft histology revealed moderate to severe rejection at T1 in all specimens. In contrast to group 1, serum levels of total bile acids increased significantly in group 2 at T1 compared with baseline (P=0.001). The increase was due to secondary bile acids which were significantly higher at T1 compared with baseline (P=0.0003). In contrast, secondary bile acids increased but not significantly at T1 in group 1 compared with baseline (P=0.056). Our study suggests that total and secondary serum bile acids may be a useful diagnostic tool for the diagnosis of acute intestinal rejection.

Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristics as a means of monitoring the development of rat small bowel allograft rejection.

This investigation used flow cytometry to monitor peripheral blood lymphocyte morphology after rat small bowel transplantation. Preliminary studies demonstrated that in vitro activated peripheral blood lymphocytes exhibited increased cell size and granularity as measured by flow cytometric analysis of forward (FSc) and side (SSc) light scatter characteristics. The formation of distinct 'activated' light scatter regions by such lymphoblastoid transformation occurred concomitantly with up-regulated p55IL-2R expression. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats without immunosuppression. Animals receiving isografts served as controls. Peripheral blood lymphocyte subsets were identified using appropriate MoAbs, and the light scatter characteristics of each cell subset were determined by backgating strategies. Increased proportions of activated alpha/beta T cell receptor (TCR)-positive cells could be detected in allografted animals as early as day 2 post-transplantation. B cells showed peak activation by day 4, at which time the proportion of activated cells was over two-fold greater than that seen in untransplanted animals--few activated B cells were detected in isografted animals. Resting natural killer (NK) cell light scatter regions only partially overlap with those of resting T and B lymphocytes, but in allografted animals almost the entire NK population fell outside the resting lymphocyte gate by day 2 post-transplantation, an activation state which was maintained until day 4. These findings associate peripheral blood cell subset lymphoblastoid transformation with developing small bowel allograft rejection. Importantly, changes were detected early and prior to the onset of overt rejection. These data suggest that analysis of peripheral blood lymphocyte light scatter properties may provide an insight into in vivo immune status after small bowel transplantation.

Posttransplant adenoviral enteropathy in patients with small bowel transplantation.

Transplant patients on immunosuppression represent a risk group for opportunistic infections, including adenoviral infections. The clinical and histopathologic findings of posttransplant adenoviral enteropathy in 3 adult intestinal transplant patients are described. The histopathologic pitfalls for the differential diagnosis between intestinal adenoviral enteropathy and acute rejection are discussed. Adenoviral enteropathy is an opportunistic infection that may follow aggressive treatment for small bowel allograft rejection, but which may require no specific treatment. It is associated with mild mixed inflammation in the lamina propria and slight increase of crypt apoptosis, resembling low-grade acute rejection. The identification of characteristic viral inclusions in the surface or crypt epithelium points to the diagnosis of adenoviral enteropathy, in spite of the increased crypt apoptosis.

Posttransplant Adenoviral Enteropathy in Patients With Small Bowel Transplantation

Transplant patients on immunosuppression represent a risk group for opportunistic infections, including adenoviral infections. The clinical and histopathologic findings of posttransplant adenoviral enteropathy in 3 adult intestinal transplant patients are described. The histopathologic pitfalls for the differential diagnosis between intestinal adenoviral enteropathy and acute rejection are discussed. Adenoviral enteropathy is an opportunistic infection that may follow aggressive treatment for small bowel allograft rejection, but which may require no specific treatment. It is associated with mild mixed inflammation in the lamina propria and slight increase of crypt apoptosis, resembling low-grade acute rejection. The identification of characteristic viral inclusions in the surface or crypt epithelium points to the diagnosis of adenoviral enteropathy, in spite of the increased crypt apoptosis.

Effect of FTY720 and Ex Vivo Graft Irradiation in Rat Small Bowel Transplantation: Apoptosis of Crypt Cells and Lymphocytes

Objective We investigated the extent of apoptosis in crypt cells and Peyer’s patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 and ex vivo graft irradiation during rejection. Materials and Methods Orthotopic small bowel transplantations (SBT) were performed from Brown Norway (BN) rats to Lewis (LEW) rats. Four groups of SBT animals were studied on days 3, 5, and 7 after operations: untreated allograft, allograft with FTY720, allograft with irradiation, and allograft with FTY720 + irradiation. Cryostat sections were prepared from the grafts, including PPs. 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/FasL. Results The graft survival was prolonged in the FTY720-treated groups. In the FTY720-treated group, the number of ISEL-positive enterocytes was significantly down-regulated on days 3, 5, and 7 compared with the untreated allograft group. The number of ISEL-positive mononuclear cells was also significantly down-regulated compared with the untreated allograft group. The FTY720 the radiation and the FTY720 + irradiation treated groups showed significantly down-regulated numbers of Fas/FasL-positive enterocytes on day 7 compared with the untreated allograft group. Fas/FasL-positive mononuclear cells were also significantly down-regulated in the allograft compared with the untreated allograft group. Conclusions FTY720 and ex vivo graft irradiation prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes, and also prolonged small bowel allograft survival. Combination FTY720 and ex vivo graft irradiation did not affect graft survival and apoptotic cell expression compared with the FTY720 only group. These findings suggest that FTY720 may prevent both rejection-associated and sepsis-induced apoptosis during the late phase of small bowel graft rejection.

Effect of FTY720 in Rat Small Bowel Transplantation: Apoptosis of Crypt Cells and Lymphocytes in Gut-Associated Lymphoid Tissues

Aim We investigated the extent of apoptosis in crypt cells and Peyer’s patches (PPs) during small bowel allograft rejection in rats to examine the effect of FTY720 during rejection. Methods Orthotopic small bowel transplantations (SBTs) were performed from BN to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, and 7 after operation: isograft, untreated allograft, allograft with FTY720. FTY720 was orally administered by gavage (1 mg/kg/d) to allograft recipients on 7 consecutive days. Cryostat sections were prepared from grafts, including PPs. 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/Fas-L. Results Graft survival was prolonged in the FTY720-treated group. The number of ISEL-positive enterocytes in the allografts increased significantly on days 3, 5, and 7 compared with the isograft group. In the FTY720-treated group, the number of ISEL-positive enterocytes in the allografts was down-regulated significantly on days 3, 5, and 7 compared with untreated allograft group. In the PPs, the number of ISEL-positive mononuclear cells increased significantly in the allografts compared with the isograft group. In the FTY720-treated groups, the number of ISEL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. The number of Fas/FasL-positive enterocytes were increased significantly in allografts compared with isograft group. In FTY720-treated groups, the number of Fas/FasL-positive enterocytes were down-regulated significantly on day 7 compared with the untreated allograft group. In the PPs, Fas/FasL-positive mononuclear cells also increased significantly on day 7 in the allografts compared with isografts. In the FTY720-treated groups, Fas/FasL-positive mononuclear cells were down-regulated significantly in the allografts compared with the untreated allograft group. Conclusions The number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes increased during small bowel graft rejection. FTY720 prevented up-regulation of the number of apoptotic enterocytes, lymphocytes, and Fas/FasL-positive lymphocytes while also prolonging small bowel allograft survival.

Novel Application of ProteinChip Technology Exploring Acute Rejection Markers of Rat Small Bowel Transplantation

Because no biomarker that reflects small bowel allograft rejection is available, we applied surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) to develop noninvasive markers required for routine diagnosis. Heterotopic small bowel transplantation (SBT) was performed in rats, and they were divided into four experimental groups: sham-operated rats (sham), syngeneic transplants (syngeneic), allogeneic transplants (allogeneic), allogeneic transplants received FK506 (allo+FK). Plasma samples were analyzed with SELDI ProteinChip arrays to detect peaks that predominated in the allogeneic model. Possible biomarkers were identified in combination with SELDI retentate chromatography mass spectrometry (RCMS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The identified protein was further analyzed by immunohistochemistry. An increase in the level of a 14.8-kDa protein, identified as lysozyme, was observed specifically in the plasma of the allogeneic group; the levels of this protein remained unchanged in the plasma of the other groups. On the other hand, the levels of a 10.1-kDa and a 13.0-kDa protein, identified as migration inhibitory factor-related proteins (MRP), MRP-8 and MRP-14, respectively, began to increase from an early stage of acute rejection. We also observed that lysozyme-positive macrophages had strongly infiltrated the lamina propria during acute rejection. We identified three plasma proteins-MRP-8, MRP-14, and lysozyme-that increased during small bowel allograft rejection. The identified proteins appeared to be markers for inflammation associated with allograft rejection. This proteomic approach will be useful for the identification of candidate biomarkers.

Effect of FTY720 in Rat Small Bowel Transplantation: Expression of Mucosal Addressin Cell Adhesion Molecule-1

Aim Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) mediates the homing of lymphocytes to gut-associated tissues (GALT). We performed a semiquantitative analysis of MAdCAM-1 expression during small bowel graft rejection in rat treated with FTY720. Methods Orthotopic small bowel transplantations (SBT) were performed from BN rats to LEW rats. Isografted animals served as controls. Three groups of SBT animals were studied on days 3, 5, 7 after operations (Isograft, untreated allograft, allograft with FTY720). FTY720 was orally administered by gavage (1 mg/kg/d) to allograft models on 7 consecutive days. 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 propia from 1 (faint), to 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 villi tip). Results The graft survival was prolonged in the FTY720-treated group. 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. In the FTY720-treated groups, MAdCAM-1 expression on HEVs in PPs was up-regulated and its expression on endothelial cells of vessels in the lamina propria was down-regulated compared with untreated allograft group. Conclusions 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 intestine immune reactivity during the early phase of small bowel allograft rejection. FTY720 was found to prevent the down-regulation of MAdCAM-1 expression on HEVs in PPs and the up-regulation of its expression on endothelial cells of vessels in the lamina propria while also prolonging small bowel allograft survival.

A clinicopathologic study of isolated intestinal allografts with preformed IgG 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.

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.

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.

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.

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.

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.