Intestinal Allograft Research Articles

Invariant natural killer T cells infiltrate intestinal allografts undergoing acute cellular rejection

Immunological responses in human intestinal allografts are poorly understood and accurate diagnosis of acute cellular rejection remains difficult. Here, human intestinal allografts were analyzed by multi-color quantitative fluorescent immunohistochemical morphometry in order to monitor the clinical course of rejection. Morphometry gave two-dimensional plots based on size and circularity, and identified phenotypes of individual cells infiltrating the allograft by fluorescent staining. Using this method, invariant TCRVα24(+) NKT (iNKT) cells were observed in the intestinal allograft during rejection. Because these were not identified in the normal donor intestine before surgery, this finding was considered to be a signature of acute cellular rejection of the intestinal allograft. Infiltrating iNKT cells released IL-4 and IL-5, Th2-related cytokines that antagonize the Th1 responses that induce acute cellular rejection. Histological observation suggested eosinophilic enteritis in the mucosa with elevation of IL-4 and IL-5. In conclusion, iNKT cells were recruited to the intestine; however, because higher levels of IL-4 and IL-5 may contribute to eosinophilic enteritis, timely steroid administration is recommended for allograft injury due to enteritis, as well as acute cellular rejection.

Innate immunity in the small intestine

This article reviews the most recent publications on innate immunity in the small intestine. We will go over the innate immune receptors that act as sensors of microbial presence or cell injury, Paneth cells as the main epithelial cell type that secrete antimicrobial peptides, and mucosal production of immunoglobulin A (IgA). In addition, we will give an update on examples of imbalance of the innate immune response resulting in clinical disease with the most relevant example being Crohn's disease. Toll-like receptors (TLRs) are involved in B-cell homing to the intestine, rejection of small intestinal allografts, and recruitment of mast cells. The TLR adaptor Toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β is necessary to activate innate immunity after Yersinia enterocolitica infection. Moreover, MyD88 is required to keep the intestinal microbiota under control and physically separated from the epithelium, and RegIIIγ is responsible for the bacterial segregation from the lining epithelial cells. In Crohn's disease, ATG16L1 T300A variant promotes a proinflammatory response; and miR-196 downregulates a protective immunity-related GTPase family M protein (IRGM) polymorphism leading to impaired clearance of adherent Escherichia coli in the intestine. The intestine is continuously exposed to dietary and microbial antigens. The host has to maintain intestinal homeostasis to keep the commensal and pathogenic bacteria under control. Some of the mechanisms to do so are by expression of innate immune receptors, production of antimicrobial peptides, secretion of IgA, or autophagy of intracellular bacteria. Unfortunately, in some cases the innate immune response fails to protect the host and chronic inflammation, transplant rejection, or other disorders may occur.

Use of 18S ribosomal DNA polymerase chain reaction–denaturing gradient gel electrophoresis to study composition of fungal community in 2 patients with intestinal transplants

Fungi form a diverse microbial community in the human intestine. Little is known about the succession of species after intestinal transplantation. We investigated the alterations of the gut fungal population in 2 patients with intestinal allografts. The ileal effluent and feces were fingerprinted using denaturing gradient gel electrophoresis, with confirmation by DNA sequencing. Analysis of 18S ribosomal DNA indicated that the phylogenetic diversity of the fungal communities was higher soon after transplantation; less diversity was observed at the later time points in patient 1. The shifts in the denaturing gradient gel electrophoresis banding patterns over time were similar in the effluent and feces in this patient. Similar changes in the fungi in the effluent and feces also were observed in patient 2. Sequence analysis of denaturing gradient gel electrophoresis bands showed that Saccharomyces cerevisiae and Kluyveromyces waltii dominated the fungal microbiota in both patients. Some species, including Candida spp, Cryptococcus neoformans, Fusarium oxysporum, Aspergillus clavatus, and Trichophyton verrucosum, were present early. We report for the first time the temporal alterations in fungal communities in patients with an intestinal allograft. This information may provide novel insight into the roles of the fungal microbiota in the pathophysiology of the transplanted intestine.

MicroRNA Signature of Intestinal Acute Cellular Rejection in Formalin-Fixed Paraffin-Embedded Mucosal Biopsies

Despite continuous improvement of immunosuppression, small bowel transplantation (SBT) is plagued by a high incidence of acute cellular rejection (ACR) that is frequently intractable. Therefore, there is a need to uncover novel insights that will lead to strategies to achieve better control of ACR. We hypothesized that particular miRNAs provide critical regulation of the intragraft immune response. The aim of our study was to identify miRNAs involved in intestinal ACR. We examined 26 small intestinal mucosal biopsies (AR/NR group; 15/11) obtained from recipients after SBT or multivisceral transplantation. We investigated the expression of 384 mature human miRNAs and 280 mRNAs associated with immune, inflammation and apoptosis processes. We identified differentially expressed 28 miRNAs and 58 mRNAs that characterized intestinal ACR. We found a strong positive correlation between the intragraft expression levels of three miRNAs (miR-142-3p, miR-886-3p and miR-132) and 17 mRNAs including CTLA4 and GZMB. We visualized these miRNAs within cells expressing CD3 and CD14 proteins in explanted intestinal allografts with severe ACR. Our data suggested that miRNAs have a critical role in the activation of infiltrating cells during intestinal ACR. These differences in miRNA expression patterns can be used to identify novel biomarkers and therapeutic targets for immunosuppressive agents.

In Brief

Purpose: The aim of this study was to analyze factors impacting on the survival of pediatric patients with intestinal failure referred for intestinal transplantation (ITx).Methods: Two hundred fifty-seven children (mean age, 3.4 ± 0.26 years) with intestinal failure were evaluated for ITx between 1990 and 1998. All patients were dependent on total parenteral nutrition (TPN) for a mean of 31 ± 2.7 months. The mean follow-up time from the date of evaluation was 9.2 ± 0.9 months.Results: Eighty-two (32%) children underwent ITx with a mean waiting time of 10.1 ± 1.3 months (simultaneous liver-intestinal allograft in 68% instances). Of the 175 patients who did not undergo transplantation, 120 died, 23 were lost to follow-up, and 32 are alive. Younger patients (≤1 year) had poorer survival rates than patients older than 1 year (P<.0001). The patients with the worse prognosis were those with necrotizing enterocolitis, and those with the best prognosis were those with Hirschsprung's disease. Patients with “surgical” causes had poorer survival rates than those with “nonsurgical” causes (P<.04). Patients with bridging fibrosis or established cirrhosis had an earlier mortality than patients with portal fibrosis (P<.003). The worst survival rate was found for patients with bilirubin levels of greater than 3 mg/dL (P<.0001), platelet counts less than 100.000/mL (P<.0001), prothrombin time greater than 15 seconds (P=.03) or partial thromboplastin time greater than 40 seconds (P<.04). Children who at the time of evaluation needed only an isolated intestinal allograft had a better prognosis than those who required a combined liver-intestine allograft (P<.00001). With multivariate analysis independent prognosis risk factors of poor outcome were hyperbilirubinemia and severity of histopathologic damage.Conclusions: Early referral for ITx should occur before the development of liver dysfunction, taking into consideration the aforementioned risk factors that would facilitate the development and ominous evolution to liver failure.

Use of canine small intestinal submucosa allograft for treating perineal hernias in two dogs

Here, we describe two dogs in which canine small intestinal submucosa (SIS) was implanted as a biomaterial scaffold during perineal herniorrhaphy. Both dogs had developed severe muscle weakness, unilaterally herniated rectal protrusions, and heart problems with potential anesthetic risks. Areas affected by the perineal hernia (PH) located between the internal obturator and external anal sphincter muscles were reconstructed with naïve canine SIS sheets. In 12 months, post-operative complications such as wound infections, sciatic paralysis, rectal prolapse, or recurrence of the hernia were not observed. Symptoms of defecatory tenesmus also improved. Neither case showed any signs of rejection or specific immune responses as determined by complete and differential cell counts. Our findings demonstrate that canine SIS can be used as a biomaterial scaffold for PH repair in dogs.

Cellular alloresponses for rejection-risk assessment after pediatric transplantation

Current immune monitoring practices detect antidonor antibodies and antibody-mediated rejection, and are less suited for the detection of acute cellular rejection (ACR), the predominant form of rejection after transplantation. We review the use of mixed lymphocyte coculture-based assays that measure cellular alloresponses, for measurement of the risk of ACR after liver, intestine, and kidney transplantation. Flow cytometry enables the rapid measurement of cellular alloresponses using dilution of the intravital dye carboxyfluorescein succinimidyl ester within 72 h or of intracellular CD154 in alloantigen-specific T-cells or B-cells within 16 h. Assay output is personalized by expressing donor-induced alloresponse as a fraction of the third-party alloresponse for each patient. The resulting parameter called the immunoreactivity index indicates increased risk of rejection if donor-response exceeds third-party response. The rejection-risk threshold immunoreactivity index predicts or associates with ACR of liver, kidney, or intestine allografts with sensitivity and specificity of 75% or more. Lifelong assessment is facilitated by using 'surrogate' donor stimulators from normal human individuals in lieu of actual donor cells, without compromising rejection-risk assessment. Cellular alloresponses can measure the risk of ACR accurately in the clinic, so that immunosuppression may be managed safely and more effectively in individual patients.

Association Between Donor-Specific Antibodies and Acute Rejection and Resolution in Small Bowel and Multivisceral Transplantation

Donor-specific antibodies (DSA) are associated with acute kidney graft rejection, but their role in small bowel/multivisceral allograft remains unclear. We carried out a prospective study to understand the impact of DSA in the setting of intestinal allograft rejection. Thirteen patients (15 grafts) were serially evaluated for DSA levels pre- and posttransplant. DSA was determined by Luminex and the results were interpreted as fluorescence intensity (FI), with FI more than 3000 considered positive. The clinical rejection episodes in allografts were significantly associated with the presence of DSA (P=0.041).We obtained 291 biopsy samples from graft ileum and date-matched DSA assay reports. Sixty-three (21.65%) of the biopsies showed acute rejection. The appearance of DSA were preformed (n=5, anti-human leukocyte antigen class II=3, anti-class I and II=2), de novo (n=4, 15.25±4.72 days after transplantation, anti-class II=1, and anti-class I and II=3) and never (n=6). Among the 63 biopsies, 30(47.6%) had significant correlations with positive DSA (kappa=0.30, P<0.001) and manifested severe rejection grade (P=0.009). In this cohort of small bowel/multivisceral transplantation patients, there was a high incidence of DSA. The presence of DSA should alert the clinical team of a higher risk of rejection, and reduction of the FI is clinically associated with resolution. Serial endoscopy guided biopsies combined with simultaneous DSA measurement in postintestinal transplantation follow-up is an effective means of screening for cellular and humoral-based forms of acute rejection.

Increased Expression of Peripheral Blood Leukocyte Genes Implicate CD14 + Tissue Macrophages in Cellular Intestine Allograft Rejection

Recurrent rejection shortens graft survival after intestinal transplantation (ITx) in children, most of whom also experience early acute cellular rejection (rejectors). To elucidate mechanisms common to early and recurrent rejection, we used a test cohort of 20 recipients to test the hypothesis that candidate peripheral blood leukocyte genes that trigger rejection episodes would be evident late after ITx during quiescent periods in genome-wide gene expression analysis and would achieve quantitative real-time PCR replication pre-ITx (another quiescent period) and in the early post-ITx period during first rejection episodes. Eight genes were significantly up-regulated among rejectors in the late post-ITx and pre-ITx periods, compared with nonrejectors: TBX21, CCL5, GNLY, SLAMF7, TGFBR3, NKG7, SYNE1, and GK5. Only CCL5 was also up-regulated in the early post-ITx period. Among resting peripheral blood leukocyte subsets in randomly sampled nonrejectors, CD14(+) monocytes expressed the CCL5 protein maximally. Compared with nonrejectors, rejectors demonstrated higher counts of both circulating CCL5(+)CD14(+) monocytes and intragraft CD14(+) monocyte-derived macrophages in immunohistochemistry of postperfusion and early post-ITx biopsies from the test and an independent replication cohort. Donor-specific alloreactivity measured with CD154(+) T-cytotoxic memory cells correlated with the CCL5 gene and intragraft CD14(+) monocyte-derived macrophages at graft reperfusion and early post-ITx. CCL5 gene up-regulation and CD14(+) macrophages likely prime cellular ITx rejection. Infiltration of reperfused intestine allografts with CD14(+) macrophages may predict rejection events.

Quantification of Intraepithelial Lymphocytes in Normal Pediatric Small Intestinal Allograft and Native Ilea

Background The small bowel acts as one of the first lines of defense against intraluminal infections and antigenic stimuli. Pediatric small bowel transplant patients are at particular risk from such agents, especially viral enteridities. Quantification of intraepithelial lymphocytes (IEL) in architecturally normal small intestinal mucosal biopsies plays an important role in the diagnosis of conditions such as celiac disease and some viral infections. No studies to date have been done to quantify IEL numbers in pediatric small bowel allografts and in native pediatric ilea. Our study investigated the IEL:enterocyte (EC) ratio in pediatric allograft and native ilea. Methods Hematoxylin and eosin slides from 50 surveillance endoscopic biopsies of small bowel allografts taken from patients <8 years of age and 50 terminal ileal biopsies from aged-matched control populations were reviewed. IEL:EC ratios were averaged from five well-oriented villi in each case. IEC numbers were compared between biopsies from proximal (afferent) and distal (efferent) limbs of double-barrel allograft stomas, as well as native terminal ilea. Results Within small bowel allografts, the average number of villus tip IELs was 1.3/20 ECs (standard deviation [SD] 0.6) in the proximal limb and 1.0/20 ECs in the distal (SD 0.6 P < .01). This value was significantly lower than in the control ilea (2.1/20 EC [SD 0.6]; P < .01, each). The overall distribution of lymphocytes was in a similar pattern throughout the villus with IEL:EC ratios on villus sides from proximal allografts, distal allografts, and native ilea being 1:20, 0.8:20, and 2:20, respectively. Conclusions The results suggest that approximately one to two IEL per 20 ECs at villus tips may represent a “normal” intraepithelial inflammatory cell population in small bowel allografts. The value is lower than that seen in age-matched native ileal biopsies. IEL numbers are significantly higher in the proximal limb compared to the distal limb of double-barrel stomas.

Tumor Necrosis Factor Alpha Inhibitors as Immunomodulatory Antirejection Agents after Intestinal Transplantation

We reported the successful administration of infliximab for late-onset OKT3-resistant rejection in two patients, who presented persistent ulcerative inflammation of the ileal graft after intestinal transplantation (ITX). Based on this experience, the present study demonstrated our long-term experience with infliximab for different types of rejection-related and inflammatory allograft alterations. Infliximab administration (5 mg/kg body weight (BW)) was initiated at a mean of 18.2 ± 14.1 months after transplantation. The number of administrations per patient averaged 8.4 ± 6.7. Repeat dosing was timed according to clinical signs and graft histology in addition to serum-levels of tumor necrosis factor alpha (TNFα), lipopolysaccharide binding protein (LBP) and C-reactive protein (CRP). Infliximab was successful in the following patients: patients with late-onset OKT3- and steroid-refractory rejection who presented persistent ulcerative alterations of the ileal graft (n = 5), patients with ulcerative ileitis/anastomositis, who did not show typical histological rejection signs (n = 2), and one patient with early-onset OKT3-resistant rejection. Infliximab was not successful in one patient with early-onset OKT3-resistant rejection that was accompanied by treatment-refractory humoral rejection. In conclusion, infliximab can expand therapeutic options for late-onset OKT3- and steroid-refractory rejection and chronic inflammatory graft alterations in intestinal allograft recipients.

Allograft survival is prolonged in the absence of NKG2D (169.19)

Abstract NKG2D is expressed by Natural Killer (NK cells) and subsets of T cells. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We have demonstrated increased expression of NKG2D ligands on rejecting grafts and enhanced infiltration of NKG2D-expressing cells in allografts during rejection. To further examine the role of NKG2D in graft survival we utilized Klrk1 mice that are deficient for NKG2D as recipients of cardiac or small intestine allografts. Groups (n=4-6) of C57BL/6 (H-2b) or NKG2D deficient Klrk1 mice (H-2b) received heterotopic donor hearts or vascularized, orthotopic small intestine grafts from BALB/c (H-2d) or BALB/c x C57BL/6 F1 mice (H-2d/b). Some groups of mice also received 200μg (heart) or 500μg (intestine) anti-CD40L mAb (MR1) at transplant. Cardiac allograft survival was similar in wild-type and Klrk1 mice although intestinal allograft survival was prolonged in Klrk1 mice. Treatment with MR1 prolonged cardiac allograft survival that was further enhanced in the absence of NKG2D. Treatment with MR1 did not prolong survival of intestine allografts in wild-type recipients. Quite dramatically, however, treatment with MR1, significantly (p=0.007) prolonged intestinal allograft survival in Klrk1 mice. Prolongation of intestinal graft survival in the absence of NKG2D and CD40-CD40L interactions suggest that the addition of therapeutics to block NKG2D has great potential for improving costimulation blockade based therapies

Natural Killer Group 2 Member D Cell Recruitment Driven by Major Histocompatibility Complex Class I Chain–Related Antigens A and B: A Possible Mechanism During Acute Intestinal Allograft Rejection in the Mouse

Intestinal allograft rejection occurs frequently despite potent T-cell depletion protocols. We investigated the interaction of major histocompatibility complex class I chain–related antigens A and B (MICA/B; a ligand for natural killer [NK] cells) and NK group 2 member D (NKG2D) cells as an alternative mechanism for acute rejection (AR) of the intestinal graft. Heterotopic intestinal allotransplantation was performed from BalbC to C57Bl mice. Samples of grafted and native intestine were obtained at days 1, 3, 6, and 8 after transplantation (n = 4–6). We performed immunostaining for MICA/B and NKG2D. Moderate AR with increased crypt apoptosis was observed at day 6 and advanced AR with crypt destruction and mucosal sloughing was present by day 8. Low MICA/B levels were observed in grafted and native intestines on day 1. MICA/B expression gradually increased in the grafts during AR but not in the native intestines. The up-regulation was found mostly in the crypts. NKG2D+ cell counts that increased in the graft colocalized with MICA/B. The increase was most prominent in the crypt and villus. Together, these results suggest that MICA/B up-regulation and its subsequent interaction with the NK cells may represent an important link between innate and adaptive immune responses early after intestinal transplantation.

Hypertrophic osteoarthropathy in intestinal transplant recipients

We report 3 patients with periosteal new bone formation consistent with hypertrophic osteoarthropathy (HOA), in the context of intestinal allograft rejection. Patient 1 developed thick periosteal new bone formation of the right arm during a prolonged episode of intestinal acute cellular rejection (ACR) 2 months posttransplant. Patient 2 developed ankle pain and swelling during an episode of severe ACR. Plain films showed periosteal new bone formation of the left ankle. In patient 3, the right wrist became swollen during an episode of moderate ACR, whereas plain films demonstrated mild periosteal reaction. Patients 2 and 3 had resolution of their symptoms once the ACR resolved with treatment. This is the first case series of HOA occurring in association with intestinal ACR. We speculate that an immune-mediated process is responsible for the bone disease. Further inquiry will help establish if HOA is related to transplant status, intestinal inflammation, or allograft rejection in general.

Treg-based therapy and mixed chimerism in small intestinal transplantation: Does Treg + BMT equal intestine allograft tolerance?

Small intestinal transplantation (SIT) is a convincing treatment selection for end-stage bowel failure, however, graft rejection and the toxicity associated with the use of non-specific immunosuppression remain the major limitations in SIT. Induction of SIT tolerance through establishment of mixed chimeras is regarded as a robust approach to solve this problem. In rodent models of nonmyeloablative recipient preparation using donor bone marrow transplant (BMT)+costimulatory blockade, deletion of alloreactive T cells played the dominant role in silencing the effector function of graft-rejecting T cells. However, such understanding of the cellular basis for allograft tolerance might be incomplete. Several recent studies demonstrated that regulatory T cells (Tregs) could promote the establishment of allogeneic mixed chimerism as well as the induction of donor-specific tolerance. Treg-based therapy in and of itself is proposed to be a promising non-toxic approach to prevent allograft from rejection. In addition, latest studies showed that, Tregs not only played a critical role in controlling both acute and chronic rejection, but also might contribute to the protection of allograft from ischemia/reperfusion injury (IRI). Taken together, we hypothesize that, co-transplant of donor bone marrow and Tregs specific for alloantigens might be one of the most effective regimens to induce transplantation tolerance, so as to improve the overall outcome of SIT. Although much efforts have been made, a deliberate protocol without toxicity still needs to be developed for the clinical application.

Acute Rejection of Small Intestine Allografts Is Associated With Increased Expression of Toll-like Receptors

Although outcomes after intestinal transplantation have steadily improved owing to advances in immunosuppressive therapy, operative techniques, and postoperative medical management, rejection of the intestinal allograft continues to be a major clinical problem and constitutes the primary reason for graft loss. Although the adaptive immune system has been the major focus of investigation regarding regulation of rejection of the intestinal allograft, the role of the innate immune system has recently become of increased interest. We hypothesized that microbial products of the microflora associated with the intestinal allograft may engage the Toll-like receptor pathway of the innate immune system to potentiate alloimmune responses and rejection of the allograft. To investigate this, we established a murine model for orthotopic intestinal transplantation and allograft rejection. Using this model, we show that the expression of Toll-like receptor 2 is increased 50-fold and the expression of Toll-like receptor 4 is increased 200-fold during rejection of the allograft. We then performed survival studies that showed increased survival of mice, which had the Toll-like receptor knocked out. These preliminary studies suggest an important role for in innate immune system in acute rejection of the small intestinal allografts, and as such represents an emerging and promising area of investigation.

Characterization of Epithelial Apoptosis in Biopsies of Small-Bowel Allografts Using Cleaved Caspase 3 Immunostaining

The diagnosis of acute cellular rejection (ACR) in small-bowel allograft biopsies depends in part on quantification of crypt apoptotic bodies (ABs). The definition of ABs varies between authors. Recently, immunoperoxidase stains specific for apoptotic material have been used in paraffin-embedded tissue. The aim was to characterize AB morphology and quantify ABs using immunoperoxidase and hematoxylin and eosin (H&E) staining. Allograft biopsies with diagnoses of negative for ACR, indeterminate for ACR, and mild ACR were selected. Sections were stained for cleaved caspase 3 (CC3). The number of ABs per 10 crypt epithelial cells was compared by H&E and CC3 staining. A total of 39 cases (15 negative, 12 indeterminate, 12 mild ACR) were obtained. CC3 staining revealed that ABs varied from well-developed "classical" exploding crypt cells to intraepithelial clusters of basophilic material. In 61%, the number detected by CC3 was higher than on H&E stains (mean 4.30 and 3.56; P < .002), with good overall correlation. With the latter definition, AB numbers by H&E were significantly higher in most cases (72%), with the diagnosis of mild ACR being downgraded in 50% if only classical ABs were counted. ABs have a range of morphology in biopsies of intestinal allografts, and H&E identifies the majority. The results suggest that AB counts should include basophilic clusters.

Exosomes Derived from Immature Bone Marrow Dendritic Cells Induce Tolerogenicity of Intestinal Transplantation in Rats

Dendritic cells (DCs) secrete exosomes bearing major histocompatibility complex I and II (MHC I /II) and co-stimulatory molecules, and play a critical role in immune regulation. Because immature DCs can induce T-cell tolerance in vitro and in vivo, we explored the possibility of using exosomes derived from immature DCs (imDex) for the induction of intestinal transplant tolerance in rats. ImDex were purified from F344 rat bone marrow immature DCs. The tolerizing capacities of imDex were analyzed in vitro and in vivo using a F344-to-Wistar intestinal transplantation model. In the context of a moderate level of MHC class II and a low co-stimulatory level expression, imDex significantly suppressed the alloreactive T-cell response with an increase in IL-10 in vitro. In vivo injection of the lower dose (20 μg) of donor (but not recipient) imDex can significantly prolong the survival of intestinal allografts. This effect was accompanied by a decrease in the anti-donor cellular response, with a significant increase in IL-10. The CD4+CD25+T cells percentage and FOXP3mRNA expression in splenic T-cells were also significantly increased in imDex treatment recipients at five days after transplantation. The results suggest that imDex can prolong the intestinal allograft survival and may be a potential strategy to facilitate induction of transplant tolerance.

MyD88 silent semi-mature DC induced immune tolerance in rat small bowel transplantation model

Objective To study MyD88 silent semi-mature DC (simDC) inducing immune tolerance in rat small bowel transplantation model. Methods All rats were randomly divided into three groups (n=6). Seven days before transplantation of intestine from F344 donors, Wistar recipient rats were injected with simDC (group A) , imDC (group B) and saline ( group C) through the penile dorsal vein respectively.Small bowel transplantation was performed and the survival time of recipients was observed. Serum IL-2 and IFN-γ levels were assayed. Results The survival time of recipients in group A was ( 13.7±1.2) days, which was significantly longer than that in groups B [(8.0±1.0) days,P<0. 05] and C [(6. 0±0. 8) days P<0. 05]. The serum levels of IL-2 and IFN-γ in group A were lower than in groups B and C (P<0. 05). Conclusion Depending on the unique phenotypic and functional features of sem-DC,they can induce the transplantation tolerance and prolong the survival of intestinal allografts after transplantatin. Key words: Dendritic cell; Immune tolerance; MyD88; Intestinal transplantation

954 Carbohydrate Digestion in Congenital Sucrase Isomaltase Deficient and Recurrent Abdominal Pain Children Assesed by 13C- Starch Breath Test

BACKGROUND: Intestinal allograft rejection is a severe complication in small bowel transplantation. Identifying involved pathogenetic mechanisms might make it possible to predict or even prevent rejection and intestinal graft failure. Rejection resembles Crohn's disease (CD) clinically and histopathologically. We investigated the role of CD-associated genetic loci, including NOD2 variants in rejection and graftfailure. METHODS: 99 recipients and 71 donors from the University of Nebraska Medical Center, USA were genotyped for three NOD2 variants and single nucleotide polymorphisms in IBD5, ATG16L1, IRGM, MST1, IL23R, STAT3 and JAK2. Genotypes were related to clinical outcomes rejection, graftfailure and patient-survival using Kaplan-Meier curves and log-rank analysis. RESULTS: Only the MST1 riskallele was more frequent in recipients compared to donors (30,8% and 20,4%, p=0,032). For the other loci, including NOD2, allelic distribution was equal within the two groups. No single locus was significantly associated with rejection, graftfailure or patientsurvival. There is no significant difference in clinical endpoints whether the recipient has a low or high number of risk alleles. Individuals receiving a donorgraft with ≤5 riskalleles in total, have decreased rejection-free survival compared to ≥6 riskalleles (p=0,040), but increased patient three-year survival (mortality: 4/19 and 16/29, p=0,016). CONCLUSIONS: In this relatively large cohort, we could not confirm the association of NOD2, with rejection and graftfailure. Furthermore we found no association of seven other CD-loci with clinical endpoints. However, patients receiving a donorgraft with six or more risk alleles have decreased three-year survival, but -surprisinglyincreased rejection-free survival.