Rejection In Small Bowel Transplantation Research Articles

139: Potential of Confocal Laser Endomicroscopy in Detecting Small Bowel Transplant Rejection Based on Current Application in Real-Time Diagnosis of GVHD

139: Potential of Confocal Laser Endomicroscopy in Detecting Small Bowel Transplant Rejection Based on Current Application in Real-Time Diagnosis of GVHD

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

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

Implant of mesenchymal cells decreases acute cellular rejection in small bowel transplantation.

The objective of the study was to show adipose tissue-derived mesenchymal stem cells (AD-MSCs) immunomodulatory effects in small bowel transplantation (SBTx). Forty Wistar Han rats (age: 10-12 weeks): were allogenic receptor rats and were allotted in 2 groups. Control group: rats undergoing orthopic SBTx ; AD-MSCs group: rats undergoing orthotopic SBTx plus AD-MSCs. Male Lewis rats were allogeneic small bowel donors. Rejection was confirmed by histological study of the explanted intestine, enterocyte apoptosis was determined in crypts and the lamina propria of the small bowel. Cytokine concentration levels (enzyme-linked immunosorbent assay) (interleukin [IL]-4, IL-10, IL-12, IL-17, IL-21, IL-23, tumor necrosis factor-alpha, and transforming growth factor [TGF]-b1) and cell percentages (flow cytometry) (CD3+ CD4+, CD8+, CD4+/25+, CD8+/25+, CD4+/25+/Foxp3+, and CD8+/25+/Foxp3+) were assessed in peripheral blood preoperatively and after death. Treatment with AD-MSCs produced a significantly lower risk of rejection in the first 7 post-operative days (five rejection cases among 20 rats in the control group and only one case in the AD-MSCs group). Treg cells and TGFb1 levels showed a significant increase in the AD-MSCs group. The local implantation of AD-MSC in the anastomosis and the intestinal lumen can induce a regulatory immune response, by increasing the percentages of Treg cells and TGb-1 levels, leading to a lower risk of acute rejection by cell mediation, in the first 7 days of the intestinal transplant. We think that the implantation of AD-MSCs, in the anastomoses and in the lumen of the donor intestine, could give rise to a chimera of donor-recipient cells.

Acute cellular rejection in small-bowel transplantation impairs NCR+ innate lymphoid cell subpopulation 3/interleukin 22 axis

Acute cellular rejection (ACR) remains as one of the main causes of graft loss and death in intestinal transplant (ITx) patients. ACR promotes intestinal injury, disruption of the mucosal barrier, bacterial translocation, and organ dysfunction. As epithelial regeneration is critical in reversing these consequences, the functional axis between the innate lymphoid cell subpopulation 3 (ILC3) and interleukin 22 plays an essential role in that process. Natural-cytotoxic-receptor–positive (NCR+) ILC3 cells have been demonstrated to induce intestinal-stem-cell proliferation along with an IL-22–dependent expansion of that population in several intestinal pathologies, though thus far not after ITx. Therefore, we intended to determine the impact of chronic immunosuppression and ACR on ILC3 cells and interleukin-22 (IL-22) production in the lamina propria after that intervention. Materials and methodsWe compared biopsies from healthy volunteers with biopsies from ITx recipients without or with mild-to-moderate ACR, using flow cytometry and the quantitative-PCR. ResultsNCR+ ILC3 cells were found to be unaffected by immunosuppression at different time points posttransplant when patients did not experience ACR, but were diminished upon the occurrence of ACR independently of the post-ITx time. Moreover, IL-22–expression levels were notably reduced in ACR. ConclusionThe NCR+-ILC3/IL-22 axis is impaired during ACR contributing to a delay in or lack of a complete and efficient epithelial regeneration. Thus, our findings reveal that IL-22 analogues could potentially be used as a new complementary therapeutic approach, in conjunction with immunosuppressant drugs, in order to promote mucosal regeneration upon ACR.

Protective effect of genes modified stem cells on the tight junction of damaged small intestinal epithelium

Objective To investigate the role of CXC subfamily chemokine receptor 3 (CXCR3) and heme oxygenase-1 (HO-1) gene-modified bone marrow mesenchymal stem cells (BMMSCs) in protecting tight junctions of transplanted small intestine epithelium. Methods BMMSCs were isolated by whole bone marrow adherence and identified by detecting Cluster of Differentiation 29 (CD29), Cluster of Differentiation 90 (CD90), Classical class Ⅰ major histocompatibility complex (RT1-A), Cluster of Differentiation 34 (CD34), Cluster of Differentiation 45 (CD45) and Classical class Ⅱ major histocompatibility complex (RT1-B) by Flow cytometry. BMMSCs modified by HO-1 gene, CXCR3 gene and CXCR3 adding HO-1 genes were obtained by adenovirus transfection. Experimental models of rat small bowel transplantation rejection were established by using Brown Norway rats as donors and Lewis rats as receptors. The pathology of intestine was analyzed by Hematoxylin-eosin staining and transmission electron microscopy. The expression of zona occludens-1 (ZO-1) in small intestine was detected by Western blotting. Results BMMSCs were long-shuttle type as well as positive for CD29 (99.6%), CD90 (99.8%) and RT1-A (99.8%) but negative for CD34, CD45 and RT1-B. Experimental models of acute rejection of small bowel transplantation were obtained successfully. Compared with the NS group (3.66±0.20), BMMSCs group (3.05±0.59), CXCR3/BMMSCs group (2.76±0.49) and HO-1/BMMSCs group (2.86±0.80), (CXCR3+ HO-1)/BMMSCs significantly alleviated intestinal damage (2.43±0.85, t=-5.996, P<0.05, 7 d). Meanwhile, (CXCR3+ HO-1)/BMMSCs could more significantly protected the tight junction structure and the level of ZO-1 protein in transplanted small intestine than other groups (t=-9.436, P<0.05). Conclusion Compared with single-gene-modified, BMMSCs modified by CXCR3 adding HO-1 could protect the structure and function of tight junctions of transplanted small intestine with better effects. Key words: Bone marrow mesenchymal; CXC subfamily chemokine receptor 3; Heme oxygenase-1; Small intestine transplantation; Zona occludens-1

Severe Late-Onset Acute Cellular Rejection in a Pediatric Patient With Isolated Small Intestinal Transplant Rescued With Aggressive Immunosuppressive Approach: A Case Report

Small intestinal transplantation is performed for patients with intestinal failure who failed other surgical and medical treatment. It carries notable risks, including, but not limited to, acute and chronic cellular rejection and graft malfunction. Late severe acute intestinal allograft rejection is associated with increased risk of morbidity and mortality and, in the majority of cases, ends with total enterectomy. It usually results from subtherapeutic immunosuppression or nonadherence to medical treatment. We present the case of a 20-year-old patient who underwent isolated small bowel transplant for total intestinal Hirschsprung disease at age 7. Due to medication nonadherence, she developed severe late-onset acute cellular rejection manifested by high, bloody ostomy output and weight loss. Ileoscopy showed complete loss of normal intestinal anatomic landmarks and ulcerated mucosa. Graft biopsies showed ulceration and granulation tissue with severe architectural distortion consistent with severe intestinal graft rejection. She initially received intravenous corticosteroids and increased tacrolimus dose without significant improvement. Her immunosuppression was escalated to include infliximab and finally antithymocyte globulin. Graft enterectomy was considered repeatedly; however, clinical improvement was noted eventually with evidence of histologic improvement and salvage of the graft. The aggressive antirejection treatment was complicated by development of post-transplant lymphoproliferative disorder that resolved with reducing immunosuppression. Her graft function is currently maintained on tacrolimus, oral prednisone, and a periodic infliximab infusion. We conclude that a prompt and aggressive immunosuppressive approach significantly increases the chance of rescuing small bowel transplant rejection.

P1.57: Severe Late Onset Acute Cellular Rejection in a Pediatric Patient with isolated small intestinal transplant rescued with aggressive immunosuppressive approach

Introduction: Small intestinal transplantation has been the standard treatment modality for pediatric patient with intestinal failure who failed other surgical and medical treatment. At its current stage, it carries its own risks including but not limited to acute and chronic cellular rejection, graft malfunctions, development of PTLD, viral and bacterial infections. Acute cellular rejection of the intestinal graft is an important and major complication secondary to its rich lymphatic supply. It is usually results from sub therapeutic immunosuppression or non-adherence to medical management. The current management includes modifying the immunosuppression to achieve therapeutic level. Late severe acute intestinal allograft rejection is associated with increased risks of sepsis, bleeding and in the majority of cases with graft loss that ends up in graft enterectomy. We present a case of a 20 year old patient who underwent isolated small bowel transplant for complete intestinal Hirschsprung Disease at age 7 years old, but due to medication non-adherence developed severe late-onset acute cellular rejection manifested by large ostomy OP, fever, weight loss. Underwent ileoscopy on presentation that showed complete loss of normal anatomical intestinal landmarks, bleeding, and ulcerated mucosa. Graft biopsies showed ulceration and granulation tissue with severe architectural distortion, and rare residual consistent with severe intestinal graft rejection. She initially received pulse doses of Intravenous corticosteroids and increased dose of tacrolimus without significant improvement. Her immunosuppression plan was escalated to include infliximab and finally had Antithymocyte globulin (ATG). Graft enterectomy option was entertained frequently during the treatment course; however clinical improvement was noted with evidence of histological improvement and salvage of the graft. The aggressive anti-rejection treatment was complicated with the development of monomorphic, plasmacytoma (PTLD) that was managed with modifying her immunosuppression. Now her graft function is maintained on tacrolimus, oral prednisone, and routine Remicade infusion. Conclusion: 1-Small bowel graft rejection is associated with increased morbidity and mortality, increased risks of sepsis, bleeding and graft loss. 2-We believe that prompt and aggressive immunosuppressive approach significantly increases the chance of rescuing small bowel transplant rejection.

Paneth and Intestinal Stem Cells are Differentially Affected during Worsening of Acute Cellular Rejection in Small Bowel Transplantation compared to Intestinal Ischemia Reperfusion Injury and Graft versus Host Disease

Instituto de Estudios Inmunológicos y Fisiopatológicos, UNLP-CONICET, La Plata, Argentina. Introduction Acute cellular rejection (ACR) is one of the leading causes of graft loss after small bowel transplantation (SBTx). Although crypt epithelial cell apoptosis is used as histopathological criteria for ACR diagnosis, it is not clear which of epithelial lineages are the target of ACR. It was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, however, no results on other cell types of the crypt have been depicted so far. The aim of our work was to elucidate integrity and functionality of Paneth and stem cells during different degrees of ACR, in comparison with other clinical conditions such as graft vs. host disease (GVHD) and ischemia reperfusion injury (IRI). Material and Methods 44 patients of SBTx and controls treated in our center between 2006 and 2016 were included in the study. Rejection diagnosis was established by an experienced pathologist according to the recommendations of the pathology workshop of the VIII Small Bowel Transplantation Symposium. We compared small biopsies from SBTx patients with no, mild, moderate or severe ACR by immunohistochemistry and quantitative PCR (n=10 per group as average) for different cell markers (Lgr5, lysozyme, defensin5, IL22R, Ki67). Other conditions such as different degree of intestinal GVHD and IRI were included as separate groups. Comparisons among groups of data were performed with the Kruskal-Wallis test and the Dunn`s post-test when more than two groups were analyzed, whereas the Mann-Whitney U test was chosen to compare data from two groups. Results and Discussion Using differential immunofluorescence counting of different markers we observed that numbers of Paneth and stem cells remain constant in all ACR groups, whereas the transit-amplifying zone is the region that concentrates most of the apoptotic bodies during ACR (p<0.05). We detected unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki-67+IL-22R+ stem cells revealing cell functionality in moderate ACR samples. Oppositely, in GVHD of moderate to severe, as well as IRI, Paneth cell number and markers were diminished (p<0.05), indicating differential mechanism that operate to trigger epithelial cell apoptosis in different situations. Conclusion Paneth and stem cells are not primary target cells during ACR. IL-22R + Ki-67+ stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients.

Gut microbiota and its implications in small bowel transplantation.

The gut microbiota is mainly composed of a diverse population of commensal bacterial species and plays a pivotal role in the maintenance of intestinal homeostasis, immune modulation and metabolism. The influence of the gut microbiota on solid organ transplantation has recently been recognized. In fact, several studies indicated that acute and chronic allograft rejection in small bowel transplantation (SBT) is closely associated with the alterations in microbial patterns in the gut. In this review, we focused on the recent findings regarding alterations in the microbiota following SBTand the potential roles of these alterations in the development of acute and chronic allograft rejection. We also reviewed important advances with respect to the interplays between the microbiota and host immune systems in SBT. Furthermore, we explored the potential of the gut microbiota as a microbial marker and/or therapeutic target for the predication and intervention of allograft rejection and chronic dysfunction. Given that current research on the gut microbiota has become increasingly sophisticated and comprehensive, large cohort studies employing metagenomic analysis and multivariate linkage should be designed for the characterization of host-microbe interaction and causality between microbiota alterations and clinical outcomes in SBT. The findings are expected to provide valuable insights into the role of gut microbiota in the development of allograft rejection and other transplant-related complications and introduce novel therapeutic targets and treatment approaches in clinical practice.

Paneth and intestinal stem cells preserve their functional integrity during worsening of acute cellular rejection in small bowel transplantation.

Graft survival after small bowel transplantation remains impaired due to acute cellular rejection (ACR), the leading cause of graft loss. Although it was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, no results of Paneth and intestinal stem cells localized at the crypt bottom have been shown so far. Therefore, we wanted to elucidate integrity and functionality of the Paneth and stem cells during different degrees of ACR, and to assess whether these cells are the primary targets of the rejection process. We compared biopsies from ITx patients with no, mild, or moderate ACR by immunohistochemistry and quantitative PCR. Our results show that numbers of Paneth and stem cells remain constant in all study groups, whereas the transit-amplifying zone is the most impaired zone during ACR. We detected an unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki-67+ IL-22R+ stem cells revealing cell functionality in moderate ACR samples. We conclude that Paneth and stem cells are not primary target cells during ACR. IL-22R+ Ki-67+ stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients.

“Snowmelt Sign” and “Corkscrew Microvessels” Predicting Epithelium Regeneration After Acute Rejection of Small-Bowel Transplantation: A Case Report

Intestinal failure characterized by inadequate maintenance of nutrition via normal intestinal function comprises a group of disorders with many different causes. If parenteral nutrition dependency develops, which is associated with higher mortality and complications, it is considered for intestine transplantation. However, the graft failure rate is not low, and acute cellular rejection is one of the most important reasons for graft failure. As a result, early identification of rejection and timely modification of anti-rejection medications have been considered to be associated with better graft and patient survival rates. The diagnostic gold standard for rejection is mainly based on histology, but hours of delay by pathology may occur. Some researchers investigated the association of endoscopic images with graft rejection to provide timely diagnosis. In this study, we present the first case report with characteristic features under magnifying endoscopy with a narrow-band imaging system to predict epithelial regeneration and improvement of graft rejection in a patient with small-bowel transplantation.

Advances in small bowel transplantation.

Small bowel transplantation is a life-saving surgery for patients with intestinal failure. The biggest problem in intestinal transplantation is graft rejection. Graft rejection is the main reason for morbidity and mortality. Rejection has a negative effect on the survival of the graft. While 50%-75% of small bowel transplantation patients experience acute rejection, chronic rejection occurs in approximately 15% of patients. Immune monitoring is crucial after small bowel transplantation. Unlike other types of transplantation, there are no non-invasive or reliable markers to predict rejection in small bowel transplantation. The diagnosis of AR is confirmed by clinical symptoms, endoscopic appearance, and pathological specimens taken by endoscopy. Thus, histopathological examinations obtained by protocol biopsies remain as the gold standard for intestinal graft monitoring; however, biopsies have some complications, especially in small grafts. In addition to the high complication rate, biopsies are non-diagnostic; thus, multiple biopsies should be performed to exclude rejection. Therefore, auxiliary assays, such as measurements of citrulline and calprotectin in the blood, cytofluorographic examination of peripheral blood immune cells, cytokine profiling, and distinct gene-set-change measurements, are increasingly being used in small bowel transplantation. Developments in the understanding of genes seem to be promising that limited gene sets, taken from blood or from intestinal biopsies, will enhance pathological diagnosis. Bone marrow mesenchymal stem cell transplantation with SBT and tissue engineering are also promising procedures.

Refinement of Histological Criteria of Acute Humoral Rejection in Small Bowel Transplantation.

Refinement of Histological Criteria of Acute Humoral Rejection in Small Bowel Transplantation.

Paneth and Stem Cells Are Not Affected During Initial Stages of Acute Cellular Rejection in Small Bowel Transplantation in Humans

Introduction: In spite of the last developments and improvements in long term outcome after intestinal transplantation, acute cellular rejection (ACR) remains as the most common cause of graft loss[1]. The diagnosis of ACR is still based on pathological findings[1][2], but it is unclear, which cell type and which crypt zone (bottom (BZ) or transit-amplifying zone (TZ)) are the main target cells. Therefore, we wanted to elucidate which crypt zone is mainly disturbed by apoptosis and whether the early target cells of ACR are Paneth cells (PC) or stem cells (SC) as has been shown in other pathologies[3]. Methods: Intestinal biopsies from ITx-patients classified by an experimented pathologist as normal (N, n=8) or with mild (MR, n=8) or moderate (MoR, n=8) ACR were used. Immunostainings were performed with antibodies against αdefensin-5 (DEFA5), interleukin-22 receptor (IL22RA1), cleaved-caspase-3 (CASP3) to identify PC, SC and apoptotic events. The expression of DEFA5, lysozyme (LYZ, PC marker), Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) and IL22RA1 genes (SC markers) was assayed by qPCR in N (n=5) and MoR (n=6). Results: Our results show that there was an increasing number of apoptotic bodies in BZ as the diagnosis worsens from N to MoR. Apoptosis per crypt in TZ during MoR exhibited statistical differences compared with the TZ in N group (N: 0.13±0.17, MoR: 0.64±0.25; P<0.05). According to this, the number of PC remained constant in N and MR groups and was not significantly lower in MoR (N: 3.57±1.0, MR: 3.56±0.85, MoR: 2.54±0.97; P:NS). Moreover, the expression of antimicrobial peptides had the same level or even higher in MoR than that in normal group (DEFA5 N: 1.23±0.94, MoR: 2.02±2.04; LYZ N: 1.20±1.08, MoR: 1.35±1.13; P:NS). Regarding SC, there were no differences in the expression level of LGR5 between groups (N: 0.89±1.10, MoR: 1.08±1.16; P:NS), whereas IL22RA1 had two-fold increase in rejection group (N: 0.46±0.33, MoR: 1.05±0.80; P:NS). In addition, no variations in the number of IL22RA1+ comparing groups were found (N: 1.74±0.32, MR: 1.55±0.44, MoR: 1.25±0.58, P:NS). Conclusion: We were able to show that cells are primarily affected in the TZ during ACR, and that PC and SC remain undamaged during MR and MoR. Our results highlight the relevance of future research in the IL22-IL22R+SC axis as a future therapeutic target to promote epithelial regeneration and avoid intestinal barrier disruption during rejection processes.

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

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

The Long and Short of It: A Case of Long-term Teduglutide for the Treatment of Intestinal Rejection in Small Bowel Transplant: 2016 ACG Presidential Poster Award

Small bowel transplantation (SBT) is a therapeutic option for those patients who suffer from short bowel syndrome, intestinal failure, and life-threatening complications related to total parental nutrition (TPN). Post-small bowel transplant rejection continues to be a major cause of morbidity and mortality. Endoscopically, graft rejection is characterized by hemorrhage, blunting, and sloughing of the intestinal villi while histologically, it is characterized by increased apoptotic bodies and decreased mitotic bodies per crypt. Teduglutide, a glucagon-like peptide 2 analogue, has been shown to improve intestinal absorption by promoting villous hypertrophy and crypt proliferation. We hypothesized that teduglutide 5 mg subcutaneously daily can promote small bowel villous growth and attenuate small bowel transplant rejection by increasing crypt division while decreasing the apoptotic rate. This novel application of teduglutide may facilitate recovery from rejection and improve nutritional absorption. We present an interesting case of a 22-year-old woman with a myofibroblastic tumor of the mesentery who underwent extensive small bowel resection and subsequent living related small bowel transplantation. She presented with fever and abdominal pain post-operative day 14 after SBT. She underwent ileoscopy revealing ulcerated and extensively denuded intestinal mucosal villi. Histology demonstrated inflammatory changes extending through the muscularis mucosa that was consistent with severe acute graft rejection. She was subsequently placed on teduglutide while continuing immunosuppressive therapy and underwent serial endoscopic evaluations with random small bowel biopsies. Endoscopically, there was serial improvement in the health of the small bowel graft, as histologically demonstrated by an increase in villous growth, overall decrease in the apoptotic rate, and overall increase mitotic rate over 9 months. Furthermore, we conducted novel fluorescent in-situ hybridization (FISH) analysis which confirmed an XY karyotype in the regenerating stem cells of the small bowel graft, indicating that her father's stem cells from the donated small bowel were indeed regenerating. In conclusion, we present a novel application of teduglutide to mitigate small bowel transplant rejection and promote villous growth. Teduglutide should be considered in these patients to help improve nutritional status, allow them to wean off TPN, and ultimately improve quality of life.

Elevated ST2 Distinguishes Incidences of Pediatric Heart and Small Bowel Transplant Rejection.

Elevated serum soluble (s) suppressor of tumorigenicity-2 is observed during cardiovascular and inflammatory bowel diseases. To ascertain whether modulated ST2 levels signify heart (HTx) or small bowel transplant (SBTx) rejection, we quantified sST2 in serially obtained pediatric HTx (n = 41) and SBTx recipient (n = 18) sera. At times of biopsy-diagnosed HTx rejection (cellular and/or antibody-mediated), serum sST2 was elevated compared to rejection-free time points (1714 ± 329 vs. 546.5 ± 141.6 pg/mL; p = 0.0002). SBTx recipients also displayed increased serum sST2 during incidences of rejection (7536 ± 1561 vs. 2662 ± 543.8 pg/mL; p = 0.0347). Receiver operator characteristic (ROC) analysis showed that serum sST2 > 600 pg/mL could discriminate time points of HTx rejection and nonrejection (area under the curve [AUC] = 0.724 ± 0.053; p = 0.0003). ROC analysis of SBTx measures revealed a similar discriminative capacity (AUC = 0.6921 ± 0.0820; p = 0.0349). Quantitative evaluation of both HTx and SBTx biopsies revealed that rejection significantly increased allograft ST2 expression. Pathway and Network Analysis of biopsy data pinpointed ST2 in the dominant pathway modulated by rejection and predicted tumor necrosis factor-α and IL-1β as upstream activators. In total, our data indicate that alloimmune-associated pro-inflammatory cytokines increase ST2 during rejection. They also demonstrate that routine serum sST2 quantification, potentially combined with other biomarkers, should be investigated further to aid in the noninvasive diagnosis of rejection.

IDO expressing dendritic cells suppress allograft rejection of small bowel transplantation in mice by expansion of Foxp3+ regulatory T cells

BackgroundIndoleamine 2,3-dioxygenase (IDO), the enzyme that catalyzes the first and rate-limiting step of tryptophan catabolism, suppresses T-cell responses by tryptophan depletion and accumulation of kynurenine metabolites. IDO prevents allograft rejection in various transplantations. MethodsDendritic cells (DC) highly expressing IDO (IDO+ DC) were cultured through transduction of adenovirus vectors carrying the IDO sequence. IDO+ DC were incubated with CD4+ CD25− T cells to detect T cell proliferation. The effects of IDO+ DC and 3-Hydroxyanthranilic acid (3-HAA) were verified in an allogeneic murine small bowel transplantation (SBT) model. Foxp3+ Treg cells of recipient mice were detected by flow cytometry and cytokines in plasma were determined by ELISA. ResultsIDO+ DC effectively suppressed proliferation of CD4+ CD25− T cells in vitro, and this effect could be enhanced by adding 3-HAA. In the SBT transplantation model, both 3-HAA (P<0.05) and IDO+ DC (P<0.01) prolonged the survival time of transplanted mice. Mice treated with IDO+ DC achieved longer mean survival time than 3-HAA administrated mice (11.5d vs. 18.5d). Grafts from IDO+ DC, 3-HAA and combination treatment group showed reduced inflammation and minimal architectural distortion. IFN-γ production was significantly inhibited by IDO+ DC and 3-HAA (P<0.05). The expression of IL-2 was slightly lower with 3-HAA or IDO+ DC treatment. However, IL-10 was higher in 3-HAA, IDO+ DC and combination treatment groups, while TGF-β was elevated in all non-control groups. ConclusionsIDO+ DC plus 3-HAA has an immunoprotective role and represents a potential strategy to suppress acute rejection and prolong survival of grafts in SBT.

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

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

Histologic Analysis of Chronic Rejection in Small Bowel Transplantation

Chronic rejection is a significant barrier to small bowel allograft survival. Although chronic rejection primarily involves vessels of the submucosa, serosa, and mesentery, some mucosal alterations have been suggested to be correlative. We retrospectively investigated explanted small bowel allografts for clinical characteristics and histological alterations in the mucosa, submucosa, and serosa. Crypt epithelial mucin loss, submucosal fibrosis, and length of time to explant were all statistically associated with chronic rejection. Medium-sized and large-sized vessels of the serosa and mesentery preferentially demonstrated histologic changes of chronic rejection. These results further define chronic vascular rejection and the relationship between the mucosal changes and chronic rejection.