| Organ transplantation is an effective and conventional treatment of the terminal stage of organ failure. The advance of transplantation technology makes many kinds of organ transplantation possible namely transplantation of cornea, heart, lung, pancreas, liver, small bowel, kidney, marrow and so on. In China, allogene organ transplantation cases make up the majority, which brings hope as well as causes trouble to patients and their family. Severe acute and chronic rejections are the main factors that lead to the death of acceptors.Small bowel transplantation is the most ideal clinical treatment of bowel failure. Clinical trial and practice of small bowel transplantation has been done for half of the contrary, but compared with the therapeutic effect of liver, kidney and heart transplantation, the small bowel transplantation is less effective. It is mainly because small bowel has redundant lymphoid tissue and histocompatibility antigen and after allogenic small bowel transplantation patients may suffer strong host versus graft reaction (HVGR), graft versus host reaction (GVHR) and severe infection. Therefore, it is crucial to have in-depth study of rejection mechanism and its treatment in order to improve prognosis.Nowadays the mechanism underlying allograft rejection remains largely unclear. Many immunocytes and cytokines are involved in the small bowel transplantation process. T lymphocytes mediated CTL, B cells and regulatory T cells (Treg) play a decisive role in immune reaction of small bowel transplantation. Cytokines involved in immune response also have a key role in this process. IL-2 and IFN-γwhich are the main cytokines secreted by Th1, have extensive function of immunoregulation and immunoenhancement. They can improve the activation of T cells, B cells, NK cells and macrophages and intensify the rejection. TNF, IL-6 and IL-8 play similar role in intensifying rejection. In contrast, IL-4 may suppress the activation of T cells and B cells so as to suppress the rejection. GF-β, IL-10 and ET can down-regulate the rejection.Th17 is a newly detected subset of CD4+ T, which differs from Th1 and Th2 subsets, mainly expresses IL-17. IL-17 is a proinflammatory factor with strong function, which can induce proinflammatory cytokine such as the expression of IL-6, IL-1β, TNF-αand IL-8 thereby causing cellular infiltration and tissue damage. Its major target cells include epithelial cells, endothelial cells and fibroblasts. Previous studies show that IL-17 is an important factor in autoimmune, disease inflammation, tumor and transplantation immunity. IL-17 exerts biological effects through combining with IL-17R and these target cells are particularly rich in small bowel tissue. IL-17 mediated inflammation is more severe in small bowel tissue. Therefore, we infer that biological function of IL-17 has close relation with small bowel rejection. FK506 is a new and highly effective immunosuppressive agent, which is extensively used in organ transplantation. It may suppress the produce of rejection related cytokines thereby suppressing the activation of T cells, as a result, suppressing the rejection. Besides, FK506 can prevent aggregation of lymphocytes in the early rejection and chemotaxis of inflammatory cells. But whether Th17 or IL-17 participates in the FK506 suppression of rejection or not remains unknown. Although FK506 is used extensively after clinical small bowel transplantation, severe side effects such as renal toxicity and neurotoxicity caused by large dose of FK506 still can not be neglected. So it is imperative to do in-depth study of rejection mechanism and develop new anti-rejection medicine.Since Th17 and IL-17 may play important roles in immune reaction, we detect the functions of Th17 and IL-17 in the process of transplantation rejection and FK506-mediated anti-rejection by using rat small bowel allograft transplantation model and biopsy specimen of human small bowel transplantation. The protective function of IL-17 monoclonal antibody in immunological rejection is further investigated after small bowel transplantation. Thus, the study may push further insight into the mechanisms underlying small bowel transplantation related rejection and lay experimental basis for clinical application of IL-17 monoclonal antibody. The major findings are as follows.1. The expression of Th17/IL-17 is positively correlated with the intensity of acute rejection after living-related small bowel transplantationIn order to examine the relation between Th17/IL-17 expression and acute rejection after living-related small bowel transplantation, we collect biopsy specimens from 4 cases of living-related small bowel transplantation from 1999 to 2003. All the clinical information is available. The expression of CD4+, IL-17+, and Th17 is further detected by LSCM. The data show that the increased expression of Th17/IL-17 is correlated with the high intensity of acute rejection after living-related small bowel transplantation. IL-17 may be considered as one of the early and sensitive warning markers for acute rejection.2. Establishment of a stable and reliable allograft animal modelWe establish one allograft model of inbred F344→LEW rat by combining the self-made controllable swivel intravenous infusion device with the technique of segmental heterotopic small intestine transplantation. The results suggest that this device may successfully ensure continuance infusion for more than 1 month in rats, improving the blood supply and nutrition of the acceptors and evidently raising the survival rate of grafts (95.0%), thus providing one valuable tool for the experimental study of small intestine transplantation immunity.3. The expression of Th17/IL-17 in grafts is positively correlated with the intensity of acute rejectionIn order to examine the relation between the expression of Th17/IL-17 in grafts and acute rejection, we detect the expression of Th17/IL-17 and relative cytokines and transcription factor by using real time quantified PCR, Western blot, LSCM and ELISA. The results show that IL-17mRNA is decreased in normal small bowel tissues. The mRNA expression of IL-17 and RORγt is found increased gradually during acute rejection period in the control group. The mRNA expression of IL-1β, IL-6, TNF-α, IL-8 and IFN-γis found increased on postoperative day 3, 5 and 7, respectively. On the contrary, the mRNA expression of IL-4, TGF-βand Foxp3 is decreased in control group, coupled with downtrend on postoperative day 3, 5 and 7.4. The role of Th17/IL-17 in FK506-related rejection suppressionIn order to investigate the role of Th17/IL-17 in FK506-related rejection suppression, we treat the rats received small bowel transplantation with FK506 and detect the expression of relative cytokines and transcription factors using the methods as described above. The results show that FK506 treatment may decrease the expression of IL-17 as well as Th17 in grafts, thus suppressing acute rejection and increasing TGF-βmRNA and Foxp3 mRNA expression in grafts. The data confirm that Th17/IL-17 may be involved in FK506-related rejection suppression.5. Establishment of IL-17 mAb and application of IL-17 mAb in suppressing acute rejection and prolonging the survival time of graftsFor the sake of exploring the function of IL-17 mAb in rejection suppression, the anti-rat IL-17 mAb is established by using hybridoma clone and is further used in the early treatment after small bowel transplantation. We found that the expression of IL-17, IL-1β, IL-6, TNF-α, IL-8 mRNA is decreased in experimental group treated with IL-17mAb, while the expression of IL-4 and TGF-βmRNA increase slowly. The results show that the survival time of experimental group treated with IL-17mAb is significantly longer than that of untreatment group. The results of histopathologic examination show no sign of rejection. The data implies that IL-17mAb treatment may effectively suppress rejection after rat small bowel transplantation and remarkably improve the survival of grafts.6. ConclusionWe found that Th17/IL-17 expression existed in grafts after human small bowel transplantation and it positively correlated with acute rejection. In order to decide whether Th17/IL-17 expression was involved in rejection, we established rat allograft small bowel transplantation model and further study confirmed that Th17/IL-17 expression also existed in grafts and it positively correlated with rejection. In addition, RORγt mRNA of grafts and expression of IL-1β, IL-6, TNF-α, IL-8 and IFN-γmRNA increased on postoperative day 3, 5, and 7. On the contrary, low expression of IL-4, TGF-βand Foxp3 mRNA had been detected in untreatment group, coupled with downtrend on postoperative day 3, 5 and 7.FK506 treatment could decrease IL-17 expression, thereby suppressing acute rejection, increase TGF-βand Foxp3 mRNA expression in grafts. It indicated that Th17/IL-17 is involved in FK506 induced rejection suppression. Likewise, early treatment with IL-17mAb after transplantation may suppress acute rejection.From the above results we may draw the conclusion that Th17/IL-17 and many cytokines participated in the acute rejection after small bowel transplantation. FK506 may suppress acute rejection through downregulating Th17/IL-17 expression. Early administration of IL-17mAb monoclonal antibody effectively inhibited acute rejection, which might pave the way for diagnosis and treatment of acute rejection and induced immunotolerance in clinic. |
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