Objective To study the mechanism of immune hyporesponsiveness of allograft rejection induced by transfusion nonpufsed allopeptide syngeneic immature dendritic cell (imDC) generated from recipient bone marrow progenitors and to explore a possible strategy for liver allograft protection in clinic. Methods Forty experimental rats were randomly divided into 4 group: control group, cyclosporine A (CsA) group, mature DC (mDC) group and imDC group. In control group, Wistar rats only received liver transplantation. In CsA group, Wistar rats underwent liver transplantation plus CsA treatment 〔10 mg/（kg·d)〕. In mDC group, recipient-derived mDC 1×106 were infused intravenously through the penile vein to Wistar rats. In imDC group, ImDC with the dose of 1×106 were injected into Wistar rats via the dorsum vein of penile. In each group, five recipients were killed on the 10th day after transplantation, the other five recipients were left to observe survival time. The levels of ALT, AST, TBIL, IL-2, IFN-γ, IL-4 and IL-10 were detected. The acute rejection and the expression of FasL/Fas in the grafts were detected by HE and immunohistochemical staining. Western blot was used to detect Scurfin protein expression of CD4+ CD25+ T cells. Results The median survival time of the liver allografts in CsA group and imDC group were significantly longer than that in control group and mDC group ( P ＜ 0.05). The levels of ALT and TBIL in control group and mDC group were significantly higher than those in CsA group and imDC group ( P ＜ 0.05). Compared with CsA group and imDC group, the levels of IL-2 and IFN-γ were higher but the levels of IL-4 and IL-10 were lower in control group and mDC group ( P ＜ 0.01). Slightly or no rejection reaction was found in CsA group and imDC group ( P ＜ 0.05). The Scurfin protein expressions of CD4+ CD25+ T cells of imDC group were significantly higher than those of other three groups. Conclusion Application of nonpufsed allopeptide syngeneic recipient-derived imDC is an effective way to induce immune hyporesponsiveness by blocking indirect recognition in rat liver transplantation model. Survival span is significantly prolonged by its protective effect. The mechanism of immune hyporesponsiveness induced by imDC transfusion might be involved in some aspects: T cell apoptosis, immune deviation of Thl/Th2 cytokine net and inhibition of T lymphocytes responsiveness by regulatory T cells.
Objective To investigate the inhibitory effect and its mechanisms of TLSFJM (JM acute T leukemia cell line derived suppressor factor) on allograf t rejection of small bowel t ransplantation in rat , and to compare the effect s and complications of TLSFJM with those of FK506. Methods One hundred male Brown Norway (BN) rats and 100 Lewis(L EW) rat s were t reated as donors and recipient s of small bowel t ransplantation , respectively. Then they were divided into five groups according to the dose of administ ration of TLSFJM and/ or FK506 : small bowel transplantation group (SBT group) ; large dose of FK506 〔0. 5 mg/ ( kg ·d) 〕group ; small dose of FK506 〔0. 25 mg/ (kg ·d) 〕group ; TLSFJM 〔10 U/ ( kg ·d) 〕group ; TL SFJM 〔10 U/ ( kg ·d) 〕associated with small dose of FK506 〔0. 25 mg/ (kg ·d) 〕group. FK506 and TLSFJM were administered through int ramuscular or int raperitoneal injection , respectively. Survival time , body weight , hepatic and renal function and histopathology of recipient s in each group were observed. Results TLSFJM took no damage effect on the recipient s’renal and hepatic functions 7 days after administ ration. When TLSFJM was administ rated associated with small dose of FK506 in small bowel transplantation , it could not only effectively suppress rejection reaction , extend recipient’s survival time , but also decreased the dosage of FK506 and prevented the side effect s. But TLSFJM may not be used as immunosuppressive agent alone for the prevention and treatment of rejection in rat small bowel t ransplantation because the rejection still existed. Conclusion As an effective immunosuppression agent , TLSFJM associated with small dose of FK506 can prolong the survival time of both recipients and graf ting small bowel , relieve intensity of rejection , and prevent the side effect s when high dosage FK506 is administ rated. TLSFJM may be used as a high-efficiency , low-toxicity immunosuppresive agent in small bowel transplantation.
Objective To investigate the roles of cell apoptosis and the gene expressions of Fas, FasL, bcl-2 and bax in acute rejection of pancreaticoduodenal transplantation and to evaluate the function of duodenum biopsy for early detection of rejection in rats. Methods Wistar and SD rats were divided into two groups: ①Wistar rats that underwent allogenic pancreaticoduodenal transplantation from the organs of SD rats; ②Wistar rats that received homogenic transplantation. The grafts were then harvested on day 3, 5 and 7 after the transplantation, and all graft samples were observed with HE staining and TUNEL was also used to detect apoptotic cells. The expressions of Fas, FasL, bcl-2 and bax were measured by immunochemical method. According to Nakhleh’s score, pathologic features of transplanted pancreas and duodenum were ranged from one to three scores in order. Results The percentage of same or different scores between the pathological scores of pancreas and duodenum in allogenic pancreaticoduodenal transplantation group were 61.1% (11/18) and 38.9% (7/18) respectively, and there were 6 specimens of pancreatic tissue got higher scores with only one higher score for duodenum. There were significant differences of histopathologic rejection scores and apoptotic indices between the two groups, respectively (P＜0.05， P＜0.01). Apoptotic indices of pancreas and duodenum both showed positive correlations with histopathologic rejection scores (r=0.965， P＜0.01; r=0.942, P＜0.01). The rejection score and apoptotic index elevated, the expression of FasL increased, bcl-2 decreased, and Fas and bax changed over time after operation. Conclusion Apoptosis maybe significantly positive correlated with the degrees of damage of the acute pancreaticoduodenal allograft rejection, and the apoptotic index maybe valuable to estimate the damage. FasL and bcl-2 were significantly related to the impairment of acute pancreatic allograft rejection as well. Duodenum biopsy may contribute to the early diagnosis of the rejecting transplanted tissues.
【Abstract】Objective This study was conducted to build experimental model of orthotopic liver transplantation in rat (ROLT) with the character of acute rejection; and to study the effect of cytotoxic T lymphocyte antigen 4 immunoglobulin G (CTLA4Ig) on prevention of rejection and the induction of immune tolerance of ROLT. Methods Build model of Wistar→SD ROLT(performed by the two cuff method) with character of acute rejection.Recipients were injected with CTLA4Ig 75 μg per ROLT into abdominal cavity after 2 days of operation. Contrast was made with no treatment group, the clinical characters, the liver function, the transplantated liver pathologic character and the concentrations of TNFα in serum were observed and measured on postoperative day 7. In treatment group, all above observation were done on postoperative month 4. Above all, determination of the effect of CTLA4Ig on preventing acute rejection and inducing tolerance in ROLT was observed.Results ①Recipients (no treatment group) died one by one within 6th～14th days; pathologic character of rejection in transplantation liver could be found; ② In treatment group, on postoperative day 7 and month 4, no clinical rejection character and no pathologic character of rejection in transplantation liver were found and serum concentration of cytokins related to TNFα found lower than that of contrast group(P＜0.05), and serum concentration of ALT、AST、TBIL、DBIL found lower too(P＜0.05); But serum concentration of TP and Alb was found higher than that of contrast group(P＜0.05). Conclusion ① CTLA4Ig treatment alone inhibits the rejection responce in ROLT. ② CTLA4Ig treatment can Prevent rejection and induce immune tolerance in ROLT model with characters of acute rejection; the serum concentration of cytokins related to TNFα can probably be used as evaluation standard of rejection in ROLT rejection.
【Abstract】ObjectiveTo explore the effects of p38 mitogenactivated protein kinase (MAPK) on apoptosis of small intestinal epithelial cells after transplantation in rats. MethodsSmall intestinal transplantation was performed in SD and Wistar rats. The recipients were divided into three groups: isograft group (Wistar→Wistar group), allograft group (SD→Wistar group) and allograft+cyclosporine A group (SD→Wistar+CsA group). The grafts were harvested on day 1, 3, 5 and 7 after operation. All graft samples were subjected to histological examination. The apoptosis of graft epithelial cells was detected by TUNEL method. p38 MAPK was measured by Westernblotting method and serum TNFα was determined by ELISA. ResultsMild, moderate and severe rejection reaction occurred in the SD→Wistar group, it was showed that the number of apoptotic cells increased with the severity of the rejection reaction by TUNEL. In SD→Wistar group, the numbers of apoptotic cells were significantly higher than those of the other two groups （P＜0.01）. The severity of rejection reaction in SD→Wistar+CsA group was less than that of SD→Wistar group and the number of apoptotic cells increased with the severity of the rejection reaction (P＜0.01). The level of serum TNFα varied with the apoptotic degree of small intestinal epithelial cells in SD→Wistar group and SD→Wistar+CsA group （P＜0.01）. The expression of p38 MAPK increased with the number of the apoptotic cells in SD→Wistar group and SD→Wistar+CsA group （P＜0.01）, but there was no evident change in Wistar→Wistar group （Pgt;0.05）. The expression of p38 MAPK and the level of serum TNFα were positively correlated with apoptosis in small intestinal rejection after transplantation （r=0.875， P＜0.01； r=0.837， P＜0.01）. p38 MAPK and TNFα were also positively correlated （r=0.826，P＜0.01）. ConclusionApoptosis plays an important role in small intestinal rejection. p38 MAPK is involved in apoptosis and is an important regulator in signal pathway of cell apoptosis.
ObjectiveTo compare tacrolumus (FK506) with cyclosporine A (CsA) in clinical application to organ transplantation.MethodsThe literature in recent years has been reviewed and compared. ResultsFK506 was a powerful immunosuppression with a mechanism of action similar to that of CsA, but significantly superiori to CsA in terms of prophylaxis and treatment of allograft acute rejection, delay of chronic rejection, and withdrawal of steroid in early period. The cardiovascular mortality and chronic graft nephropathy (CGN),such as hypertension and hyperlipidemia were less frequently seen in FK506treated patients and FK506 also had an acceptable safety profile, including a low incidence of hypertrichosis,gingival hyperplasia and infections.However, CsA had been showed a better result in prevention of posttransplantation diabetes mellitus (PTDM ) and more economic agent than FK506. Pharmacokinetic studies showed CsA in the form of Sandimmun Neoral showed less inter an intrapatient variability than FK506.Meanwhile, the combination of MMF and FK506 or CsA has been proved effectively with excellent graft and patients survival. Conclusion FK506 and CsA are safe and effective long term maintenance immunosuppressive agents in organ transplantation with wonderful prospect.
【Abstract】Objective To evaluate the pathological diagnosis of liver allograft rejection. Methods Literatures about diagnosis of liver transplantation rejection in recent ten years were reviewed.Results Humoral rejection was rare. The main features were graft blood vessel thrombosis and liver damage and necrosis about some days or one week after transplantation. The humoral rejection of liver graft occurred later than that of kidney and heart transplantation. The diagnosis of acute liver graft rejection was based on Banff Schema. During chronic rejection intrahepatic bile ducts among hepatic lobules in portal area disappeared, and inflammation, fibrosis and stricture of hepatic arteries and veins were found, but the morbidity was less than that of kidney， heart, lung and pancreas grafting. Conclusion Banff standard is the most important base of diagnosing liver graft rejection.
Objective To insure early detection and hence efficient prevention of allograft rejection in transplanted heart, investigate possible applications of NAD(P)H fluorescence components analysis at the level of living cardiac cells to propose new approaches for diagnosis of rejection. Methods NAD(P)H was studied for noninvasive fluorescent probing of the mitochondrial function. Human cardiomyocyte were isolated from one additional endomyocardial biopsy (EMB) of 14 pediatric patients with heart ransplantation. Rat cardiomyocyte (n=5, 13-14 week old) were also isolated by the same approach for human myocytes. Autofluorescence(AF) was recorded in living cardiomyocytes following excitation with 375 nm UVlight and detection by spectrallyresolved time correlated single photon counting (TCSPC), based on the simultaneous measurement of the fluorescence spectra and lifetimes. Rat cardiac cells were divided into four groups: normoxic condition, normoxia with Rotenone, ischemic condition and ischemia with Rotenone. Comparison of cardiomyocyte AF between human and rat; compared kinetics of rat cardiomyocytes AF in normoxic conditions to ischemiamimicking ones, induced at physiological temperatures by reducing cell pH and oxygen content; comparison of cardiomyocyte AF dynamic changes in transplanted pediatric patients presenting either no rejection (R0) or mild rejection (R1). Results We have achieved appropriate isolation of living cardiomyocytes from human biopsies, as well as from rat cardiac tissues and determined their AF. At least a 3-exponential decay with 0.5-0.7ns, 1.9-2.4 ns and 9.0-15.0 ns lifetime pools is necessary to describe human cardiomyocyte AF within 420560 nm spectral range. Rat cardiomyocyte steadystate AF in ischemiamimicking condition was significantly increased when compared normoxic ones (Plt;0.05); application of Rotenone induced a significant increase in AF intensity in ischemic and normoxic condition, however no significant difference between the two groups (Plt;0.05).Human cardiomyocyte AF was found significantly lower in comparison to experimental rat model in the same condition(Plt;0.05). A correlation between changes in steadystate NAD(P)H fluorescence and rejection grades was found when comparison of R1 to R0. R1 showed significantly increased fluorescence intensity (Plt;0.05), without change in the spectra shape, results can be comparable to the effect of ischemiamimic conditions. Conclusion Our studies clearly demonstrated that spectrallyresolved fluorescence spectral analysis coupled to fluorescence lifetime are high sensitive approaches to examine mitochondrial metabolic oxidative state directly in living human cardiomyocytes with good reproducibility. Human cardiomyocytes are more metabolically active than the rat ones, while this activity (and thus ATP production) seems lowered during rejection process. In perspective, the advantage of this method is the possibility of its combination to multiphoton confocal microscopy, which can result in the adaptation of this approach directly to tissue biopsy, as well as in vivo directly via cardiac catheterization without the necessity of cell isolation. This approach provides promising new tool for clinical diagnosis and treatment of allograft rejection, and will enhance our knowledge about cardiomyocyte oxidative metabolism and/or its dysfunction at a cellular level.
Abstract:Objective To investigate the expression and significance of Voltage-gated Cl channel-3 （ClC-3） in acute cardiac allograft rejection in rats. Methods The model of heterotopic cardiac allograft of SD to Wistar rats was established. The rats were divided into two groups： control group and cyclosporin A（CsA） treated group （CsA group）. Living span of the transplants in eight rats of each group were observed. Allograft samples were harvested separately on the day 1, 3, 5, 7 after operation （n = 6）. The rejection was evaluated by routine pathological examinations. The myocardial apoptosis by terminal deoxylnucleotidyl transferase mediated-dUTP nick end labeling （TUNEL） method and the local expression of ClC-3 were detected by reverse transcriptase polymerase chain reaction （RT-PCR）. Results The allografts survival time was significantly longer in CsA group compared with that in control group （15.4±5.1dvs. 7.6±1.5d, P〈0.05）. There was lesser pathological changes in CsA group than that in control group. The apoptosis index were significantly higher in control group and the expression of ClC-3 was significantly lower（P〈0.05）. CsA could inhibit the rise of apoptosis index and the decrease of the ClC-3 expression. Conclusion The ClC-3 expression is closely related with the severity of myocardial necrosis and apoptosis index, which indicates that ClC-3 plays a very important role in the necrosis and apoptosis during acute cardiac allograft rejection of rat.
Objective To investigate the effect of interleukin-10 （IL-10） gene transfer on expression of CD44, selectin-E, lymphocyte function associated antigen-1 （LFA-1）, vascular cell adhesion molecule-1 （VCAM-1） in mice heart transplantation rejection. Methods Model of mice cervical heterotopic heart transplantation was set up, 96 mice were divided into three groups with random number table, control group： heart transplantation between C57 mice; transplant group： heart from BALB/C mice transplant to C57 mice; IL-10 group： IL-10 was transfected on BALB/C mice isolated heart for 1 hour, then transplanted to C57 mice. The messenger ribonucleic acid （mRNA） level expression of CD44 ,selectin-E ,LFA-1 ,VCAM-1 and IL-10 were measured by reverse transcription-polymerase chain reaction （RT-PCR） at the 5th day after transplantation. Results The mRNA level expression of CD44, selectin-E ,LFA-1 ,VCAM-1 in transplant group were significantly increased than those in control group （P〈0.01）. The mRNA level expression of CD44, selectin-E, LFA-1 ,VCAM-1 in IL-10 group were significantly decreased than those in transplant group （P〈0.01）. Conclusion IL-10 gene transfer is able to decrease the expression of CD44, selectin-E,LFA-1 ,VCAM-1 and suppress the heart transplantation rejection in mice.