| Liver transplantation(LT) has become the most effective treatment for patients with end-staged liver diseases, however, infections is still common after LT and seriously decreases the recepients and graft survival. As the severity of illness of recepients pretransplant, as well as the particularity of LT, posttransplantive infections charactered various of pathogen species, severity, and high morbidity and mortality. Moreover, infection related with rejection posttransplant. Viral infection has been shown to promote chronic rejection posttransplant, whilethe relationship between bacterial infection and rejection are not entirely definited. Severe infections, accompanied with imbalance and functional impairment of T cell subsets, showed "immune paralysis" of the immune system. DCs play an immunosuppressive effect role during this course via their heterogeneity. There has been growing evidences to support this. The effection of immune regulation treatments to sepsis has been more explicit. However, it is controversial in organ transplant due to fear of rejection. In this study, the intraabdominal infection was induced by E. coli after orthotopic liver transplantation in rats(ROLT), and thymosin-alpha1 was injected intraperitoneally then, the changes of cellular immune response was monitored.ObjectiveTo momitor the immume function changes of T cells and the proliferation and biological effect of dendritic cells derived from spleen and to explore the role of thymosinα1 in the immune conditioning of recepients by established a model of intraabdominal bacterial infection following orthotopic liver transplantation in rat.Methods 1. Established the orthotopic liver transplantation model of closed colony rats: based on the classical two-cuff technique by Kamada, 120 cases of rat orthotopic liver transplantation were performed with fast resecting the donor liver, continuous sutureing suprahepatic inferior vena cavum(SHVC) to observe the time of surgical procedures, postoperative complications and recepients survival rate.2. Establishment of rats orthotopic liver transplantation with acute rejection models: according to the different donor-recipient combinations,the research animals were divided into three groups: SD→SD group (Isograft group), SD→Wistar group and DA→Lewis groups, each group with 32 cases. Six rats killed randomly to observe the changes in blood liver function (ALT and TBIL) and liver pathological changes in 3, 5, 7, 10 days respectively after transplantation. The residual rats were retained for survival analysis.3. Peritoneal catheter was retented after rats orthotopic liver transplantation with acute rejection model. E. coli ATCC25922 was perfused through the tube 3 days and 5 days posttransplant. The research groups were randomly divided into five infection groups, including 2×108cfu/ml, 2×107cfu/ml, 5×106cfu/ml, 1×106cfu/ml, 5×105cfu/ml, and one control group (perfused sterile saline 2ml intraperitoneally) at each time point. Rats were sacrificed in 1 day before perfusion, 1 day, 3 days, 5 days, and 7 days after perfusion resceptively, each time point with four rats, another eight rats involved in each group was retained for survival analysin. To observed recepients'general condition, ascites bacterial culture and leukocyte count, peripheral blood white blood cell count, TCO2, HCO3-, PH, ALT, TB, as well as liver and kidney lung HE staining to evaluate the status and severity of infection.4. Used DA or LEW rats as donors and LEW rats as recepients, established rat liver transplantation model. Based on whether induction of infection or no, the experimental animals were divided into four groups: G1 group (LEW→LEW, non-infection group), G2 group (LEW→LEW, infection group), G3 group (DA→LEW, non-infection group), G4 group (DA→LEW, infection group). The bacteria were perfused through peritoneal catheter with concentration of 1×106cfu/mL three day after transplantation. Rats were sacrificed in 1 day before perfusion, 1 day, 3 days, and 5 days after perfusion resceptively, each time point with four rats. Through three means: liver pathology, CXCR3 and its ligand CXCL10 changes detected by immunohistochemistry and fluorescence quantitative PCR, and apoptosis of liver cells assayed by TUNEL to evaluate the change of the graft immune rejection responses.5. Experimental animal groups, disposition, and time of specimen collection were similar with step 4. The proportion of peripheral blood T lymphocyte subsets and its function were identified by flow cytometry (FCM) and one-way mixed lymphocyte culture respectively. Serum cytokine changes were detected by ELISA to evaluated the T lymphocyte subsets, function and differentiation after bacterial infection.6. Experimental animal groups, disposition, and time of specimen collection were similar with step 4. The spleen dendritic cells(DCs) of recepients were isolated freshly by magnetic cell sorting technique. Their phenotype and function were detected by flow cytometry and one-way mixed lymphocyte culture to evaluate the effects on spleen DCs by bacterial infection on recepients.7. Used DA rats as donors and LEW rats as recepients to established rat liver transplantation model. According to whether induction of infection and/or drug intervention or no, rats were divided into four groups: G1 group was the control group, G2 group was drug intervention based on G1, G3 group was infected group, G4 group was drug intervention based on G3. Infection-induced method was similar to the step4. Thymosinα1 with a dose of 2mg was injected into the abdominal cavity in a one-time six days after transplantataion (72h after infection). Rats were sacrificed 1 day pre-infection and 2 days after drug intervention (eight days posttransplantation) respectively. Each time point and each group included four rats. Six rats were retained to survival analysis in each group. The general conditions and survival rate were observed. The pathological changes in liver grafts, the proportion of peripheral blood T lymphocyte subsets and their functional changes were inditified to evaluate the effection of immune regulation on rat liver transplantation with intra-abdominal bacteria infection.8. The data were analyzed through SPSS16.0 software. One-way ANOVA, t test, rank sum test methods and Kaplan-Meier survival analysis were applied. P<0.05 was considered statistically significant.Results1. In the stabile stage of rat orthotopic liver transplantation, the time of the main steps of operation were as follow: donor operation 30.2±2.5min, donator liver trimming 5.7±1.6 min, suprahepatic inferior vena cava(SHVC) anastomosis 9.1±0.8 min, portal vein (PV) reconstruction 1.6±0.5 min, infrahepatic vena cava(IHVC) reconstruction of 1.5±0.4 min, anhepatic period 15.4±1.1min, biliary duct reconstruction 1.1±0.3min, recepients operation 39.5±1.4 min. Surgical success rate was 100%, one-week survival rate was 97.7%, late complications such as biliary obstruction incidence was 3.3%.2. The survival time of SD→SD group was more than 120d; the median survival time of SD→Wistar group was 100d, its 95%CI was (83.369~116.631)d, no statistical difference was found between the two groups(P=0.317). The median survival time of DA→Lewis Group was 12d, its 95%CI was (10.125 ~13.848)d, the difference had remarkable statistical significance (vs SD→Wistar group, P=0.000). In regard to serum ALT and TBIL levels, downward trend was found in SD→SD group gradually, the same was seen in SD→Wistar group but slower during the first five days posttransplant, while in DA→Lewis Group they increased progressively. At 10 days after transplantation, ALT and TBIL presented SD-SD < SD-Wistar < DA-Lewis simultaneously, (58.38±12.44 vs 100.96±16.50 vs 1162.25±84.34) and (15.99±5.92 vs 76.65±9.45 vs 175.06±16.67) respectively, the difference all had statistical significance (DA→Lewis Group vs SD→SD group and SD→Wistar group, P<0.05). There was no acute rejection episodes in SD→SD group with rejection activity index (RAI) score 1 to 2 score. The same representations was found in SD→Wistar group except that 1 rat showed severe acute rejection with RAI 8 score at 10 days postoperation. In DA→Lewis group, acute rejection episodes appeared in some rats at three days after operation and the pathological grading of damages all intensified quickly with RAI score 8 to 9 at 10 days posttransplant. And the result of Kruskal-Wallis H test in day 10 showed:χ2=20.107,ν=2, P=0.000, obviously statistical significance was found between three groups.3. The survival time were extremely short in all intra-abdominal injection groups induced in 5d postoperative (mostly of the 1~3 days after infection). Among the intra-abdominal injection groups induced in 3d posttransplant, the survival time after infection were most of 2~3 days in G1, G2 and G3 group, 5~9 days in G4, 9~10 days in G5. The result of Log-Rank analysis were follow: (χ2=13.322,ν=1,P = 0.000, G4 vs G5), (χ2=11.829,ν=1,P = 0.001,G4 vs G0) and (χ2=0.013,ν=1,P = 0.909,G5 vs G0). In regard to ascites white blood cell count, a continuously increased trend was found in G4, while G5 showed increased firstly and then decreased. Remarkly statistical difference was found with P<0.01 in 7 days after infection (G4 vs G5). Ascites bacterial culture was negative in G5 group during the late phase of infection, it might be that the body resisted bacterial spontaneously. While in G4, it was continuously positvie. As time goes by, peripheral WBC, ALT and TBIL increased continuously, PH, HCO3- and TCO2 value decreased, showing serious acid-base balance disturbances and abnormal liver function in G4 group. Statistical difference was found with P<0.05 in end point of study (G4 vs G0). There were abnormalities during earlier phase of infection in G5, but normalities during later phase, statistical difference was also found with P<0.05 in end point of study (G4 vs G5). HE staining showed that kidneys and lung were normal in pathology in each group at the end of the observation.4. HE staining showed that all the four rats presented severe acute rejection in G3 group. While only one recepient showed severe acute rejection pathologically in G4 group, but parenchyma of liver graft was damaged more seriously than G3. The RAI score of this two groups were (8.0±0.8 vs 6.5±1.3,P<0.05). In regard to CXCL-10 and its ligand CXCR-3 either in immunohistochemistry results or the relative expression of mRNA levels, a continuously increased trend was found in G3 group, while G4 showed increased firstly and then decreased. statistical difference was found with P<0.05 in end point of study (G4 vs G3). The amount of apoptotic cells in liver graft increased continuously in all groups, they distributed mainly in the hepatic parenchyma in G3 but mainly in portal area in G4. Statistical difference was found with P<0.05 in terminal point of study (G4 vs G3).5. The result of one way MLR showed that T lympholeukocyte function elevated slightly in G1, increased in G2, elevated remarkably in G3, increased constituously in G4. The increased magnitude of G2 and G4 were lower than G3. Statistical differences were found between G1, G2 and G3 (0.3727±0.05479 vs 0.7795±0.00995 vs 0.9550±0.03594, P<0.05) at day 5 after infection. The difference was seen between G3 and G4 (0.7560±0.00787 vs 0.9550±0.03594, P<0.05). Correspondingly, the trends of T lympholeukocyte subpopulation and CD4+/CD8+ ratio in various group was different. No obvious changes were found in G1 group. Rising trend was found in G2 group except day 5 postinfection. The subpopulations of T lympholeukocyte decreased in the first three time point in G3 except day 8, the CD4+/CD8+ ratio elevated continuously ( day 8 vs day 2, P<0.05). Obvious decreased trend of CD4+ and CD8+ T cells and CD4+/CD8+ were seen in G4 with a ratio of 1.43~1.46 at day 5 after infection (P <0.05, vs G1 and G3). Regarding to serum cytokines, the trends of IL-10 level were unchanged in G1, mildly elevated in G2, significantly reduced in G3, and remarkably elevated in G4. Pairwise comparisons between groups differences were obviously statistical significant (P <0.01) in day 5 after infection. Contrastively, IFN-γlevel was significantly increased in G3 group (and acute rejection occurred synchronously), but reduced notedly in G2 and G4 at day 5 after infection (vs day 3, P<0.01). Pairwise comparisons between groups differences were remarkably statistical significant (P<0.01) in day 5 after infection. Another serum cytokines we examined was IL-12, the trends of it in all experimental groups were similar to IFN-γlevel, but the level decreased more significantly in day 5 post-infection in G4 than in G2 (P<0.05).6. Rats spleen DCs were with typical dendritic features isolated using immunomagnetic beads freshly. About 1.5~3×106 cells were available from each spleen. The expression trends of MHC-Ⅱ, CD80 and CD86 remained almost coherence at all time points in all group. G1 was high expression postoperative, G2 group decreased after infection, G3 increased continuously during the whole process, the expression levels in G4 were significantly lower, DCs oriented toward immature. Pairwise comparisons between groups differences were significantly statistical significant (P<0.01) in day 8 after surgery. Differences were all statistical significant on three surface molecules in G2 and G4 groups at day 8 vs day 2 posttransplant (P<0.05). The same results were seen in G3 except CD80. In regard to the function (to stimulate allogeneic T cell proliferation capacity) of DCs at day 8 after operation, decreased in G4 group but increased in G3 group (0.27±0.05 vs 0.93±0.02, P<0.01).7. The median survival time were 12 days in both G1 and G2 group, 95% CI was (10.400~13.600) and (10.868 ~13.132), respectively, the difference was not statistical significant (P>0.05). The median survival time were 9 days (95% CI, 7.400~10.600) in G3 and 10 days (95% CI, 8.868～11.132) in G4, the difference was not statistical significant (P>0.05). At the terminal of research, HE staining result showed severe acute rejection with RAI score of 8~9 in G1 and G2, and moderate to severe acute rejection with RAI score of 6~8 in G3 and G4. Difference was not statistical significant (P>0.05) between G3 and G4. The subsets of T cells and CD4+/CD8+ ratio both increased in treatment groups G2 (vs G1) and G4 (vs G3) (P<0.05).Conclusions1. A high survival rate, stable and reliable closed colony rat orthotopic liver transplantation was established in this study. Futhermore, a inbred rat orthotopic liver transplantation with acute rejection model was successful established too.The results were stable, reliable, reproducible, can be used for basic research in related fields.2. The model of intraabdominal infection induced by E. coli after liver transplantation in the inbred rat was feasible and reliable and repeatable with following advantages e.g. more precise confirmation of the existence of intraabdominal infection and reducement of trauma to the rats. The model could meet the needs for experimental study.3. Intraperitoneal bacterial infection after liver transplantation in the rats worsened the hepatic parenchymal damage in some extent, but released the pathological manifestations of acute rejection of allograft partly. The decreased expression of chemokine CXCL-10 and its receptor CXCR-3 in the late stage of infection, as well as the augmentation of apoptosis of infiltrating lymphocytes around the centrilobular vein and periportal space of allograft may be one of the causes contributed to the improvement of liver acute rejection.4. The T lymphocyte function increased during the early stage of acute rejection and/or infection. It reduced significantly in the late phase of acute rejection accompanied with infection. The ratio of CD4+/CD8+ T cells increased in the acute rejection model, and increased during the early stage of infection model but decreased in the later period, and it dropped dramatically in the infection folling acute rejection model. The levels of serum IFN-γelevated when acute rejection occurred; the serum levels of IL-10 increased in the infection model. The synergistic action of these two factors promoted the change from Th1 cells to Th2 cells. Serum IL-12 secretion reduction may have contributed to this change. Anti-inflammatory response and systemic immunosuppression took the place during this time.5. The spleen DCs presented rapid mature and full function to stimulate allogeneic T cell proliferation during early stage of acute rejection and/or infection. But it was immaturing and lost the function during the late phase of infection, in particular under the comorbid state of infection and rejection, which may be involved in local and systemic immune suppression of the recepients.6. Immune regulating treatment did not significantly prolong the survival time of recepients and not augmented acute rejection during the short-term of rats liver transplantation complicated with abdominal bacterial infection. But it improved the subproportions, ratio and function of T lymphocyte according to our small size samples. A large scale of sample size was needed to enhance the reliability of this results.
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