Effect And Mechanism Of Abdomimal Cavity Bacterial Infection On The Immune Rejection Of Liver

BackgroundInfection is the most common complication of liver transplantation, which is one of the major reasons for death in patients postoperative. However, Most of the serious recipients infection caused by bacterial or fungal ,and most occurred within 2 weeks after transplantation. Studies have confirmed that the virus and fungal infection are promoted to the rejection.In the clinical ,the therapy of immunosuppressive and the anti-infection is conflict. In this conditions, requirs reducting or stopping use immunosuppressive drug.Studys shows:while sever infection occurred ,for protecting mechanism,immunosuppressive can be priminged.Treg can secreted cytokine IL-10 and TGF-β,and play suppressive effect.Based on the above- mentioned results, In our research,we will investigate the immune status change with bacterial infection after liver transplantation ,and the proliferative changes of Treg cells. Therefore explored the relationship between bacterial infection and rejection will contribute to a new therapeutic strategy.Objective1. To construct a rat liver transplantation model with abdominal cavity bacterial infection,and investigate the effect to the immune rejection.2. To study and research multiplication on the lympholeukocyte subgroups and function ,and effect of Treg cells in the condition of bacterial infection and rejection. Methods and Results: Part I Effect of abdomimal cavity bacterial infection on the rejection model of rat liver transplantationMethods:1. orthotopic liver transplants (OLT,DA into Lewis) were established by modified Kamada two-cuff technique, applied Bacterium coli to induce abdominal cavity infection ,two injection time was dividied:3 or 5 days ,and 6 groups were divided trabant Postoperative:5×10⁵cfu/ml group,2×10⁵cfu/ml group,2×10⁶cfu/ml group,2×10⁷cfu/ml group,2×10⁸cfu/ml group and control group。five time points were predetermined as 1d preinfection,1d,3d,5d,7d postinfection, 6 animals were used per time-point, Rat were sacrificed at each time-point and the liver specimens and blood samples were collected. The infecion evaluated by Tm,ALT,TB,WBC,TCO₂, HCO₃^-,PH levels . 2. DA or Lewis into Lewis orthotopic liver transplants were established , 4 groups was divided after operation:isotransplant group G1, isotransplant infection group G2,allotransplant group G3 and allotransplant infection group G4.The bacterial concentration was 5×10⁵cfu/ml, five time points were predetermined as 1d preinfection,1d,3d,5d,7d postinfection, 6 animals were used per time-point, Through technology of RT-PCR,immunohistochemistry and liver pathology to evaluated rejection. The data were analyzed with SAS 6.0 .Multiple-groups were compared using MANOVA followed by pairwise comparison with Tukeys post-hoc analysis P<0.05 was considered statisticallysignificant.Results:1. The group of 5d injectioned postoperation was gived up because of the longest subsistence was 5d postinfection,which disadvantage to research. 2 or 5×10⁵cfu/ml in 3d injection postoperation,the survival incidence of 7d post infectionem were 37.5% and 100%.But the group of 2×10⁵cfu/ml,the infection can be rescured 5d later infection. In 5×10⁵cfu/ml group ,the Tm,WBC,ALT,TB increased and disturbance of acid-base balance. which compared with control group was significant difference(P < 0.01). Without multi-organic pathological lesion .2. The expression of CCR5mRNA increased and CCR3,MCP-1mRNA decreased in G4,from 5d postinfection, Compared with G3 group , every index had statistical significance.Banff scored manifest rejection lessened in G4 and had statistical significance compared G3.Partâ…¡Effect of bacterial infection posttransplantationon Treg, T leukomonocyte multiplication,function and differentiatedMethods: Rats were divided into 4 groups : isotransplant group G1, isotransplant infection group G2,allotransplant group G3 and allotransplant infection group G4.The bacterial concentration was 5×10⁵cfu/ml, five time points were predetermined as 1d preinfection,1d,3d,5d,7d postinfection, 6 animals were used per time-point. Applied flow cytometry,mixed lymphocyte culture(MLC),RT-PCR technology to determinat T lympholeukocyte subpopulation, lympholeukocyte function, cytokine interleukin-2, interleukin-6, interleukin-10, interleukin-4, interferon-γ,tumor necrosis factor-α,transforming growth factor-β.Meanwhile applied flow cytometry and RT-PCR to determinat multiplication of Treg and expression of Foxp3mRNA.Results:1. The cell population of T lympholeukocyte decreased,lopsided development, CD4+/CD8+ ratio degraded.Compared G3 every time-point postinfection had statistical significance(P<0.01);MLC results showed lympholeukocyte function rised in G4,but infra G3,from 3d postinfection,compared with G3 every time-point had statistical significance.2. Our data showed that the expression of interleukin-10, interleukin-4,transforming growth factor-βmRNA increased, But the three are not totally consistent with the expression, which was significantly higher after the former; Expression of Interleukin-2, interleukin-6 mRNA increased at first,but decreased 5d postinfectio;Interferon-γ,tumor necrosis factor-αmRNA expression level was lowered. Compared with G3,from 3d postinfection,every index had statistical significance.3. Treg multiplication quiet in G1,G2,G3, and had no statistical significance each other.In G4, Treg multiplicated predominance,especially at 5d postinfection,compared with other groups had statistical difference. Expression of Foxp3 mRNA in G4 increased ,but infered to Treg multiplication,had significance with G3.Partâ…¢Effect of Treg on immunologic derangement post transplant infectionMethods:1. Construct DA to LEW OLT,divided into 4 groups postoperation: acute rejection group G1,infection group G2,G3:based on G2 added to anti-IL-10 and anti-TGF-βmonoclonal antibody,each dosage was 1.2mg/kg/d and 0.9mg/kg/d, G4:based on G2,added to anti-CD25 monoclonal antibody, dosage was 1.0mg/kg/d. five time points were predetermined as 1d preinfection,1d,3d,5d,7d postinfection, 6 animals were used per time-point. Applied flow cytometry,mixed lymphocyte culture(MLC),RT-PCR technology to determinat T lympholeukocyte subpopulation, lympholeukocyte function, cytokine interleukin-10, interleukin-4, interferon-γ,transforming growth factor-βmRNA.And evaluated rejection level through expression of CCR3,CCR5 mRNA,Banff scored of liver pathology.Results:1. The cell population of T lympholeukocyte decreased in G3 and G4,but CD4+/CD8+ ratio rised,especially in G3,Compared with G1,G2,G4,from 5d postinfection had statistical significance. T lympholeukocyte function partial recoveried ,Compared with G1,G2,from 5d postinfection had statistical significance. Expression of interleukin-4, interferon-γmRNA increased in G3 and G4, Compared with G2 every time-point had statistical significance postinfecion;2. Expression of CCR5,CCR3mRNA increased in G3,G4,and had statistical significance compared with G2;rejection was partial recoveried in G3 and G4.Conclusion:1. Rat abdominal cavity bacterial infection after OLT which Induced by injecting E. coli is stable and suitable for study ;Which has favourable repeatability and without multi-organic impairment,profitable for research;2. Synergistic action of bacterial infection and immunological rejection after liver transplantation deceased the expression of CCR5 mRNA,but increased expression of CCR3 and MCP-1mRNA, immunological rejection inhibited in part ;3. Synergistic action of bacterial infection and immunological rejection after liver transplantation decreased T lympholeukocyte function, CD4⁺/CD8⁺ ratio degraded,promoted Th2 differentiated.4. Bacterial infection after liver transplantation promting Treg multiplication and expression Foxp3 mRNA,but partial Treg can expression Foxp3 mRNA;5. Inhibited multiplication of Treg or blochaged effect of interleukin-10, transforming growth factor-β,can promoting Th2 differentiated,partial recoveried T lympholeukocyte function and immunological regection; Treg is not the only inhibitory factor.