| BackgroundAfter organ transplantation an immunosuppressive regimen is required to prevent graft rejection. Immunosuppressive drugs inhibit immune function by targeting both T-and B-cell responses through blockage of cellular proliferation induced by alloantigen stimulation, and by inhibition of the cytokine production necessary for such stimulation. However, the absence of discrimination between the immune response against alloantigen from the transplanted organ and the immune response against environmental antigens renders transplanted patients strongly immunodeficient and susceptible to bacterial and viral infection. Optimising the immunosuppressive drug regimen to balance mandatory immunosuppression and preserving immunity is a difficult challenge for clinicians in charge of transplanted patients. Preserving immunity by minimizing immunosuppression or inducing tolerance is one of the major goals of the transplant immunologist. Several strategies are exciting, but further work is necessary to find the best protocol to induce tolerance. Defining the ideal strategies for inducing tolerance or minimizing the role of immunosuppressive drugs, and development of assays to measure tolerance and immunity, are among the most important challenges in organ transplantation over the next few years.A reliable index of immune status could result in customization of immunosuppressive drugs, not only in the context of rejection/tolerance, but also in the context of a strong increase in susceptibility to infections. Such an objective is highly desirable, given the morbidity and mortality associated with long-term administration of immunosuppressive therapy. However, although monitoring of immune function (with the use of analysis of blood or graft specimens) has helped delineate the host’s response to the graft (including the phenotype and function of infiltrating cells, alterations in the T-cell repertoire, and the types of cytokines and antibodies produced), no test has yet proved to be a reliable method in detecting rejection. Therefore, the combination of clinical assessment and a graft biopsy is still a useful and prevalent pattern to determine the immune status of the recipent. However, the biopsy is a harmful approach for the graft and the host.In vivo donor specific cytotoxicity assay has been performed, in which the donor cells with complex antigens were used to detect the immune intensity of the recipients. In recipients, the donor cells were attacked not only by cytotoxic lymph cells but also by antibody and complement. The quantitative determination to complex antigen of donor can reflect the activity of CTL and reflect the immune state of recipients. In cytotoxicity assay, the target cells came from donor(splenocytes, PBMA and so on), which could reflect the antigenicity of donor in the maximal degree. Internal references were self cells from recipient. Internal references and target cells were same in immune state when they were obtained and treated, so it eliminated the nonspecific effect such as natural death or homing by physical and chemical injury in the treatment process. The ratio of cells can eliminate the nonspecific error. Analysis the attacking situation of allogeneic splenocytes complex antigen by flow cytometry can achieve single cell level and achieve the goal of quantitative detection.In vivo donor specific cytotoxicity assay was carried out in mice. C57BL/6j and Balb/c splenocytes were harvested, labeled by5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), and a skin transplantation model was established with the recipients BALB/c mice and the donors C57BL/6mice. After intravenous transfusion of the CFSE-labelled donor/recipient splenocytes mixture, the following examinations are carried out at different time points. The assay could quantitatively detect the immune status of skin transplant recipients in vivo.According to the principle of in vivo donor specific cytotoxicity assay, we describe a method to perform the assay in New Zealand rabbits. CFSE followed donor splenocytes rejection by flow cytometry. and recipients splenocytes as control in vivo. Labelled spleen cell mixtures were injected intravenously into various recipients and blood samples were taken at different time points to follow the transferred cells. We found that the labelled recipient cells could be readily detected by flow cytometry for up to8hours, and the loss of these labelled donor cells in various transfusion experiments with different major and minor histocompatibility differences followed the rejection kinetics of skin transplants.ObjectiveTo establish a method of donor specific cytotoxicity cytotoxicity assay in vivo in New Zealand rabbits for detecting the immune status of the recipient using flow cytometry. To provide new basis for the clinical application of in vivo donor specific cytotoxicity assay.MethodsTotally5female New Zealand rabbits and5male New Zealand rabbits of clean grade were selected and paired into5groups, in which the female rabbits were donors and the male rabbits were recipients. In model group, a4cm×2cm skin cut down from the back of female rabbit were transplanted to male rabbit, and the skin transplantation model was established with the donor female rabbit and the recipient male rabbit two weeks later. Totally5female New Zealand rabbits and5male New Zealand rabbits were selected and paired into5groups as negative control without skin transplantation. The spleens of the rabbits were takeout and grinded in Hanks’ solution, and then the splenoncytes were harvested after erythrocytes lysis by7.5g/L ammonium chloride. The splenoncytes of female rabbits were labelled by5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) at a concentration of0.24uM, and the splenoncytes of male rabbits were labelled by CFSE at a concentration of6uM. The splenoncytes of rabbits paired were mixed. Trypan blue exclusion displayed that cell survival rate above95%. After intravenous transfusion of the CFSE-labelled donor/recipient splenocytes mixture, the following examinations are carried out at different time points(1h,2h,4h,8h).The percent specific lysis was calculated using the equation:%specific lysis=1-donor cells/recipient cells×100%All statistics were conducted by software SPSS13.0, and differences among groups were compared with t test.Results:1、All experimental animals involved in the result analysis.2、The average survival times of the skin transplantation in rabbits of the model groups were15days.3、Compared with the control groups, the percent specific lysis of donor cells in the rejection model mice1h,2h,4h,8h, after transfer were all significantly higher than those of normal female rabbits (t=25.284,13.624,24.215, and15.146, all P<0.01). In the control groups, the percent of specific lysis rapidly increased 47.39%±2.53%at1h after transfer, and at2h the percent was61.38%±7.23%.Conclusion:We demonstrate that the method is simple, precise, reliable and well suited for quantitatively detecting the immune status of skin transplant recipients in vivo. Consecutive surveillance of the percent of specific lysis is helpful in the early detection of acute rejections, and in judgment of the therapeutic effect of immunosuppressive drugs and possibility of infection. This method offers a simple and effective tool to test the immune status of the host in transplantation experiments in which donor and host are not completely syngeneic. |
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