| In the past two decades, the field of organ transplantation has witnessed unparallel advances. Much of this success can be attributed to the development of potent immunosuppressive and immune induction agents, one of which is the first humanized monoclonal antibody, Campath-1H. Campath-1H was officially approved for the treatment of B-cell chronic lymphocytic leukemia, but it was increasingly off-label used in allotransplantation and showed promise in the short/medium-term patient and allograft survival.Campath-1H based immunomodulation solutions were recognized by the major transplantation centers. However, the exact effect of Campath-1H induction was not elaborated studied. Thus, understanding the mechanism of action of immne induction and combination of immunosuppressive agents were still in debate.In our studies, we choose cynomolgus monkeys as animal model to elucidate the unique role of Campath-1H induction, in the absence of confounding maintenance immunosuppression. We exammed the quantities and phenotypes alteration of immune cells in different lymphatic compartments, and observed various cell depletion and repopulation before and after induction, in order to demostrate possible mechanism of action of Campath-1H on immune system.Overall, our study provides significant insight into the in vivo biological activities of Campath-1H and will be useful in the optimal use of Campath-IH in clinical settings.Chapter IIdentification of a nonhuman primate model for the in vivo study of Campath-1HBackground:Campath-1H induction has been increasingly used in allotransplantation, however, the exact biologic activities and pharmacokinetic characteristics are still unknown. Much of this inadequacy could be attributed to the lack of reliable animal model which Campath-1H can be administered safely. We aim to explore the immune cell depletion and repopulation profile after Campath-1H administration. And we also try to clarify the unique pharmacokinetic process in vivo.Methods:Male healthy cynomolgus monkeys,3-5 years old and 3 to 7 kilogram of body mass, were obtained from Academy of Military Medical Science institute. Venous blood samples were drawn and purified erythrocytes were collected by Ficoll separation. Via cytometric analysis, cynomolgus monkeys were divided into two categories, which is CD52 positive or negative expression on erythrocytes. After screening, fifteen CD52- erythrocyte monkeys were enrolled and randomly assigned to five groups. After administration of Campath-1 H (3.0mg/kg, intravenous infusion), venous blood samples were taken as scheduled. Blood routine and biochemical examination were done to evulate the biologic and toxic effect of Campath-1 H. We used a simple enzyme-linked immunosorbent assay to determine the serum concentration of Campath-1H, and some crucial pharmacokinetic parameters were obtained. Data were analysed via commercial software package Graphpad 5.0. All data were analyzed by One-Way ANOVA (LSD) and Dunnett t test. The significance level was accepted at P< 0.05.Results:According to CD52 expression screening, about 40% cynomoglus monkeys were CD52neg on erythrocytes. Administration of Campath-1 H at the level of 3.0mg/kg in screened monkeys did induce rapid and sustained immune cells depletion without significant toxicity. Peripheral lymphocytes were the primary target of depletion and were greatly reduced to minimum at day 9 (1.03±0.31×109/L vs baseline level 5.04±0.48×109/L, p<0.01); The repopulation phase was slow and it returned to predose level at day 35. Contrastint to lymphocytes, monocytes and NK cells were less sensitive to depletion and the repopulation was faster. Further, we found that there are far more amount of expression of CD52 on lymphocytes than monocytes and NK cells. Pharmacokinetic studies of Campath-1H showed that it is a biphasic clearance in blood, the mean peak concentration was 8.8±1.8ug/ml (1hr postdose), the maximal clearance rate was 35.525ng/ml/hr, and the terminal half-life in cynomolgus monkeys was 9.89 days.Conclusions:We demostrated that screened CD52neg-erythrocyte cynomolgus monkeys were reliable nonhuman primate model for in vivo study of Campath-1H. Campath-1H is able to induce rapid and profound immune cell depletion as seen in clinical allotransplantation, whereas the efficacy of depletion and the duration of suppression is less potent. The differential effect on various immune cells is relevant to the amount of CD52 expression on target cell types.Chapter ⅡThe efficacy and duration of Campath-1H induction in major lymphatic compartments in cynomolgus monkeysObjectives:As great amount of immune cells locate in lymphatic tissues, therefore, depletion and suppression of these cells were crucial to tolerance-induction therapies. However, it is not clear that the impact of Campath-1H on these lymphatic tissues. In this part of investigation, we originally observed and described the lymphodepletion and immune reconstitution profiles in primary and secondary lymphatic tissues.Methods:Fifteen prescreened cynomolgus monkeys were randomly assigned to five groups. After infusing Campath-1H, monkeys were accordingly killed at preset time interval (3d,9d,14d,21d and 35d) for blood and tissue collection. Besides that, each monkeys received same-volume of normal saline and an abdominal operation, by which bone marrow, spleen specimen and lymph node were taken to serve as predose baseline. Lymphocytes in spleen, lymph node and intestine were isolated, counted and phenotyped. Bone marrow specimens were also analysed by FACS, to determine whether Campath-1H had detrimental effect on hemaetologic progenitor cells. Compararions of these data in different time points were made for the better understanding of the effect of Campath-1H on various lymphatic tissues. Data were analysed via commercial software package Graphpad 5.0. All data are presented as Mean±SD and data were analyzed by One-Way ANOVA (LSD) and Dunnett t test. The significance level was accepted at P<0.05.Results:Cytometric analyses of bone marrow specimens showed that Campath-1H had little or no effect on pluripotent or committed hemaetologic stem cell. However, Campath-1H could deplete T cell-committed hematologic progenitor cell (T-HPC). In contrast to circulating lymphocytes, splenocytes and lymph node (LN) lymphocytes were less sensitive to depletion (the minimal mean value was approximate to 32% and 40% of the baseline level, respectively). FACS analysis showed that the majority part of the surviving and reconstitutional lymphocytes were memory-like cells. Upon day 35, lymphocytes in spleen and LN recovered to baseline level.Conclusions:Campath-1H has impact on primary and secondary lymphatic tissues of cynomolgus monkeys. The depression on T-HPC repopulation could be an important factor reasonable for the delay recovery of peripheral T cells. And some memory cells in secondary lymphatic tissues relatively resisted against depletion.Chapter ⅢMechanism of action involved in Campath-1H immune-induction activity in cynomolgus monkeysBackground:The balance of regulatory T cell (Treg) and effector T cells (Teff) determine the immune status of the receptor. However, it is unknown whether Campath-1H has effect on these subsets. In this part of study, we originally investigated the alteration of Treg:Teff ratio and the balance of cytokines after Campath-1H administration.Methods:The monkeys were injected with Campath-1H as indicated in Part II. Monkeys were anethesized for blood and tissues collection in preset time intervals. Phenotypes of Treg (CD4+CD25+Foxp3+) and Teff (CD4+CD25-/low) subsets were analysed by FACS. And also, we detected serum concentration of IL-2, IL-10 and IFN-y by ELISA determination. Data were analysed via commercial software package Graphpad 5.0. All data are presented as Mean±SD and data were analyzed by One-Way ANOVA (LSD) and Dunnett t test. The significance level was accepted at P<0.05.Results:Campath-1H could significantly influence the phenotypes of lymphocytes. In the early stage of depletion, the proportion of Treg increased significantly, accompanying with the fall of Teff. However, the ratio of Treg:Teff showed the trend of decline over time. Furthermore, the data showed an outgrowth of IL-10 over IFN-y indicates a stage of immune hyporesponsiveness. Further investigation showed that memory-like phenotypes were the dominant cell types surviving Campath-1H mediated depletion. Moreover, the subpopulation of memory effector T cells was the priority part of postdepletional lymphocytes homestasis.Conclusions:Campath-1H exerts different impact on Treg and Teff, and also, it is capable to increase the inhibitory ingredients of cytokine network. In such a sense, Campath-1H could probably reduce the intense of alloactivity. Eventually, the resurrection of memory cells (especially the memory effector T cells) broke the established balance of immune hyporesponse. |
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