| Due to the rapid and better therapecutic effects and bioavailability characteristics, Tranditional Chinese Medicine Injection (TCMI) was widely used in clinical critical care, cardiovascular and cerebrovascular diseases and cancer treatment and so on. However, in recent years, the reports of adverse reactions had been gradually increasing, in particular the serious averse events such as anaphylactic shock caused by Houttuynia, Shuanghuanglian, Qing Kai Ling Injections, severely restricting the development of Chiniese medicine in China. The major of adverse reactions included anaphylactic reactions and anaphylactoid reactions. Nowadays, the guidline of evaluation of immunotoxicity of drugs domestic and abroad still confined to the evaluation of animal models, only focusing on immune suppression and contact allergic, without covering the systemic allergic and autoimmune diseases induced by drugs. Anaphylactic shock caused by TCMIs has not been found any positive results in the preclinical safety evaluation studies. In view of this, the evalution methods available might be not sensitive enough for prediction immunotoxicity, or there might be a big difference in immune system between the experimental animals and human. It was a great challenge for secientists that how to predict immune toxicity such as alleric reactions in preclinical safety evaluation.1. ObjectiveBecause of their physiology and biological evolution closed to human, nonhuman primates became one of the most commonly used animal models in the preclinical drug safety evaluation, especially in evaluating biotech drugs such as humanized monoclonal antibody and human vaccines. However, in2006, the healthy volunteers suffered multiple organ failure after receiving TGN1412(anti-CD28antibody super activator), while the cynomolgus in preclinical toxicity study did not show any abnormality. In addition, although most of TCMI indicated safe in preclinical studies, some allergic reactions even shock or death were reported when those TCMIs entered into the market. Our previous studies showed that the degree of anaphylactoid reactions induced by Tween80, an adjuvant of TCMI, were different in different animalspecies. It was suggested that the immune response to the exogenous allergenic substances were different in animal species. Therefore, it was one of the key techonologies of drug immunotoxicity evaluation that we studied on the differences and similarities of immune system in different animal species, eapeclially between human and animals. It will contribute to choosing suitable animal species, extrapolating animal toxicity data and objectively and accurately evaluating the impact of drug on human health.In vitro immunotoxicology has been paid more and more attention. Firstly, it was increasingly desirable to reduce the number of animals used in toxicity studies. Secondly, immunotoxicology could be conducted using human peripheral blood, so we could gain information from human and compare these data with that from animal studies. Furthermore, toxicogenomics microarray was considered a valuable tool for the data translation from animal to human. Pharmacokinetic including the distribution and metabolism of drug was the basis of translation among species, while the key gene changed could be the end of drug metabolism. Therefore, comparing the differences of expression profiles of genes related to toxic among different animals and human tissues, finding the significant key genes expressed both in animals and human would be helpful to scientifically and accurately transfer animal toxic data to human.In this paper, based on the previous results of our laboratory, we discussed the morphological similarities and differences, the distribution and number of immune cells, in thymus and lymph nodes among human, cynomolgus and rhesus using the method of immunohistochemistry. We further explored the functional similarities and differences of peripheral blood mononuclear cells of human and cynomolgus using in vitro immunotoxicology test,. It would establish some available in vitro systems for preclinical evaluation of drug immunotoxicity and provide a scienctific basis for selecting animal models and transferring animal data to human. Finally, we studied gene expression profiles in the peripheral blood mononuclear cells after dosing T/B cells activators using toxicogenomics technology. We analysed the similarities and differences of immune activation response mechanism between human and cynomolgus on gene level, in order to find the possible bridge biomarkers.2. Methods2.1Comparative study for the type and number of different immune cells in lymph node and thymus between cynomolgus/rhesus monkeys and humanThe lymph node and thymus were collected from the control and placebo cynomolgus (43males,61females) and rhesus (37males,39females) of toxicological preclinical safety studies from2004to2011in National Center for Safety Evaluation of Drugs (NCSED), National Institutes for Food and Drug Control (NIFDC), China. The thymus (4cases) and lymph nodes (11cases) of human were derived from the cross-reactivity studies human tissue bank of NCSED. All tissues were examined for the distribution and number of B cells (immunolabled with CD20), T cells (immunolabled with CD3, CD4, CD8), and macrophages (immunolabled with CD68) using the method of immunohistochemistry.2.2Comparative study for the function of T and B cells between human and cynomolgus(1) Monoclonal antibody induced cytokine release:Human and Cynomolgus peripheral blood mononuclear cells (PBMCs) were used. Two groups were designed, including negative control group (Isotype control antibody IgG) and ANC28.1antibody group. Cell culture medium from each group was collected at2hours and24hours and48hours. The antibody of interest was immobilized by wet coating or air-coating according to the methods established by Lucy Findlay and the cytokines (TNF-α、 IFN-γ) were quantified using the method of ELSIA.(2) T cells proliferation and B cells proliferation:Human and Cynomolgus PBMCs were used. In T cells proliferation, four groups were designed, including negative control group (cell culture medium) and PHA groups (2μg/ml、5μg/ml、10μg/ml). Similar to T cells proliferation, in B cells proliferation study, megative control groups and LPS groups (2μg/ml、5μg/ml、10μg/ml) were designed.PBMCs incubated with different substances for72hours in37℃incubator. All samples were examined cell proliferate by adding10μl cck-8accordign to the instruction of the cell counting kit (Dojindo).(3) B lymphocytes antibody-secreting assay:Human and Cynomolgus PBMCs were used. In experiment1(Cynomolgus PBMCs), six groups were designed, including negative control group1(cell culture medium, coated with Fluzone1:30), negative control group2(cell culture medium, coated with Fluzone1:90), Fluzone group1(Fluzone1:50, coated with Fluzone1:30), Fluzone group2(Fluzone1:50, coated with Fluzone1:90), Fluzone inhibit group1(Fluzone+CD20, coated with Fluzone1:30), Fluzone inhibit group2(Fluzone+CD20, coated with Fluzone1:90).In experiment2(Human PBMCs), eight groups were designed, including negative control group1(cell culture medium, coated with Fluzone1:30), negative control group2(cell culture medium, coated with Fluzone1:90), Fluzone low-dose group1(Fluzone1:50, coated with Fluzone1:30), Fluzone low-dose group2(Fluzone1:50, coated with Fluzone1:90), Fluzone high-dose group1(Fluzone1:10, coated with Fluzone1:30), Fluzone high-dose group2(Fluzone1:10, coated with Fluzone1:90), Fluzone inhibit group1(Fluzone+CD20, coated with Fluzone1:30), Fluzone inhibit group2(Fluzone+CD20, coated with Fluzone1:90).PBMCs incubated with different substances for6days in37℃incubator. Antibody-secreting cells (ASC) was quantitated by ELISPOT assays. 2.3Gene expression profiles of the peripheral blood mononuclear cellsHuman and Cynomolgus PBMCs were used. Five groups were designed, including negative control group (PBS),2μg/ml PHA group,10μg/ml PHA group,2μg/ml LPS group and10μg/ml LPS group. All samples were collected for RNA isolation and gene expression analysis. Significance analysis of microarrays was used to find different expression genes (DEGs) with threshold set at fold change>1.5and P value<0.05. The DEGs were further analyzed on significant enrichment of Gene Ontology (GO) terms and KEGG pathway analysis.3. Results3.1Characteristics of distribution and number of T cells/B cells/macrophages in thymus and lymph node of human, cynomolgus and rhesusB cells primarily were located in the lymphoid follicle of lymph nodes and the medulla of thymus. Anti-human CD20antibody reacted with cynomolgus/rhesus monkeys indicating strongly positive expression on the membrane of B cells, suggesting the close relationship of human and monkeys.T cells were found diffusely throughout the paracortical area of lymph node and cortical area of thymus. Anti-human CD3antibody reacted with cynomolgus/rhesus, indicating positive or mild positive expression on the membrane of T cells. Anti-human CD4indicated weak positive expression on the T cells of cynomolgus/rhesus, whereas anti-human CD8indicated no cross-reactivity with cynomolgus/rhesus. It was suggested that there are huge differences between human and monkey in T cells, especially CD8+cytotoxic T cells.Macrophage cells were observed in medulla of lymph node and in cortical and medulla of thymus. Anti-human CD68antibody reacted with cynomolgus/rhesus monkeys, indicating strongly positive expression on the membrane of macrophage cells. It showed a close relationship between human and monkeys as for the similar staining degree and distribution.3.2The differences of T cells and B cells proliferation ratio, cytokines and antibodies secretion between human and cynomolgus(1) Monoclonal antibody induced cytokine release:ANC28.1, anti-CD28super activator, induced the activation of PBMCs without CD3antibody co-stimulation. With the extension of time, the release of TNF-α and INF-γ were gradually increased from human PBMCs. There was no diffenence between wet coating and air-coating. ANC28.1could not induce any secretion of TNF-α and INF-γ in cynomolgus PBMCs. (2) T cells proliferation and B cells proliferation:The number of PBMCs from human and cynomolgus was significantly increased as the concentration of PHA (T cells activators) increased. However, at the same concentration of PHA, PBMCs growth rate of human was significantly higher than that of cynomolgus. The number of human PBMCs was also significantly increased as the concentration of LPS (B cells activators) increased. The proliferation trend between cynomolgus and human PBMCs were similar under the stimulation of different concentration of LPS.(3) B lymphocytes antibody-secreting assay:The number of antibody-secreting cells (ASC) in cynomolgus and human in Fluzone groups was significantly higher than that in negative control groups. The number of ASC was decreased in Fluzone inhibit groups after given CD20antibody. Under the same condition, the increasing ASC in group of coating with Fluzone1:90was higher than that in group of coating with Fluzone1:30. The secretion of antibody from cynomolgus ASC was similar to human ASC under the same conditions.3.3The differences of gene expression profiles in the peripheral blood mononuclear cells between human and cynomolgusAfter dosing PHA (T cells activator), the microarray data showed that the number of differentially expressed genes (DEGs) in low-dose group and high-dose group were115and1510in human and21and166in cynomolgus, respectively, compared to control group. In PHA stimulation, there were some similar immune mechanism between human and cynomolgus, such as the DEGs were involved in DNA replication, immune response, chemotaxis and inflammatory response. The differences between human and cynomolgus were as follows:1) The numbers of DEGs involved were far higher in human than that in monkey;2) The cell proliferative response was strong in human, especially much more genes involved in mitosis or M phase of mitotic cell cycle, while that changes were seldom observed in cynomolgus, shown by the fact that the DEGs involved in DNA replication, anti-apoptosis and regulation of cell shape;3) The altered genes in human were mainly associated with immune response and inflammatory response. It was noteworthy that the DEGs in human involved in antigen processing and presentation are mainly MHC Ⅱ class. On the contary, the immune response in cynomolgus was mainly associated with antigen processing and presentation, but DEGs involved in that processing are mainly MHC Ⅰ class.After dosing LPS (B cells activator), the microarray data showed that the number DEGs in low-dose group and high-dose group were341and810in human and588and562in cynomolgus, respectively, compared to control group. In LPS stimulation, there were same immune mechanism between human and cynomolgus. For example, the DEGs were involved in immune response and antigen processing and presentation of peptide or polysaccharide antigen via MHCⅡ class. In contrast to PHA, there was no significant difference in the number of DEGs between human and cynomolgus. The differences between human and cynomolgus were as follows:1) The DEGs were mainly involved in inflammatory response and immune response, and secondly involved in antigen processing and presentation via MHC Ⅱ and T cell costimulation in human. However, the DEGs in monkey were mainly involved in antigen processing and presentation via both MHC Ⅰ and MHC Ⅱ class.4. ConclusionsMorphologically, in this study, there are huge differences between human and monkey in T cells, especially CD8+T cells in thymus and lymph nodes. But there are consistency of B cells and macrophages between human and monkeys in the staining degree and distribution, manifesting high degree of genetic similarity between species. Although the number of T cells or B cells of lymph nodes was higher in human than that in cynomolgus, the ratio of T cells to B cells of lymph nodes in human was similar to the ratio in cynomolgus/rhesus.Functionally, in this study, the release of cytokines, TNF-α and INF-γ, was observed in human PBMC with dosing ANC28.1but not observed in cynomolgus PBMC. At the same concentration of T cells activators PHA, the human PBMCs growth rate significantly higher than that of cynomolgus, suggesting that human T cells is more sensitive and susceptible to the antigen activation than cynomolgus T cells. However, the proliferation trend in cynomolgus and human PBMCs were similar under the stimulation of LPS. In B lymphocytes antibody-secreting assay, cynomolgus ASC was similar to human ASC under the same Fluzone stimulation. We also found out that cynomolgus B cells has similar antibody secreting function compared with human B cells.From gene level, in this study, the number of differentially expressed genes in human PBMCs was higher than that in cynomolgus PBMCs after dosing T cells activator (PHA). The DEGs were associated with inflammatory response, immune response, antigen processing and presentation via MHC Ⅱ and cell cycle and proliferation, and so on. In LPS, the DEGs were associated with inflammatory response, immune response, antigen processing and presentation and T cell costimulation, and so on. In contrast to PHA, the number of DEGs was similar between monkey and human. Moreover, although there were some differences in gene expression profiles, some DEGs with same function were found in these two species.In short, there were significant difference in molecular homology of T cell phenotype and function of T cell proliferation and cytokine secretion induced by antibody between human and cynomolgus. The gene expression profiles data further confirmed that the DEGs involved in immune regulation and response in human are more complex and sensitive than that in monkey after dosing T cell activator. The results may provide a scientic basis for the low sensitivity of cynomolgus in prediction immunotoxicity in preclinical studies. It was suggested that the evaluation of toxicity related with T cells in animal study be paid more cautious when transferring these data to human, especially in immune activation evaluation of TCMI and biotechnology drugs, and in vitro tests used human peripheral blood be a useful method to predict immunotoxicity in the future. Secondly, there was similarity between human and cynomolgus, such as the molecular homology of B cell phenotypelt and function of B cell proliferation and antibody induced by B cells. Finally, according to the analysis of gene expression profiles, some DEGs with same function were found in different treated groups in human and cynomolgus. Therefore, these DEGs may be closely related to immune activation and may be bridge biomarkers for transferring toxic data among species. |
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