Effect of Yiqi Jiedu Prescription-containing Serum on Proliferation of mTEC and Regulatory T Cells in Myasthenia Gravis Patients with Thymus Hyperplasia / 中国实验方剂学杂志

ObjectiveTo investigate the effect of Yiqi Jiedu prescription-containing serum on the proliferation of medullary thymic epithelial cells （mTEC） and regulatory T （Treg） cells in myasthenia gravis （MG） patients with thymus hyperplasia. MethodAccording to serological methods，35 SD rats were adaptively fed for one week and randomized into the low-，medium-， and high-dose Yiqi Jiedu prescription groups，control group， and prednisone group，with seven rats in each group， which were then gavaged with the corresponding drugs for one week for preparing the drug-containing serum. The effect of Yiqi Jiedu prescription-containing serum at different concentrations on the proliferation of mTEC and Treg cells were determined by cell counting kit-8 （CCK-8） assay. Besides， the effect of mTEC and Yiqi Jiedu prescription-containing serum on Treg cell proliferation were observed through co-culture. ResultThymocytes were cultured for a period of time. Their mean positive rate revealed by flow cytometry using mTEC characteristic marker Ulex europaeus agglutinin Ⅰ （UEAI） was 92.54%. Treg cells were sorted by magnetic beads. The purity of Treg cells after repeated magnetic bead sorting was as high as 92%. mTEC and Treg cells showed high positive expression rates，and their cell purity met the requirements of subsequent experiments. When the concentration of Yiqi Jiedu prescription-containing serum was 2.5%-15%，it exhibited an inhibitory effect against mTEC and Treg cells. When the concentration was equal to or greater than 20%，it promoted cell proliferation，which was further enhanced with the extension of action time. The results after 48 h of culture showed that compared with the control group，prednisone and low-dose Yiqi Jiedu prescription had no significant effect on the proliferation of these two kinds of cells，but the medium- and high-dose Yiqi Jiedu prescription remarkably reduced their proliferation inhibition rate （P<0.01）. After co-culture with mTEC， the control group was not significantly different from the prednisone group and the low-dose Yiqi Jiedu prescription-containing serum group in the proliferation of Treg cells，while the medium- and high-dose Yiqi Jiedu prescription-containing serum groups significantly lowered the proliferation inhibition rate （P<0.01）. ConclusionYiqi Jiedu prescription-containing serum affects the proliferation of mTEC and Treg cells in MG patients with thymus hyperplasia. Compared with the solely cultured Treg cells isolated from MG patients，the Treg cells co-cultured with mTEC exhibit enhanced proliferation in MG patients，suggesting that mTEC can regulate the proliferation of Treg cells. This effect becomes more obvious after the intervention with Yiqi Jiedu prescription-containing serum，indicating that intervention effect of Yiqi Jiedu prescription on Treg cells can be produced during its treatment of mTEC， which may be one of the mechanisms of Yiqi Jiedu prescription-containing serum in alleviating MG.

Establishment of a animal model of allogeneic bone marrow transplantation plus thymic epithelial cells transplantation / 中华器官移植杂志

Objective To explore the establishment of animal model of allogeneic bone marrow transplantation plus thymic epithelial cells transplantation,and then examine the feasibility and effects of thymic epithelial cells transplantation applied in allogeneic bone marrow transplantation.Methods One day before transplantation the recipient BALB/C mice were given total-body irradiation,then transplanted with bone marrow cells from donor C57BL/6 mice and thymic epithelial cells from E14-16 embryonic thymus of donor C57BL/6 mice.In order to explore the appropriate irradiation dose,we set up three different dose groups:7 Gy;6.5 Gy;6 Gy.The recipient mice transplanted with BMT plus TCT served as experimental group,and those transplanted with BMT only served as control group (n =8 each).Then in vivo imaging in mammals was done to observe the thymic epithelial cells transplantation.Thymus index was measured.The thymus in each group was collected for histological examination and immunohistochemical staining of K5 and K8.Flow cytometry was used to examine the T cells subsets in peripheral blood of recipient mice 4 weeks after thymus transplantation.Results The recipient mice with 6.0 Gy TBI had long-term survival but implantation was done unsuccessfully,and those with 6.5 Gy had lower survival rate but implantation was done successfully.6.5 Gy was the minimum lethal dose and could be used as the appropriate irradiation does in this study.In vivo imaging in mammals detecting system showed the experimental group obvious fluorescent signals could be detected in the experimental group,but no fluorescence was found in the control group.Four weeks after transplantation,the thymus was bigger and thymus index was higher in the experimental group than in the control group.And the chiemra thymus of the experimental group also had normal cortex and medulla histological structure.Four weeks after transplantion,the percentages of CD4+ and CD8+ T cells of the peripheral blood in experimental group were significantly higher than in control group (P＜ 0.05).Conclusion Thymic epithelial cells can be transplanted in the thymus of the recipient mice with allogeneic bone marrow transplantation and promote the reconstitution of T lymphocytes of peripheral blood in the recipient mice.

Growth hormone in the presence of laminin modulates interaction of human thymic epithelial cells and thymocytes in vitro

BACKGROUND: Several evidences indicate that hormones and neuropeptides function as immunomodulators. Among these, growth hormone (GH) is known to act on the thymic microenvironment, supporting its role in thymocyte differentiation. The aim of this study was to evaluate the effect of GH on human thymocytes and thymic epithelial cells (TEC) in the presence of laminin. RESULTS: GH increased thymocyte adhesion on BSA-coated and further on laminin-coated surfaces. The number of migrating cells in laminin-coated membrane was higher in GH-treated thymocyte group. In both results, VLA-6 expression on thymocytes was constant. Also, treatment with GH enhanced laminin production by TEC after 24 h in culture. However, VLA-6 integrin expression on TEC remained unchanged. Finally, TEC/thymocyte co-culture model demonstrated that GH elevated absolute number of double-negative (CD4-CD8-) and single-positive CD4+ and CD8+ thymocytes. A decrease in cell number was noted in double-positive (CD4+CD8+) thymocytes. CONCLUSIONS: The results of this study demonstrate that GH is capable of enhancing the migratory capacity of human thymocytes in the presence of laminin and promotes modulation of thymocyte subsets after co-culture with TEC.

Fibronectin modulates thymocyte-thymic epithelial cell interactions following Trypanosoma cruzi infection

Developing thymocytes interact with thymic epithelial cells (TECs) through cell-cell interactions, TEC-derived secretory moieties and extracellular matrix (ECM)-mediated interactions. These physiological interactions are crucial for normal thymocyte differentiation, but can be disrupted in pathological situations. Indeed, there is severe thymic atrophy in animals acutely infected with Trypanosoma cruzi due to CD4+CD8+ thymocyte depletion secondary to caspase-mediated apoptosis, together with changes in ECM deposition and thymocyte migration. We studied an in vitro model of TEC infection by T. cruzi and found that infected TEC cultures show a reduced number of cells, which was likely associated with decreased proliferative capacity, but not with increased cell death, as demonstrated by bromodeoxyuridine and annexin-V labelling. The infected TEC cultures exhibited increased expression of fibronectin (FN), laminin (LM) and type IV collagen. Importantly, treatment with FN increased the relative number of infected cells, whereas treatment with anti-FN or anti-LM antibodies resulted in lower infection rates. Consistent with these data, we observed increased thymocyte adhesion to infected TEC cultures. Overall, these results suggest that ECM molecules, particularly FN, facilitate infection of the thymic epithelium and that the consequent enhancement of ECM expression might be associated with changes in TEC-thymocyte interactions.

Can thymic epithelial cells be infected by human T-lymphotropic virus type 1?

The human T-lymphotropic virus type-1 (HTLV-1) is the cause of adult T cell leukaemias/lymphoma. Because thymic epithelial cells (TEC) express recently defined receptors for the virus, it seemed conceivable that these cells might be a target for HTLV-1 infection. We developed an in vitro co-culture system comprising HTLV-1+-infected T cells and human TECs. Infected T cells did adhere to TECs and, after 24 h, the viral proteins gp46 and p19 were observed in TECs. After incubating TECs with culture supernatants from HTLV-1+-infected T cells, we detected gp46 on TEC membranes and the HTLV-1 tax gene integrated in the TEC genome. In conclusion, the human thymic epithelium can be infected in vitro by HTLV-1, not only via cell-cell contact, but also via exposure to virus-containing medium.

Expression of IL-17A during Thymus Regeneration in the Rat / 체질인류학회지

IL-17A is a pro-inflammatroy cytokine secreted by activated T cells. The IL-17 family consist of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F. IL-17A and IL-17F are produced primarily in activated T cells. In contrast, IL-17B, IL-17C, IL-17D and IL-17E are expressed in a wide assortment of tissues. Their functions partially overlap those of IL-17A, although they have not been as thoroughly investigated. The receptor for IL-17A (IL-17R) is widely expressed in a variety of tissues. IL-17A and IL-17E mRNAs were expressed in only EL4 cells. IL-17C mRNA expression was observed in the thymic subcapsular/cortex epithelial cells (SNEC), cortex or cortical reticular cells (CREC), medullary epithelial cells (MEC), medullary interdigitating-like cells (MDC), thymocytes and EL4 cells. However, IL-17C mRNA was not expressed in RAW 264.7 cells. Immunohistochemical study also demonstrated not only the presence of IL-17A mainly in the thymic epithelial cells, but also the upregulated expression of IL-17A in the thymic epithelial cells of the regenerating thymus. Thus, the results of the present study suggest that IL-17A expressed in the thymocytes and thymic epithelial cells could play an important role in the development of new T cells to replace T cells damaged by cyclophosphamide treatment during thymus regeneration.

Cytotoxicity of zearalenone for thymic epithelial cells in mice / 中国兽医学报

To elucidate the effects of Zearalenone(ZEA) on proliferation and cell cycle of cultured thymic epithelial cells in mice,trypan blue staining and flow cytometric analysis were performed.At the concentrations from 1 to 25 mg/L,ZEA displayed a significant inhibitory action to proliferation of thymic epithelial cells in its dose-and timedependent manner.Higher doses(10-25 rag/L)ZEA could induce a profound increase in G2/M phase with arrest of thymic epithelial cells in the G2/M phase in a dose-dependent manner.In conclusion,ZEA could be assumed that there were toxic effects on the thymie epithelial cells of mice in vitro.

Ultrastructure of Different Types of Thymic Epithelial Cells in the Rat Thymus / 대한해부학회지

Thymic epithelial cells constitute a major component of the thymic microenvironment. The thymus is involved in the regulation of the proliferation, maturation and differentiation of thymocytes. There is some controversy about the classification of thymic epithelial cell types. Traditionally, thymic epithelial cells have been divided into cortical and medullary epithelial cell types. In general, the thymic epithelium can be broadly subdivided into subcapsular, cortical and medullary epithelial cells, and Hassall's corpuscles by immunocytochemical methods. Although a few studies were performed on the ultrastructural characteristics of the different types of thymic epithelial cells, there is still some controversy about the classification of thymic epithelial cell subsets. Thus, the present study was performed to investigate the ultrastructural features of thymic epithelial cell subsets in adult male Sprague-Dawley rats, which are the most commonly used species of rat for biological researches, using transmission electron microscopy to shed more light on the heterogeneity of thymic epithelial cells. On the basis of ultrastructural features, we could identify and classify eight subsets of epithelial cells in normal rat thymus. In particular, this study provided a clear and easy way to identify the type 3 epithelial cells by their characteristic 'perinuclear arrangement pattern of relatively short bundles of tonofilaments'. This is an important finding since the type 3 epithelial cells has been considered to be the most difficult type to identify among various thymic epithelial cell types. The results of the present ultrastructural study of thymic epithelial cells provided more insight into the heterogeneity of thymic epithelial cells, and can contribute to the understanding of roles played by different types of thymic epithelial cells.

Immunohistochemical Study of TrkA Neurotrophin Receptor Expression during Thymic Regeneration / 대한해부학회지

Numerous studies have demonstrated interactions between the nervous/endocrine and the immune system. Increasing evidence suggests that some members of neurotrophins such as nerve growth factor (NGF) are involved in the control of immune system. Recent studies have demonstrated that the TrkA receptor, which serves as the high affinity receptor for NGF and neurotrophin-3 (NT-3), is expressed in thymic epithelial cells. In the present study, we investigated the expression of the TrkA receptor in the rat thymus from a model of thymic involution and regeneration induced by cyclophosphamide. After single dose of cyclophosphamide (150 mg/kg) was administered to Sprague-Dawley rats by intraperitoneal injection, the rats were sacrificed at 3, 7 and 14 days. The immunocytochemical characterization of the thymus was carried out using cryostat-cut sections. We found an increased expression of TrkA immunoreactivity in the thymic epithelial cells, especially in the subcapsular epithelial cells in cyclophosphamide-treated rats. The cortical epithelial cells also showed an increased expression of TrkA immunoreactivity after cyclophosphamide treatment, although the expression level was lower than that of the thymic subcapsular epithelial cells. However, there was no significant alteration of TrkA immunoreactivity in the medullary epithelial cells of the thymus from cyclophosphamide-treated rats. In general, most of these phenomena disappeared two weeks after cyclophosphamide administration and thus, the immunohistochemical features became to be similar to those of normal thymus. In conclusion, it may be speculated that TrkA receptor via interaction with their ligands provides an important signal to the thymic epithelial cells, especially to the subcapsular epithelial cells, for the thymic regeneration during recovery from acute thymic involution. Thus, our results support the proposed immunoregulatory role of neurotrophins.