Effects of CIITA antisense RNA on the expression of HLA class Ⅱ molecules

To study the effect of the major histocompatibility complex class Ⅱ (MHCⅡ) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class Ⅱ molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-Ⅱ was constructed. HeLa cells transfected with pcDNA-Ⅱ and pcDNA3 were induced by IFN-g for 3 d. The expression of HLA class Ⅱ molecules on HeLa/pcDNA-Ⅱ cells was significantly decreased, while it has no effect on the expression of HLA class Ⅰ molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class Ⅱ molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.     

Distribution of α-MSH-like immunoreactive cells in the nervous system, Hatschek''''s pit and other tissues of amphioxus, Branchiostoma belcheri

Immunohistochemical localization of a-melanocyte-stimulating hormone (α-MSH) in the nervous system, Hatschek's pit and other tissues of amphioxus (Branchiostoma belcheri) was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek's pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek's pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek's pit and for the origin and evolution of vertebrate adenohypophysis.     

P15——a new tumor suppressor gene

In addition to the tumor suppressor genes such as Rb and p53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumorigenesis and the tumor progression. In the KIP and INK4 families of CKIs, p15 shares extensive homology with p16. Findings in many tumors and their cell lines show that the inactivation of p15 (deletion, mutation, rearrangement, etc.) is very frequent, and inactive p15 is involved in the progress of some tumors. These studies provide evidence that the p15 is a new tumor suppressor gene. Furthermore, the research on the molecular mechanism of p15 in regulation of cell proliferation shows that p15 can inhibit the growth of some kinds of tumor cells, and p15 is the mediator of TGF-β-induced cell arrest. Investigations on p15 in cell differentiation suggest that increased p15 is related to the change of malignant phenotype. These results supply clues for further interpretation about the molecular mechnism of cell cycle control and cell tumorigenesis. And they may provide theoretical and experimental basis for application of p15 to clinical therapy of tumors.     

Distribution of a-MSH-like immunoreactive cells in the nervous system,Hatschek's pit and other tissues of amphioxus,Branchiostoma belcheri

Immunohistochemical localization of α-melanocyte-stimulating hormone （α-MSH） in the nervous system, Hatschek＇s pit and other tissues of amphioxus （Branchiostorna belcheri） was performed using the antibody against synthetic α-MSH. The results revealed that α-MSH-like immunoreactive cells were distributed at the dorsal side and ventral side of brain vesicle, the dorsal side and the surrounding of nerve tube, and in the epithelial cells of Hatschek＇s pit, the zone 1, 3, and 6 of endostyle and gut. The immunoreactive substance was also found in the primary oocytes of the small and large growth stage of ovary and early stage spermatogenic cells in testis. These findings indicate that α-MSH is an ancient and highly conserved hormone and it is extensively distributed in amphioxus. Although Hatschek＇s pit in amphioxus does not have a structure of the intermediate lobe of vertebrate adenohypophysis, it has already hosted α-MSH-like endocrine cells, implying that the functional differentiation of α-MSH-like cells occurred earlier than the differentiation of the tissue structure. The results of the present study provided a new evidence for the endocrinology of Hatschek＇s pit and for the origin and evolution of vertebrate adenohypophysis.     

N-Terminal Truncation of TACO Inhibits PMA-Induced U937 Cell Adhesion

The effect of TA001-299, the N-terminal truncation of TACO, on phorbol 12-myristate 13-acetate （PMA）-induced U937 cell adhesion was investigated. Full-length TACO and several truncations were overexpressed in U937 cells. The effects of the expressed proteins on U937 cell adhesion mediated by PMA-induced differentiation were observed by fluorescence microscopy. The results show that the overexpression of TACO1-299 inhibits cell adhesion while overexpressions of the other proteins do not have this effect. The actin-binding capability of TACO1-299 was investigated and the results show that TACO1-299 lacks the ability of TACO to bind F-actin. The inhibitive effect of TACO1-299, the functional domain of TACO, suggests that TACO may play a role in cell differentiation mediating adhesion of monoblastic leukemia cells.     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Pathogen-induced human TH17 cells produce IFN-γ or IL-10 and are regulated by IL-1β

IL-17-producing CD4+ T helper cells (TH17) have been extensively investigated in mouse models of autoimmunity. However, the requirements for differentiation and the properties of pathogen-induced human TH17 cells remain poorly defined. Using an approach that combines the in vitro priming of naive T cells with the ex vivo analysis of memory T cells, we describe here two types of human TH17 cells with distinct effector function and differentiation requirements. Candida albicans-specific TH17 cells produced IL-17 and IFN-γ, but no IL-10, whereas Staphylococcus aureus-specific TH17 cells produced IL-17 and could produce IL-10 upon restimulation. IL-6, IL-23 and IL-1β contributed to TH17 differentiation induced by both pathogens, but IL-1β was essential in C. albicans-induced TH17 differentiation to counteract the inhibitory activity of IL-12 and to prime IL-17/IFN-γ double-producing cells. In addition, IL-1β inhibited IL-10 production in differentiating and in memory TH17 cells, whereas blockade of IL-1β in vivo led to increased IL-10 production by memory TH17 cells. We also show that, after restimulation, TH17 cells transiently downregulated IL-17 production through a mechanism that involved IL-2-induced activation of STAT5 and decreased expression of ROR-γt. Taken together these findings demonstrate that by eliciting different cytokines C. albicans and S. aureus prime TH17 cells that produce either IFN-γ or IL-10, and identify IL-1β and IL-2 as pro- and anti-inflammatory regulators of TH17 cells both at priming and in the effector phase.     

STAT1 signaling is required for optimal Th1 cell differentiation in mice

Although IFN-γ alone does not prime type I T helper cell (Th1) differentiation, the loss of IFN-γ signaling leads to impaired Th1 phenotype: IFN-γ receptor-deficient (Ifngr-/-) Th1 cells fail to permanently repress IL-4 expression. They can differentiate into IL-4-producing cells under Th2-inducing conditions. These observations suggest that IFN-γ signaling plays a critical role in si- lencing Il4 gene in Th1 cells and stabilizing Th1 phenotype. IFN-γ signaling has been further shown to inhibit IL-4 express...     

Effects of CIITA antisense RNA on the expression of HLA class II molecules

To study the effect of the major histocompatibility complex class II (MHC II) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class II molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-II was constructed. HeLa cells transfected with pcDNA-II and pcDNA3 were induced by IFN-γ for 3 d. The expression of HLA class II molecules on HeLa/pcDNA-II cells was significantly decreased, while it has no effect on the expression of HLA class I molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class II molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.     

Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells

On activation, T cells undergo distinct developmental pathways, attaining specialized properties and effector functions. T-helper (T(H)) cells are traditionally thought to differentiate into T(H)1 and T(H)2 cell subsets. T(H)1 cells are necessary to clear intracellular pathogens and T(H)2 cells are important for clearing extracellular organisms. Recently, a subset of interleukin (IL)-17-producing T (T(H)17) cells distinct from T(H)1 or T(H)2 cells has been described and shown to have a crucial role in the induction of autoimmune tissue injury. In contrast, CD4+CD25+Foxp3+ regulatory T (T(reg)) cells inhibit autoimmunity and protect against tissue injury. Transforming growth factor-beta (TGF-beta) is a critical differentiation factor for the generation of T(reg) cells. Here we show, using mice with a reporter introduced into the endogenous Foxp3 locus, that IL-6, an acute phase protein induced during inflammation, completely inhibits the generation of Foxp3+ T(reg) cells induced by TGF-beta. We also demonstrate that IL-23 is not the differentiation factor for the generation of T(H)17 cells. Instead, IL-6 and TGF-beta together induce the differentiation of pathogenic T(H)17 cells from naive T cells. Our data demonstrate a dichotomy in the generation of pathogenic (T(H)17) T cells that induce autoimmunity and regulatory (Foxp3+) T cells that inhibit autoimmune tissue injury.     

IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells

On activation, naive T cells differentiate into effector T-cell subsets with specific cytokine phenotypes and specialized effector functions. Recently a subset of T cells, distinct from T helper (T(H))1 and T(H)2 cells, producing interleukin (IL)-17 (T(H)17) was defined and seems to have a crucial role in mediating autoimmunity and inducing tissue inflammation. We and others have shown that transforming growth factor (TGF)-beta and IL-6 together induce the differentiation of T(H)17 cells, in which IL-6 has a pivotal function in dictating whether T cells differentiate into Foxp3+ regulatory T cells (T(reg) cells) or T(H)17 cells. Whereas TGF-beta induces Foxp3 and generates T(reg) cells, IL-6 inhibits the generation of T(reg) cells and induces the production of IL-17, suggesting a reciprocal developmental pathway for T(H)17 and T(reg) cells. Here we show that IL-6-deficient (Il6-/-) mice do not develop a T(H)17 response and their peripheral repertoire is dominated by Foxp3+ T(reg) cells. However, deletion of T(reg) cells leads to the reappearance of T(H)17 cells in Il6-/- mice, suggesting an additional pathway by which T(H)17 cells might be generated in vivo. We show that an IL-2 cytokine family member, IL-21, cooperates with TGF-beta to induce T(H)17 cells in naive Il6-/- T cells and that IL-21-receptor-deficient T cells are defective in generating a T(H)17 response.