OBJECTIVEBy using the RNAi method to inhibit Itk protein expression specificity, to observe lymphocytes proliferation and cytokines production, verify its function as a drug target.

METHODSDesigned siRNA aims at Itk sequence according to its sequence and solid structure, then electrotransfected into mouse spleen lymphocytes, We validated the decrease of Itk protein by Western-Blot, and detected the change of the cell proliferation by MTS and the change of inflammatory cytokines by ELISA.

RESULTSItk protein can be suppressed by Itk-siRNA, there were significantly reduced compared to its control group on cell proliferation as well as cytokine secretion such as IL-2, IL-4, IL-5, IFN-gamma. They all have statistical difference (P < 0.05).

CONCLUSIONItk has an important immunomodulatory effect in mouse spleen lymphocytes proliferation and secretion of inflammatory cytokines.This can supply an experimental basis to regard Itk as drug target for inflammation therapy.

Animals ; Cell Differentiation ; Cell Proliferation ; Cytokines ; genetics ; immunology ; Lymphocytes ; cytology ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Protein-Tyrosine Kinases ; genetics ; immunology ; Spleen ; cytology ; immunology

OBJECTIVETo observe the specific cellular and humoral immune responses after immunization with recombinant adenovirus Ad5F35-LMP2 in rhesus monkeys.

METHODSSixteen rhesuses were immunized with Ad5F35-LMP2 through intra-muscular injection in three groups: high dosage group (1.5 x 10(10) TCID(50)/rhesus), medium dosage group (1.5 x 10(9)TCID(50)/rhesus), low dosage group (1.5 x 10(8)TCID50/rhesus) and the last group was control (PBS 4 ml/rhesus). They were totally immunized three times at intervals of one month. The EBV-LMP2 specific cellular immune responses were tested during the 0, 4, 8, 12 weeks by Elispot after immunization respectively. And the titers of anti-LMP2 antibody were tested by EIA at the same time.

RESULTSEBV-LMP2 specific cellular and humoral immune responses which were induced by recombinant adenovirus Ad5F35-LMP2 can be found in all the three dosage groups. The potency of immune responses was related with the dosage of immunization. Higher dosage elicited more potent immune response.

CONCLUSIONThe recombinant adenovirus Ad5F35-LMP2 could elicit LMP2 specific cellular and humoral immune responses in rhesus.

Adenoviruses, Human ; genetics ; Animals ; Cell Differentiation ; Herpesvirus 4, Human ; genetics ; Immunity, Cellular ; immunology ; Immunization ; methods ; Macaca mulatta ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Matrix Proteins ; genetics ; immunology

Cell Differentiation ; Cytodiagnosis ; Gastrointestinal Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Stromal Cells ; pathology

OBJECTIVETo explore the role of indirect antigen presentation pathway on the immunogenecity of epidermal cells.

METHODSHuman epidermal cells (HEC), allogeneic human peripheral blood lymphocytes (PBL) and mononuclear cells (PBM, including monocytes) were isolated and cultured in vitro. HECs were transfected by human-originated CTLA4Ig-adenovirus vector. The CTLA4Ig expression was observed. Allogeneic PBLs or PBMs were added to the transfected and non-transfected HECs with simple cultured PBLs and PBMs as the control. The proliferation of PBL and PBM was determined by (3)H-TdR incooperation.

RESULTSHECs could be successfully transfected by CTLA4Ig-adenovirus vector and expressed corresponding proteins. The non-transfected HECs could stimulate slight proliferation of allogeneic PBLs (P < 0.05) and stimulate remarkable proliferation of PBMs (including monocytes) (P < 0.05). The proliferation reaction of PBLs and PBMs decreased significantly (P < 0.05) after being stimulated by HEC which was modulated by CTLA4Ig genes.

CONCLUSIONIndirect antigen presentation pathway might play important roles in the HEC immunogenicity which could be evidently inhibited by CTLA4Ig.

Adenoviridae ; genetics ; Antigen Presentation ; immunology ; physiology ; Antigens, CD ; Antigens, Differentiation ; genetics ; immunology ; CTLA-4 Antigen ; Cell Division ; immunology ; Cells, Cultured ; Coculture Techniques ; Epidermis ; cytology ; immunology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocytes ; cytology ; immunology ; Signal Transduction ; Transfection

CD4+ T lymphocytes play a major role in regulation of adaptive immunity. Upon activation, naive T cells differentiate into different functional subsets. In addition to the classical Th1 and Th2 cells, several novel effector T cell subsets have been recently identified, including Th17 cells. There has been rapid progress in characterizing the development and function of Th17 cells. Here I summarize and discuss on the genetic controls of their differentiation and emerging evidence on their plasticity. This information may benefit understanding and treating immune diseases.
Animals ; CD4-Positive T-Lymphocytes/cytology/*immunology ; Cell Differentiation ; Cell Lineage ; Cytokines/*genetics ; Epigenesis, Genetic ; Gene Expression Regulation ; Humans ; Interleukin-17/immunology/metabolism ; T-Lymphocytes, Regulatory ; Th1 Cells/immunology ; Th17 Cells/*immunology ; Th2 Cells/immunology ; Transcription Factors/*genetics ; Transcription, Genetic

Immunosuppressive regulatory T lymphocytes (Treg) expressing the transcription factor Foxp3 play a vital role in the maintenance of tolerance of the immune-system to self and innocuous non-self. Most Treg that are critical for the maintenance of tolerance to self, develop as an independent T-cell lineage from common T cell precursors in the thymus. In this organ, their differentiation requires signals from the T cell receptor for antigen, from co-stimulatory molecules, as well as from cytokine-receptors. Here we focus on the cytokines implicated in thymic development of Treg, with a particular emphasis on the roles of interleukin-2 (IL-2) and IL-15. The more recently appreciated involvement of TGF-β in thymic Treg development is also briefly discussed. Finally, we discuss how cytokine-dependence of Treg development allows for temporal, quantitative, and potentially qualitative modulation of this process.
Animals ; Cell Differentiation ; genetics ; Cytokines ; immunology ; Forkhead Transcription Factors ; genetics ; immunology ; Gene Expression Regulation ; Immune Tolerance ; genetics ; Interleukin-15 ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Mice ; Receptors, Antigen, T-Cell ; genetics ; immunology ; T-Lymphocytes, Regulatory ; immunology ; Transforming Growth Factor beta ; genetics ; immunology

BACKGROUNDEnhanced green fluorescent protein (EGFP) has been an important reporter gene for gene therapy. Human mesenchymal stem cells (hMSCs) are ideal target cells in cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated by PLEGFP-N1 retroviral transduction.

METHODShMSCs were isolated from human bone marrow by density gradient fractionation and adherence to plastic flasks. Individual colonies were selected and cultured in tissue dishes. Packaging cells PT67 were transfected by PLEGFP-N1 retroviral vector, and hMSCs were transduced by viral supernatant infection. Meanwhile, hMSCs-EGFP were identified by immune phenotypes and whether it could differentiate into osteoblasts or adipocytes under conditioned media was investigated.

RESULTSThe rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morphological features. Flow cytometric analyses showed that hMSCs-EGFP were positive for CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced into osteocytes or adipocytes under conditioned media. These biological features of hMSCs-EGFP were consistent with those of hMSCs.

CONCLUSIONShMSCs transduced by PLEGFP-N1 retroviral vector can be used in vivo securely because they can maintain their biological characteristics and differentiation. It is a simple and reliable way to trace the changes of hMSCs in vivo by EGFP during cell transplantation and gene therapy.

Cell Cycle ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Genetic Therapy ; Green Fluorescent Proteins ; genetics ; Humans ; Immunophenotyping ; Mesenchymal Stromal Cells ; cytology ; immunology ; metabolism ; Retroviridae ; genetics

Tissue and organ differentiation is tightly controlled to ensure proper development and function of the growing embryo as well as cells such as lymphocytes that differentiate throughout the adult stage. Therefore it is vital that the genes and the protein they encode that are involved in these processes function accurately. Hence, any mutation or error that occurs along the way can result in extensive damage, which is expressed in various ways in the embryo and can result in immune pathogenesis, including immunodeficiency and autoimmune diseases, when lymphocyte development is altered. A number of studies have been carried out to look at the genes regulating transcription in tissue differentiation, including the transcription factors Pbx1. This gene is of particular interest to us as we have identified that it is associated with systemic lupus erythematosus susceptibility (Cuda et al., in press). This perspective summarizes the known roles of Pbx1 in tissue differentiation as well as our recent findings associating genetic variations in Pbx1 to lupus susceptibility, and we will speculate on how this gene controls the maintenance of immune tolerance in T cells.
Animals ; Cell Differentiation ; Chromatin Immunoprecipitation ; DNA-Binding Proteins ; genetics ; immunology ; Genetic Loci ; immunology ; Genetic Predisposition to Disease ; Homeodomain Proteins ; genetics ; immunology ; Humans ; Immune Tolerance ; Lupus Erythematosus, Systemic ; genetics ; immunology ; Lymphocyte Activation ; Mice ; Mice, Transgenic ; Pre-B-Cell Leukemia Transcription Factor 1 ; Protein Structure, Tertiary ; Proto-Oncogene Proteins ; genetics ; immunology ; Signal Transduction ; T-Lymphocytes, Regulatory ; cytology ; immunology ; Transcription Factors ; genetics ; immunology ; Tretinoin ; metabolism

In order to assess the number and function of macrophages in the placenta of pregnancy complicated with gestational diabetes mellitus (GDM) as well as those of normal pregnancies, placenta samples were collected from 15 GDM patients (GDM group) and 10 normal pregnant women (control group). The expression levels of macrophage markers (CD68/CD14) and inflammatory cytokines (IL-6/TNF-α) in placenta were detected using immunohistochemistry and PCR. The results showed that the number of CD68+ or CD14+ cells in the GMD group was remarkably higher than that in the control group (P<0.05), indicating that the number of macrophages in the GDM group was significantly greater than that in the control group. The mRNA expression levels of CD68+, IL-6 and TNF-α were higher in the GMD group than in the control group. In conclusion, more macrophages accumulate in placenta of pregnancy complicated with GDM, and the expression levels of pro-inflammation factors are also increased in GDM pregnancies, suggesting that macrophages and inflammatory mediators (IL-6 and TNF-α) may play an important role in GDM.
Adult ; Antigens, CD ; Antigens, Differentiation, Myelomonocytic ; Cell Count ; Cytokines ; genetics ; immunology ; metabolism ; Diabetes, Gestational ; genetics ; immunology ; metabolism ; Female ; Humans ; Immunohistochemistry ; Inflammation Mediators ; immunology ; metabolism ; Interleukin-6 ; genetics ; immunology ; metabolism ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Macrophages ; immunology ; metabolism ; pathology ; Placenta ; immunology ; metabolism ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Necrosis Factor-alpha ; genetics ; immunology ; metabolism

Antibodies, Neoplasm ; blood ; immunology ; Carcinoma, Hepatocellular ; pathology ; Cell Differentiation ; Female ; Humans ; Liver Neoplasms ; pathology ; Male ; Tumor Suppressor Protein p53 ; blood ; genetics ; immunology