Objective To identify and verify the distribution of Telocytes derived from heterogeneous interstitial cells in the vital organs of ApoE mice.Methods Heart,kidney,and liver tissues were harvested from ApoE adult mice. Immunohistochemical assays were performed by using different immunobiological markers.Results Telocytes were found in these vital organs. The expressions of immunobiological markers differed among different organs. CD34,CD117,and CD28 were positively expressed in Telocytes in cardiac tissue;CD117 and plateled-derived growth factor-Α were negatively expressed in Telocytes in renal tissue;and CD117 and plateled-derived growth factor receptor-Α had negative expression in Telocytes in hepatic tissue. Furthermore,the distribution of Telocytes also differed in the same organ.Conclusions Telocytes exist in the vital organs of ApoE mice,as demonstrated by immunohistochemisty assay. The expressions of immunobiological markers differ among Telocytes in different organs.
Animals ; Antigens, CD34 ; metabolism ; CD28 Antigens ; metabolism ; Kidney ; cytology ; Liver ; cytology ; Mice ; Mice, Knockout, ApoE ; Myocardium ; cytology ; Proto-Oncogene Proteins c-kit ; metabolism ; Telocytes ; cytology

OBJECTIVE: To explore the role of DNAM-1 in the activation, proliferation and function of type Ⅰ regulatory T cells (Tr1 cells). METHODS: Anti-CD3/CD28 antibodies were used to stimulate mouse T cells derived from the spleen of wild-type (WT) mice, and the expression level of DNAM-1 in resting and activated Tr1 cells was evaluated with flow cytometry. Na?ve CD4⁺ T cells isolated by magnetic cell sorting from the spleens of WT mice and DNAM-1 knockout (KO) mice were cultured in Tr1 polarizing conditions for 3 days, after which CD25 and CD69 expressions were measured using flow cytometry. The induced Tr1 cells were labelled with CFSE and cultured in the presence of anti-CD/CD28 antibodies for 3 days, and their proliferative activity was analyzed. The expressions of IL-10 and p-STAT5 in DNAM-1-deficient Tr1 cells were detected before and after IL-2 stimulation. RESULTS: The expression level of DNAM-1 was significantly upregulated in CD4⁺ T cells and Tr1 cells after stimulation with anti-CD3/CD28 antibodies (P < 0.05). DNAM-1 knockout did not cause significant changes in the number or proportion of Tr1 cells, but but significantly increased the expression levels of the activation markers CD69 and CD25 (P < 0.05). Compared with WT Tr1 cells, DNAM-1-deficient Tr1 cells exhibited reduced proliferative activity in vitro (P < 0.05) with downregulated IL-10 production (P < 0.05) and decreased expressions of Il-10 and Gzmb mRNA (P < 0.05). In DNAM-1-deficient Tr1 cells, IL-2 stimulation significantly reduced IL-10 secretion level and the expression of p-STAT5 as compared with WT Tr1 cells. CONCLUSION DNAM-1 participate in the activation and proliferation of Tr1 cells and affect the biological functions of Tr1 cells through the IL-2/STAT5 pathway.
Animals ; Antigens, Differentiation, T-Lymphocyte ; CD28 Antigens/metabolism* ; Cell Proliferation ; Cells, Cultured ; Interleukin-10 ; Interleukin-2/metabolism* ; Mice ; RNA, Messenger ; STAT5 Transcription Factor/metabolism* ; T-Lymphocytes, Regulatory

The objective of study was to investigate the expressions of CD80, CD86 and its ligand CD28 on peripheral lymphocytes in patients with idiopathic thrombocytopenic purpura (ITP), to explore the effect of interleukin 18 (IL-18) and its clinical significance in ITP. The expressions of co-stimulatory molecules (CD80, CD86 and its ligand CD28) on peripheral lymphocytes from 34 ITP patients and 34 normal humans were detected by immunofluorescence and flow cytometry. The IL-18 in the plasma was detected by using enzyme linked immunosorbent assay (ELISA). The results showed that the expressions of CD80 and CD86 on peripheral lymphocytes from ITP patients were higher than that of the normal control (4.21 +/- 2.27%, 7.19 +/- 5.16% vs 2.34 +/- 0.87%, 4.08 +/- 1.96%) (P < 0.01); the concentration of IL-18 in plasma of ITP patients was (538.31 +/- 111.33) pg/ml, but the concentration of IL-18 in plasma of controls was (489.44 +/- 49.07) pg/ml. The level of IL-18 negatively correlated with the platelet counts in peripheral blood (r = -0.395, P < 0.05). It is concluded that the CD28/CD80 and CD28/CD86 costimulatory molecules are overexpressed, when the IL-18 level in ITP patients is obviously higher than that in normal controls. When ITP occurred, and the co-stimulatory molecules CD80 and CD86 are closely associated with ITP, it seems that IL-18 may play an important role in ITP pathogenesis.
B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; CD28 Antigens ; metabolism ; Humans ; Interleukin-18 ; blood ; Lymphocytes ; metabolism ; Purpura, Thrombocytopenic, Idiopathic ; blood ; immunology

AIMTo construct recombinant retroviruses expressing anti-CD20 scFv/CD80/CD28/zeta gene and detect its expression in Jurkat cells.

METHODCD28-zetacDNA were amplified from plasmids pBULLET and inserted into pLNCX vector that contained anti-CD20 scFv/CD80 gene. The recombinant plasmids were transfected into PA 317 cells. Retroviruses were harvested from culture medium of PA 317 cells. Then NIH 3T3 were transfected with retroviruses. Objective gene expression was determined by PCR and FACS. Jurkat cells were transfected with high titer of retroviruses and resistant clones were obtained by G418 selection. Objective mRNA was determined by RT- PCR.

RESULTSThe recombinant eukaryotic vector was constructed successfully by PCR and enzyme digestion analysis and objective gene was amplified from NIH 3T3 cells transfected with retroviruses by PCR; FACS showed that objective protein could be expressed in NIH 3T3 cells. Objective gene was amplified from Jurkat cells transfected with retroviruses by RT-PCR.

CONCLUSIONRecombinant retrovirus expressing anti-CD20 scFv/CD80/CD28/zeta gene was successfully constructed and objective protein could be expressed in Jurkat cells.

Antigens, CD20 ; genetics ; metabolism ; B7-1 Antigen ; genetics ; metabolism ; CD28 Antigens ; genetics ; metabolism ; Genetic Vectors ; Humans ; Immunoglobulin Fab Fragments ; genetics ; metabolism ; Immunoglobulin Variable Region ; genetics ; metabolism ; Jurkat Cells ; Recombinant Fusion Proteins ; genetics ; metabolism ; Retroviridae ; genetics ; T-Lymphocytes ; metabolism ; Transfection

OBJECTIVE: To investigate the expression of costimulatory molecules CD28/B7 and CD40/CD40L in T and B lymphocytes as well as its relations with total IgE (TIgE), eosinophil cationic protein (ECP) in serum and nasal allergic symptoms in patients with allergic rhinitis (AR). The effect of specific immunotherapy (SIT) on them were also investigated. METHOD: Thirty allergic allergic rhinitis patients were chosen as observation group, and 30 healthy patients as control group. Cytofluorometric analysis was used to compare the expression level of CD28/B7-1, B7-2 and CD40/CD40L on T cells and B cells in the two groups. The relationship between the CD28/ B7-1, B7-2 and CD40/CD40L expression level and serum Total IgE, ECP level were analyzed. RESULT: The expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells in allergic rhinitis patients were significantly higher than in the healthy, and serum level of TIgE has a positive relationship with the expression level of CD40L on T cells. ECP has a positive relationship with the expression level of B7-2 on B cells. The expression level of B7-1 showed no significant difference between the two groups. After specific immunotherapy for 6 months, the expression level of CD28/B7-2 and CD40/CD40L on T cells and B cells were decreased in allergic rhinitis patients but still higher than in healthy. CONCLUSIONS The upregulated level of costimulatory molecules CD28/B7-2 and CD40/ CD40L on T cells and B cells may play an important role in the pathogenesis of allergic rhinitis, specific immunotherapy can downregulate the expression level of CD28/B7-2 and CD40/CD40L, and decrease the serum level of TIgE, it may be a possible mechanism in the treatment of allergic rhinitis.
Adult ; B-Lymphocytes ; immunology ; metabolism ; B7-2 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CD40 Antigens ; metabolism ; CD40 Ligand ; metabolism ; Case-Control Studies ; Desensitization, Immunologic ; Female ; Humans ; Immunoglobulin E ; blood ; Male ; Rhinitis, Allergic, Perennial ; blood ; immunology ; metabolism ; therapy ; T-Lymphocytes ; immunology ; metabolism

This study was purposed to investigate the molecular mechanism of 4-1BBL reverse signals in the human acute monocytic leukemia cell line of U937. The U937 cell line was used as target cells, and stimulated by the mouse anti-human 4-1BBL monoclonal antibody 1F1. The nuclear translocation of NF-κB and the co-location of 4-1BBL and CD28i molecules in U937 cells were observed with confocal laser scanning microscopy. The protein and m-RNA expression levels of 4-1BBL and CD28i were detected by flow cytometry and RT-PCR respectively. The results showed that the significant nuclear translocation of NF-κB and co-localization of 4-1BBL and CD28i on membrane of U937 cells appeared after being stimulated by mAb1F1. It is concluded that the 4-1BBL reverse signals transduction mediating the growth of U937 cells relates with the nuclear translocation of NF-κB. CD28i may be involved in intracellular 4-1BBL reverse signaling pathways.
4-1BB Ligand ; immunology ; metabolism ; Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; metabolism ; Coculture Techniques ; Humans ; NF-kappa B ; genetics ; Signal Transduction ; U937 Cells

OBJECTIVETo investigate the effect of in vivo administration of rhG-CSF and/or rhIL-11 on mice immune system function.

METHODST cell subgroups, suppressor T cells (CD8+ CD28-, CD4+ CD25+, CD3+ CD4- CD8- T cells), expression of CD28 on T cells, and spleen T cells intracellular IL4/IFN-gamma secretion were determined by multicolor flow cytometry. MTT was used to determine the T cell proliferation capacity and mixed lymphocyte reactions.

RESULTSIn vivo administration of cytokines decreased the percentage of lymphocytes (P < 0.05), rhIL-11 and rhG-CSF in combination significantly decreased the CD4+/CD8+ ratio and increased the percentage of CD8+ CD28- suppressor T cells compared to either cytokine alone (P < 0.01). There was no difference in the percentage of CD3+ CD4- CD8- and CD4+ CD25+ suppressor T cells between either of the cytokines. Furthermore, cytokines treatments significantly decreased the capacities of splenic T cells proliferation and the response to alloantigens compared with the PBS treatment (P < 0.05), the combination group being more significantly decreased (P < 0.01). And cytokines treatment significantly decreased the production of IFN-gamma and increased the production of IL-4 compared with the PBS treatment(P < 0.05). The ratio of IFN-gamma/IL-4 were significantly decreased after the combination compared with either of them alone.

CONCLUSIONThe combination of rhIL-11 and rhG-CSF is potentially synergistic in the induction of immune tolerance by their effects on the proliferation capacity and function of T lymphocytes.

Animals ; CD28 Antigens ; metabolism ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Immune Tolerance ; Interleukin-11 ; pharmacology ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins ; T-Lymphocytes ; drug effects ; immunology ; metabolism

Antigens, CD ; metabolism ; B7-1 Antigen ; metabolism ; CD28 Antigens ; metabolism ; CTLA-4 Antigen ; Child ; Humans ; Interleukin-2 ; metabolism ; Mucocutaneous Lymph Node Syndrome ; immunology ; metabolism ; RNA, Messenger ; genetics ; Receptors, Immunologic ; metabolism

OBJECTIVETo investigate the correlation of the changes in CD8(+)CD28(-) T cell percentage with platelet (PLT) and D-dimer (D-D) levels in patients with multiple injuries (MI).

METHODSTwenty-six patients with MI, 31 with a single injury (SI group) and 26 healthy individuals were examined for peripheral blood CD8(+)CD28(-) T cells and intracellular transformation growth factor-β1 (TGF-β1) and interleukin 10 (IL-10) contents using flow cytometry at 24, 48, and 72 h after the injuries. PLT and D-dimer levels were compared among the 3 groups.

RESULTSCD8(+)CD28(-) T cells, TGF-β1 and IL-10 were significantly higher in MI group than in SI group and healthy control group (P<0.05) without significant differences between the latter 2 groups. The levels of PLT and D-D differed significantly among the 3 groups, the highest in MI group and the lowest in the control group. In MI group, CD8(+)CD28(-) T cells, TGF-β1 and IL-10 significantly increased at 48 h after the injury (P<0.05) but decreased significantly at 72 h (P<0.05) compared with the measurements at 24 h. The levels of PLT and D-D trended to decrease with time after the injuries and showed significant differences among the 3 groups at any of the 3 time points (P<0.05). CD8(+)CD28(-) T cells, TGF-β1 and IL-10 were all positively correlated with the levels of PLT and D-D in MI patients (r>0.70, P<0.05 for all comparisons).

CONCLUSIONIn MI patients, CD8(+)CD28(-) T cell percentage and their cytokines tend to increase early after the injury but decrease significantly at 72 h in close relation with the changes of the coagulation function following the injuries.

CD28 Antigens ; metabolism ; CD8 Antigens ; metabolism ; Case-Control Studies ; Fibrin Fibrinogen Degradation Products ; metabolism ; Flow Cytometry ; Humans ; Interleukin-10 ; metabolism ; Multiple Trauma ; immunology ; T-Lymphocyte Subsets ; cytology ; Transforming Growth Factor beta1 ; metabolism

BACKGROUNDThe B7/CD28 pathway provides critical costimulatory signals for complete T cell activation, and members of this pathway have served as useful targets for immunotherapeutic strategies. In this study, we investigated the RNA interference (RNAi) effect induced by small interfering RNA (siRNA) targeting CD28 mRNA on human lymphocytes and its specificity.

METHODSAccording to CD28 gene sequence, we designed and synthysized three different siRNAs (siRNA-1, siRNA-2, siRNA-3) containing 21 bases using Silencertrade mark siRNA construction kit. These siRNAs were transfected into freshly isolated human lymphocytes with Lipofectamine 2000 reagent. At 24-hour, 48-hour and 72-hour post transfection, these cells were collected and analyzed. The changes of surface expression of CD28 gene were detected by flow cytometry, and the changes of CD28 mRNA levels were determined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The cell viability of transfected lymphocytes was determined by methyl thiazolyl tetrazolium (MTT) assay and trypan blue dye exclusion assay.

RESULTSThree siRNAs (siRNA-1, siRNA-2, siRNA-3) specifically targeting CD28 mRNA were successfully designed and constructed. Flow cytometry analysis showed that a decrease in CD28 expression was detectable at 24-hour post transfection. Different siRNA showed different inhibition effects on CD28 expression. At 48-hour post transfection, the degrees of reduction with siRNA-1, siRNA-2 and siRNA-3 were 22.10% +/- 1.63%, 73.50% +/- 1.02% and 42.90% +/- 0.89% respectively compared with the control (P < 0.001). Neither of the groups transfected only with siRNA or lipo showed marked reduction in CD28 expression (3.15% +/- 0.75% and 4.55% +/- 0.80%) (P > 0.05). Moreover, lymphocytes treated with siRNA-co showed no marked reduction in CD28 expression (5.07% +/- 0.96%) (P > 0.05). The results of semi-quantitative RT-PCR assay indicated CD28 mRNA level was inhibited after transfection of specific siRNAs. At least 4-fold of reduction in siRNA-2 group occurred at 48-hour post transfection compared with the control (P < 0.001). MTT assay and trypan blue dye exclusion assay demonstrated that the viable cell rations of transfected lymphocytes were significantly reduced in siRNA-1, siRNA-2 and siRNA-3 groups at 48-hour post transfection (P < 0.01). The control groups showed no marked reduction in cell viability (P > 0.05).

CONCLUSIONSThree different siRNAs were synthesized and transfected into lymphocytes. They could reduce the expression of CD28 and the CD28 mRNA level. siRNA-2 was the most efficient. The cell viability reduced correspondingly. Therefore, the silencing effect on CD28 mRNA induced by siRNA may contribute to costimulatory blockade. This result show that siRNA may be useful for further study on graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT).

Adolescent ; Adult ; CD28 Antigens ; genetics ; Cell Survival ; Cells, Cultured ; Flow Cytometry ; Gene Silencing ; Humans ; Lymphocytes ; metabolism ; RNA, Small Interfering ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction