The aim of this study was to separate and amplify CD4(+)CD25(+)Treg cells from splenocytes of sensitized mice. The percentage of CD4(+)CD25(+)Treg cells was detected by flow cytometry in sensitized and normal control mice. CD4(+)T, CD4(+)CD25(+)Treg and CD4(+)CD25(-) T cells were isolated from mouse splenocytes by MACS. CD4(+)CD25(+)Treg cells were expanded in vitro cultures in addition of CD3/CD28 MACSiBead and IL-2. The activity of cells was detected with 0.4 trypan blue staining. The purity of cells after sorting, the main surface marker and the level of Foxp3 were detected by flow cytometry. The results showed that CD4(+)CD25(+)Treg cell proportion was higher in sensitized mice than normal control mice (P < 0.05). The average purity of CD4(+)CD25(+)Treg cells was 87. The activity of these cells was more than 97, and the expression of Foxp3 in these cells was high. The amplification multiples achieved 42 times after 2 weeks in vitro. The percentage of CD4(+)CD25(+) regulatory T cells was 85.32, and the expression of Foxp3 decreased from (76.92 ± 1.72) to (75.33 ± 2.11) (P > 0.05). It is concluded that the sorting of CD4(+)CD25(+)Treg cells is isolated successfully by MACS without affecting the vitality of target cells. The amplification of CD4(+)CD25(+)Treg cells is successful in vitro. Expression of surface markers and Foxp3 gene does not obviously change after amplification, so that to establish a practical method to recover and enlarge the amount of CD4(+)CD25(+)Treg cells in good condition.
Animals ; CD4 Antigens ; biosynthesis ; Flow Cytometry ; Forkhead Transcription Factors ; metabolism ; Immunomagnetic Separation ; methods ; Interleukin-2 Receptor alpha Subunit ; metabolism ; Lymphocyte Count ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; cytology

Human immunodeficiency virus (HIV) infects the host cells by the fusion of viral and cell membranes. Blocking the combining between HIV and the receptors can prevent HIV from entering the host cells. We designed an invasion-inhibitor for HIV-1 targeting dendritic cell (DC), including 2 important HIV-1 receptors CD4 and CCR5, and 2 molecules Flt3-L and Mip-3alpha. With the synthetic gene of the inhibitor, 2 eukaryotic expression vectors pABK-CKR5-CD4/Flt3L-Mip3alpha (pABK-HIV-MF) and pABK-CKR5-CD4 (pABK-HIV-MT) were constructed and transfected into HEK 293 cells for expression. Results from RT-PCR, immunofluorescent assay, ELISA and Western blot approved that the invasion-inhibitor for HIV-1 was successfully and exactly expressed in the eukaryotic cells. Current study formed a solid base for the further research on the function of inhibitors for HIV-1 and elimination targeting DC.
Artificial Gene Fusion ; CD4 Antigens ; biosynthesis ; genetics ; Chemokine CCL20 ; biosynthesis ; genetics ; Dendritic Cells ; immunology ; metabolism ; Genetic Vectors ; genetics ; HEK293 Cells ; HIV Envelope Protein gp120 ; genetics ; HIV-1 ; physiology ; Humans ; Receptors, CCR5 ; biosynthesis ; genetics ; Receptors, HIV ; antagonists & inhibitors ; Transfection ; fms-Like Tyrosine Kinase 3 ; biosynthesis ; genetics

Increasing importance is being given to the stimulation of Th1 response in cancer immunotherapy because its presence can shift the direction of adaptive immune responses toward protective immunity. Based on chemokine receptor expression, CXCR3+CCR4-CD4+ T cells as Th1-type cells were investigated its capacity in monocyte-derived dendritic cell (DC) maturation and polarization, and induction of antigen specific cytotoxic T lymphocytes (CTL) in vitro. The levels of IL-4, IL-5 and IL-10 were decreased to the basal level compared with high production of IFN-gamma, TNF-alpha, and IL-2 in CXCR3+CCR4-CD4+ T cells stimulated with anti-CD3 and anti-CD28 antibodies. Co-incubation of activated CD4+ or CXCR3+CCR4-CD4+ T cells with DC (CD4+/DC or CXCR3+CD4+/DC, respectively) particularly up-regulated IL-12 and CD80 expression compared with DC matured with TNF-alpha and LPS (mDC). Although there was no significant difference between the effects of the CXCR3+CCR4-CD4+ and CD4+ T cells on DC phenotype expression, CXCR3+CD4+/DC in CTL culture were able to expand number of CD8+ T cells and increased frequencies of IFN-gamma secreting cells and overall cytolytic activity against tumor antigen WT-1. These results demonstrated that the selective addition of CXCR3+CCR4-CD4+ T cells to CTL cultures could enhance the induction of CTLs by DC in vitro, and implicated on a novel strategy for adoptive T cell therapy.
Antigens, CD4/*immunology ; Cell Line ; Cells, Cultured ; Cytokines/immunology ; Cytotoxicity, Immunologic ; Dendritic Cells/cytology/*immunology ; Humans ; Interferon-gamma/biosynthesis ; Receptors, CCR4/*immunology ; Receptors, CXCR3/*immunology ; T-Lymphocytes, Cytotoxic/*cytology/immunology ; Th1 Cells/*immunology

BACKGROUNDT cell immune abnormalities in patients with dilated cardiomyopathy (DCM) has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator (ANT) result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules.

METHODSThe ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice (n = 6 in each group) simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-gamma and interleukin-4 producing cells among splenic CD4(+) lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR.

RESULTSTreatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P56lck and Zap-70 and a lower level of CD45 expression by spleen T cells. Also, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies (mAb) group. Whereas irrelevant antibody (rat anti-mouse IgG) did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group.

CONCLUSIONThe results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor (TCR) signal transduction in mouse autoimmune dilated cardiomyopathy.

Adenine Nucleotide Translocator 1 ; immunology ; Animals ; Antibodies, Monoclonal ; therapeutic use ; Autoantibodies ; blood ; Autoimmune Diseases ; therapy ; CD4 Antigens ; immunology ; Cardiomyopathy, Dilated ; immunology ; therapy ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Leukocyte Common Antigens ; analysis ; Mice ; Mice, Inbred BALB C ; Receptors, Antigen, T-Cell ; antagonists & inhibitors ; physiology ; Signal Transduction

OBJECTIVETo study the influence of percutaneous microwave ablation (PMA) and surgical resection for patients with small primary hepatocellular carcinoma (PHC) on dissemination of tumor cells in peripheral blood determined by AFP mRNA.

METHODSForty patients with small PHC (The maximal diameter < or = 5 cm) confirmed histologically were included in this study. All the patients had single tumor nodule only without metastasis. Of the 40 patients, 19 were treated by PMA and 21 by surgical resection. Blood samples were collected and tested immediately before treatment, 30 min after the mass ablated/resected, 1 d and 7 d later by RTD-Nested-RT-PCR for AFP mRNA. The CD3, CD4, CD8 and CD4/CD8 in blood, and hepatic function were tested at the same time points as well.

RESULTSAfter treatment, ALT and AST in peripheral blood increased in both groups, but more intensely in the surgical group. The CD3, CD4 and CD4/CD8 in peripheral blood decreased at 30 min, 1 day and 7 days after surgical resection, and the lowest value was at 30 min after surgery. The immune function was kept at the same level as pre-treatment in the PMA group. AFP mRNA copies in blood could be detected in 27 of 40 patients (67.5%) in two groups before treatment, and the copy number was increased after treatment. There was no significant difference between the two groups. The patients were followed up for 1 - 16 months. AFP mRNA copies in blood could be detected persistently in the 4 patients with extrahepatic metastasis or liver recurrence.

CONCLUSIONSurgical resection and microwave ablation may cause PHC cells dissemination into the blood circulation in patients with small PHC, and there was no difference between the two treatment groups. The cellular immune function in peripheral blood is decreased after surgical resection, but is maintained at the same level as pre-treatment in the PMA group. The impairment of liver function is less severe after PMA treatment than surgical resection. PMA may provide certain value for clinical management of small hepatocellular carcinoma.

Adult ; Aged ; CD3 Complex ; blood ; CD4 Antigens ; blood ; CD4-CD8 Ratio ; CD8 Antigens ; blood ; Carcinoma, Hepatocellular ; blood ; surgery ; therapy ; Catheter Ablation ; methods ; Female ; Follow-Up Studies ; Hepatectomy ; Humans ; Liver Neoplasms ; blood ; surgery ; therapy ; Male ; Microwaves ; therapeutic use ; Middle Aged ; Neoplasm Recurrence, Local ; RNA, Messenger ; biosynthesis ; genetics ; alpha-Fetoproteins ; biosynthesis ; genetics

OBJECTIVETo probe the effect and mechanism of Compound Glycyrrhizin in treating AIDS.

METHODSForty AIDS patients were randomly divided into a treatment group and a control group, both treated with HAART. In addition, the former was given Compound Glycyrrhizin for 6 months, and the CD4+ T count and the expressions of CD8+ and HLA-DR on the surface of peripheral blood lymphocytes (PBL) were studied before and after the treatment.

RESULTSAfter 6 months of treatment, the expressions of CD8+ and CD38+ of PBL in the treatment group [(6.6 +/- 2.1)%] were found lower than in the control [(11.4 +/- 3.8)%] (t = 5.043, P < 0.01) and CD4+ T count [(243.6 +/- 91.2) x 10(6)/L vs (170.8 +/- 55.7) x 10(6)/L] rose more significantly (t = 3.045, P < 0.01).

CONCLUSIONCompound Glycyrrhizin can lower the expression of active T-lymphocyte subset, inhibit HIV and help immune reconstitution.

ADP-ribosyl Cyclase 1 ; biosynthesis ; Acquired Immunodeficiency Syndrome ; drug therapy ; immunology ; Adolescent ; Adult ; Anti-HIV Agents ; therapeutic use ; Antiretroviral Therapy, Highly Active ; CD4 Lymphocyte Count ; Female ; Glycyrrhizic Acid ; therapeutic use ; HLA-DR Antigens ; biosynthesis ; Humans ; Male ; Middle Aged ; T-Lymphocyte Subsets ; drug effects ; immunology

Blockade of signal 1 or 2 for T-cell activation by the use of anti-CD45RB and anti-CD154 monoclonal antibodies (mAb) (two-signal blockade) has been proven effective in preventing or delaying graft rejection. However, the mechanisms of its immunomodulatory effects are clearly unknown and the present studies were performed to determine how the two-signal blockade modulate allogeneic immune responses, especially T-cell mediated cellular immunity, in a murine skin allograft model. We now report on the profound inhibition of alloreactive T cells by two-signal blockade via CD4-dependent mechanisms. C57BL/6 mice of BALB/c skin allograft were treated with anti-CD45RB, anti-CD154, CTLA4-Ig, or their combinations. For depletion of CD4 or CD8 T cells, the recipients received CD4-depleting or CD8-depleting mAb. We confirmed that survival of skin allograft was markedly prolongated in the two-signal blockade-treated group. In depletion study, anti-CD45RB, anti-CD154 and CD4-depleting mAb-treated group showed acute rejection of skin allograft in contrast to CD8-depleting group treated with the two-signal blockade. In the group treated with the two-signal blockade, the proportions of CD4+CD45RB(low)and CD8+CTLA-4 regulatory T cells were increased while effector CD8+ T cells, including IFN-gamma-secreting and CD8+CD62L(low)T cells, were decreased when compared with non-treated group. In contrast, the CD4-depleted group treated with the two-signal blockade resulted in recovery from immunoregulatory effects of two-signal blockade. In addition, results of IL-4 and IL-10 production were also showed CD4-dependence. Therefore, the two-signal blockade is accompanied by CD4-dependent mechanisms in allogeneic skin transplantation.
Transplantation, Homologous ; T-Lymphocytes, Regulatory/cytology/immunology ; Skin Transplantation/*immunology ; Signal Transduction/drug effects/immunology ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Male ; Lymphocyte Depletion ; Lymphocyte Activation/immunology ; Interleukin-4/biosynthesis ; Interleukin-10/biosynthesis ; Graft Rejection/*immunology/prevention & control ; Flow Cytometry ; Cytotoxicity, Immunologic/immunology ; CD8-Positive T-Lymphocytes/cytology/immunology/metabolism ; CD40 Ligand/*immunology ; CD4-Positive T-Lymphocytes/cytology/immunology/metabolism ; Antigens, CD45/*immunology ; Antigens, CD4/*immunology ; Antibodies, Monoclonal/administration & dosage/*pharmacology ; Antibodies, Blocking/administration & dosage/pharmacology ; Animals

Adult ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Female ; Hepatitis B, Chronic ; immunology ; metabolism ; Humans ; Leukocyte Common Antigens ; biosynthesis ; genetics ; Liver Neoplasms ; immunology ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Middle Aged

BACKGROUNDThe vaccination of mice with DNA encoding single candidate antigens has failed to induce significant protection against Schistosoma japonicum (S. japonicum) challenge infections. In this study, we evaluated the feasibility of using a multivalent DNA vaccine which co-expressed S. japonicum integral membrane protein Sj23 and murine cytokine IL-12 to induce protective immune responses.

METHODSThe plasmid pVIVO2-IL12-Sj23, a eukaryotic expression vector expressing Sj23 and murine IL-12 simultaneously, was constructed, identified, and tested for expression in vitro. Its ability to protect against S. japonicum challenge infections was analyzed according to worm reduction rate and egg reduction rate after vaccination of BALB/c mice. The serum levels of specific IgG antibody were determined by enzyme-linked-immuno sorbent assay (ELISA) and Western blot analysis. Using cultured spleen cells, IFN-gamma and IL-4 post-stimulation were quantified by ELISA. The phenotypes of splenocyte populations were analyzed by flow cytometry (FCM).

RESULTSThe plasmid DNA pVIVO2-IL12-Sj23 was proven to express well in vitro by transient transfection of HEK-293 cells. Immunization resulted in a worm reduction rate of 45.53% and egg reduction rate of 58.35%. ELISA and Western blot analysis indicated that immunized mice generated specific IgG against Sj23. Spleen cells showed significant increases in IFN-gamma but decreases in IL-4. No significant differences in CD4+ and CD8+ subgroup ratios were observed after the challenges.

CONCLUSIONSThe multivalent DNA vaccine pVIVO2-IL12-Sj23 is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infections. Cytokine IL-12, as a gene adjuvant, was able to enhance the Th1 responses and, hence, the protective immunity.

Animals ; Antibodies, Helminth ; blood ; Antigens, Helminth ; genetics ; immunology ; CD4-CD8 Ratio ; Cytokines ; biosynthesis ; Helminth Proteins ; genetics ; immunology ; Interleukin-12 ; genetics ; immunology ; Male ; Membrane Proteins ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Schistosoma japonicum ; immunology ; Th1 Cells ; immunology ; Vaccination ; Vaccines, DNA ; immunology

OBJECTIVETo investigate the effect of mitogen Phorbol 12-myristate 13-Acetate (PMA) on CD3, CD4 and CD8 expression of human T-lymphocytes.

METHODSPeripheral blood mononuclear cells from 37 blood samples stimulated in vitro with PMA at different concentrations (2,5,10,20 and 50 ng/ml for 4 hours) and time (10 ng/ml for 2,4 and 6 hours) were analyzed by 4-color flow cytometry (FCM).

RESULTSUnder different PMA stimulation protocols,significant CD4 down-regulation was observed,which was negatively correlated with intracellular cytokine secretion (r= 0.601,P<.001), except for PMA stimulation at 10 ng/ml for 2-hours which showed no significant intracellular cytokine secretion. The expressions of CD3 and CD8 molecules after PMA activation were not significantly affected as compared with pre-activation. Among CD3 positive T lymphocytes, CD4/CD8 double-negative cells only account for 5.52%.

CONCLUSIONPMA has a significant down-regulation effect on CD4 molecules of Th cells, without altering the CD3 and CD8 expression. For quantitative analysis of Th1/Th2 variation, indirect method such as CD3(+)CD8( ) T cells can be used to define CD4(+) Th cells stimulated by PMA in the future.

CD3 Complex ; blood ; CD4 Antigens ; blood ; CD8 Antigens ; blood ; Child ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; biosynthesis ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; chemistry ; drug effects ; Tetradecanoylphorbol Acetate ; pharmacology ; Th1 Cells ; immunology ; Th2 Cells ; immunology