OBJECTIVETo observe the regulating effect of hepatitis C virus (HCV) envelop (E) 2 gene immunization-induced immune responses by adenovirus mediated interleukin 12 (IL-12).

METHODSHCV E2 protein was expressed and purified from NIH 3T3 and then used as an antigen to detect antibodies against HCV E2. With 51Cr release, SP2/0 expressing HCV E2 was used as target cell to detect specific cytotoxic T lymphocytes (CTL) response; adenovirus recombined IL-12 was propagated by 293 cell. HCV E2 recombinant and adenovirus recombined IL-12 were injected into the quadriceps femoris muscles and abdominal cavities of 6-8 weeks old BALB/C mice. Sera were collected at 2, 3, and 4 weeks and detected for antibodies for E2. Spleen cells isolated at 4 weeks were analyzed for specific CTL response.

RESULTSIt was found that expression of IL-12 at an undetectable level did enhance HCV E2 gene immunization-induced CTL activity and there was no effect on its hormonal immune response.

CONCLUSIONUsing adenovirus to express interleukin 12 was helpful for regulation of HCV E2 gene immunization-induced immune response. Combined HCV E2 and IL-12 can render a strong anti-HCV CTL activity and may be of use in the development of HCV gene vaccine in the future.

Adenoviridae ; physiology ; Interleukin-12 ; biosynthesis ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; Viral Envelope Proteins ; genetics ; immunology

OBJECTIVETo study the impact of an agonist anti-CD(40) monoclonal antibody 5C11 on the induction and biological characteristics of leukemic dendritic cells.

METHODSCombinations of 5C11 and different cytokines were used to induce differentiation of leukemic blasts into dendritic cells. Morphology was observed by light microscopy. Surface antigens of the induced cells were analyzed by fluorescence-activated cell sorting (FACS), the yields of dendritic cell by cell counting, the levels of IL-6 and IL-12 by ELISA, T cell proliferating activity by allo-mixed lymphocyte reaction (MLR) in vitro. Allogeneic T cells were stimulated with leukemic dendritic cells and T-cell cytotoxicity was measured by MTT assay.

RESULTSWhen cultured with combinations of 5C11 and different cytokines, the leukemic cells isolated from the patients could differentiate into dendritic cells. The morphology showed typical features of dendritic cells, which expressed high levels of CD(40), CD(80) and CD(86). In comparison with the original leukemia cells, the leukemic dendritic cells secreted less IL-6 but more IL-12 (P < 0.05). The leukemic dendritic cells were potent to stimulate the proliferation of allogeneic T cells, and the latter was able to lyse the original leukemia cells.

CONCLUSIONLeukemic blasts could be induced to differentiate into functional dendritic cells. It may be of great value in the adoptive immunologic therapy of leukemia.

Antibodies, Monoclonal ; immunology ; CD40 Antigens ; physiology ; Cell Differentiation ; Dendritic Cells ; immunology ; Humans ; Immunophenotyping ; Immunotherapy ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Leukemia ; immunology ; pathology ; therapy

Adolescent ; Adult ; Cells, Cultured ; Dendritic Cells ; immunology ; Female ; Heparin ; pharmacology ; Hepatitis B, Chronic ; immunology ; Humans ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Male ; Middle Aged ; Phenotype

Bacille Calmette-Guerin (BCG) induces potent Th1 responses with the help of interleukin (IL)-10 and IL-12 released from dendritic cells (DCs), and suppresses Th2- associated allergic reactions. However, there are still some controversies on therapeutic effects of BCG in asthmatics. This study investigated whether BCG administration to DCs suppresses IL-5 production from T cells in atopic asthmatics. DCs derived from peripheral blood of subjects were cultured with or without BCG and Dermatophagoides farinae extract. Some DCs were co-cultured with T cells in the presence of BCG or the above culture supernatants. In the atopic asthmatics, BCG significantly increased IL-10 and IL-12 production from DCs. In the presence of D. farinae extract, BCG further increased IL-10 production. BCG-induced IL-10 production was significantly higher in the atopics (n=14) than in the non-atopics (n=9). Both BCG and the BCG-treated DCs culture supernatant significantly increased IFN-gamma production from T cells. Both BCG and the supernatant from DCs+BCG+D. farinae co-cultures significantly decreased IL-5 production (all p<0.05), but the supernatant from DCs+BCG co-cultures did not. In conclusion, administration of BCG together with D. farinae extract effectively decreased IL-5 production from T cells, probably through the action of IL-10 and IL-12 released from DCs in D. farinaesensitive asthmatics.
Adult ; Asthma/*immunology ; BCG Vaccine/*immunology ; Dendritic Cells/*immunology ; Female ; Humans ; Interferon-gamma/biosynthesis ; Interleukin-10/biosynthesis ; Interleukin-12/biosynthesis ; Interleukin-5/*biosynthesis ; Lymphocyte Activation ; Male ; T-Lymphocytes/*immunology

OBJECTIVETo study the effects of Lipopolysaccharide (LPS) on the maturation and secretion of human peripheral dendritic cells (DCs).

METHODSDCs from healthy human peripheral monocytes (PBMCs) were induced in vitro with rhGM-CSF, rhIL-4, Flt3-L and TNFalpha. The subjects were divided into 3 groups: the long-term group stimulated with LPS 1 microg/ml at day 1, 4, 7, 9 post culture; the short-term group stimulated with LPS 1 microg/ml at day 7 and 8 post culture, and the DCs without LPS stimulation was control group. After 10 days of culture, the morphologic features of DCs were observed by light and electron microscopes, the phenotypic patterns were characterized by flow cytometry, the proliferation of T cell were evaluated with mixed leukocytes reaction (MLR) and the levels of IL-12 and IFNgamma produced by DCs were analyzed with ELISA.

RESULTSCompared with the short-term group, the expressions of HLA-DR (65.81%+/-10.96%), CD86 (48.81%+/-18.13%), CD80 (13.56%+/-5.48%), CD83 (11.52%+/-5.09%), the secretions of IFNgamma(15.60+/-5.83 pg/ml) and IL-12 (51.77+/-11.02 pg/ml) by the DCs in long-term group were decreased obviously (P is less than 0.05) and the proliferation of homogenic lymphocyte cells (1.548+/-0.365) stimulated by DCs was also impaired (P < 0.05).

CONCLUSIONLong-term LPS stimulation can suppress the maturation and secretion of DCs, which might be the reason of poor immunity in the patients with intestinal endotoxemia.

Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Interleukin-12 ; biosynthesis ; Lipopolysaccharides ; pharmacology ; Monocytes ; cytology ; metabolism

OBJECTIVESevere pneumonia is one of the common severe diseases in children. Increasing evidences show that immune response greatly contribute to severe pneumonia. Dendritic cells (DC) are the important antigen presenting cells in the lung. To study the role of dendritic cells in development of severe pneumonia in children, the authors measured the number of mature DC in bronchoalveolar lavage fluid (BALF), and evaluated the relationship among IL-12, pro-inflammatory cytokines and clinical scores.

METHODSThe following 3 groups of children were enrolled in this study: severe pneumonia group: 27 children with severe pneumonia treated between November 2002 and May 2003 in PICU; mild pneumonia group: 30 children with mild pneumonia in department of pulmonology; control group: 29 children without pneumonia but receiving ventilator treatment for chest surgery. Mature DC in BALF was determined in severe pneumonia group and the control group on the day of tracheal intubation for mechanical ventilation. Acute lung injury scores and severe disease scores were evaluated in children with severe pneumonia and mild pneumonia. All children's serum levels of TNF-alpha, IL-6 and IL-12 were measured by using ELISA within 24 hours after admission. SPSS version 11.5 was used for statistical analysis.

RESULTS(1) The percent of mature DC in children with severe pneumonia was significantly higher when compared with the control group on the first day after ventilation [14.2 (3.9 - 51.8)] vs. [1.3 (0.2 - 22.5)] (Z = 5.44, P < 0.01). (2) In severe pneumonia group, the concentration of serum IL-12 [117.0 (79.9 - 159.4) ng/L], TNF-alpha [90.6 (52.2 - 185.9) ng/L], IL-6 [128.7 (73.3 - 793.8) ng/L] were significantly higher than those in mild pneumonia group where the values were [71.6 (19.4 - 196.8)], [26.6 (2.5 - 113.9)], and [39.9 (7.8 - 82.5)] (P < 0.01), and the control group [6.4 (12.2 - 92.0)], [6.4 (1.8 - 91.9)], and [23.0 (6.4 - 54.2)] (P < 0.01). Serum IL-12, TNF-alpha and IL-6 levels in children with mild pneumonia were higher than those of control group (P < 0.01). (3) The percent of mature DC was increased with the serum level of IL-12 (r = 0.48, P < 0.01), TNF-alpha (r = 0.58, P < 0.01), IL-6 (r = 0.51, P < 0.01) and lung injury scores (r = 0.39, P < 0.05), but it did not correlate with severe disease scores (r = -0.11, P > 0.05).

CONCLUSIONSThere is excessive production of pro-inflammatory cytokines and over-stimulation of lung dendritic cells in children with severe pneumonia. Over-stimulation of lung dendritic cells, the increased serum levels of IL-12, TNF-alpha, IL-6 and the severity of pneumonia may suggest that DC plays an important role in pathogenesis of severe pneumonia in children.

Bronchoalveolar Lavage Fluid ; cytology ; Child, Preschool ; Cytokines ; biosynthesis ; blood ; Dendritic Cells ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Infant ; Interleukin-12 ; biosynthesis ; Interleukin-6 ; biosynthesis ; Male ; Pneumonia ; immunology ; pathology ; physiopathology ; Severity of Illness Index ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo clone the p40 and p35 subunit cDNA of porcine IL-12(pIL-12) and construct the fusion gene of recombinant porcine single-chain interleukin-12 (pscIL-12).

METHODSThe total RNAs were extracted from porcine peripheral blood mononuclear cells (PBMCs) and porcine splenic lymphocytes for cloning pIL-12 p35 and p40 cDNA by RT-PCR. A hydrophobic polypeptide linker (Gly4Ser)3 was used for splicing two different gene fragments (pIL-12) p40+linker+p35 (pscIL-12) by recombinant PCR to construct pscIL-12 fusion gene. The pscIL-12 fusion gene was then inserted into pcDNA3.1(+) eukaryotic expression plasmid, and the resulted pcDNA3.1(+)-pscIL-12 was transfected into CHO-K1 cells via lipofectin. The expression of pscIL-12 mRNA in the transfected cells was identified by RT-PCR.

RESULTSThe sequence of the cloned porcine IL-12 p40 and p35 cDNA and the constructed pscIL-12 fusion gene were verified by PCR, restriction enzyme digestion and sequencing. The mRNA of pscIL-12 fusion gene was detected in the transfected CHO-K1 cells by RT-PCR.

CONCLUSIONThe constructed pcDNA3.1(+)-pscIL-12 eukaryotic expression plasmid allows expression of pscIL-12 in CHO-K1 cells, thus facilitating further study of the biological activity and adjuvant effect of pscIL-12 fusion protein.

Animals ; Base Sequence ; CHO Cells ; Cloning, Molecular ; Cricetinae ; DNA, Complementary ; genetics ; Interleukin-12 ; biosynthesis ; classification ; genetics ; Molecular Sequence Data ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Swine

OBJECTIVETo observe the functional changes of dendritic cells (DCs) after infection by recombinant retrovirus carrying human telomerase reverse transcriptase (hTERT) gene fragment.

METHODSInterleukin-12 (IL-12) levels in DC culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA). The abilities of DCs infected with recombinant retrovirus carrying hTERT gene (hTERT-DCs) and non-infected DCs (N-DCs) to stimulate allogeneic lymphocyte proliferation were evaluated with mixed leukocytes reaction (MLR), and the surface markers of DCs including CD80, CD83, CD86 and HLA-DR were detected by flow cytometry. Cytotoxic T lymphocyte (CTL) assay was performed with CytoTox 96 non-radioactive cytoxicity assay.

RESULTSCompared with N-DCs, hTERT-DCs showed no significant changes in IL-12 secretion and capacity to stimulate allogeneic lymphocytes reaction, but had significantly lower CD83 expression. Specific CTLs induced by hTERT-DCs resulted in higher cytotoxicity against telomerase-positive target cells than that against the negative target cells.

CONCLUSIONInfection with the recombinant retrovirus carrying hTERT fragment may jeopardize the maturation of DCs, which, however, still retain their capacity to activate and stimulate lymphocyte proliferation and to prime autologous T lymphocytes to generate specific CTL against hTERT.

Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; virology ; Genetic Vectors ; Humans ; Interleukin-12 ; biosynthesis ; Recombination, Genetic ; Retroviridae ; genetics ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Telomerase ; biosynthesis ; genetics

The relationship between T cell immunoglobulin domain and mucin domain protein 3 (Tim-3)/Galectin (Gal)-9 pathway and recurrent spontaneous abortion (RSA) was studied. Thirty-one pregnant women with RSA and 27 normal early gravidas were investigated to detect the levels of Tim-3 and Gal-9 in villi and deciduas by Western blotting. Meanwhile, the concentration of interleukin (IL)-4 and IL-12 in peripheral blood plasma was determined by ELISA in 25 healthy fertile non-pregnant controls, the normal early gravidas and pregnant women with RSA mentioned above, respectively. It was found that the relative expression levels of Tim-3 and Gal-9 in villi and deciduas were significantly increased in pregnant women with RSA as compared with those in the normal early gravidas. The concentration of IL-4 in peripheral blood plasma of pregnant women with RSA was lower than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05), but that of IL-2 in pregnant women with RSA was significantly higher than that of the normal early gravidas (P<0.05) and healthy fertile non-pregnant controls (P<0.05). It was suggested that the overexpression of Tim-3/Gal-9 pathway may be related to the pathogenesis of RSA.
Abortion, Spontaneous ; metabolism ; pathology ; Adolescent ; Adult ; Chorionic Villi ; metabolism ; pathology ; Female ; Galectins ; biosynthesis ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Interleukin-12 ; blood ; Interleukin-4 ; blood ; Membrane Proteins ; biosynthesis ; Pregnancy ; Pregnancy Proteins ; biosynthesis ; Up-Regulation

OBJECTIVETo Explore a two-step culture system to generate a large number of dendritic cells (DC) differentiated from cord blood (CB) CD(34)(+) cells.

METHODSEnriched CB CD(34)(+) cells with immunoadsorption were primarily cultured in the presence of stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (Tpo) and interleukin-3 (IL-3) for 7 (group I), 10 (group II) or 14 days (group III) respectively, and then further cultured with GM-CSF, IL-4 and TNF-alpha for 5 - 8 days to induce DC. The expansion and cell function were evaluated by flow cytometry (FCM) and mix-lymphocyte reaction (MLR), and detection of IL-12 in the supernatant by using ELISA.

RESULTSThe total nucleated cells with 53.39 +/- 20.59-, 307.17 +/- 119.59- and 1117.25 +/- 335.49-folds expansion could be respectively obtained after 7 - 14 days of expansion culture. After DC induction, CD(1a)(+) cells were 21.40 +/- 16.70-, 143.2 +/- 60.35- and 150.8 +/- 42.16-fold increase as compared to the initial nucleated cells. Comparing with that in group I, the CD(1a)(+) cells were much more in groups II and III; but there was no difference between the latter two groups (P > 0.05). The cultured cells in the three groups showed almost the same allo-stimulatory capability and IL-12 excretion when the second culture duration maintained 8 days, while the capability and excretion were greatly decreased when the duration shortened to 5 days (P < 0.05).

CONCLUSIONA plenty of functionally mature DC could be obtained from the CD(34)(+) cells in the two-step culture system of 7 - 10 days HSC expansion followed by 8 days DC induction.

Antigens, CD34 ; analysis ; Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; physiology ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; cytology ; Humans ; Interleukin-12 ; biosynthesis ; Lymphocyte Activation