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

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

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

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

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 study the changes of IL-12 produced by dendritic cells in peripheral blood in children with Henoch-Schonlein purpura (HSP), and to explore its influence on TH1/TH2 balance in order to elucidate its significance in the pathogenesis of HSP.

METHODSThe levels of interferon-gamma (IFN-gamma), interleukin-4 (IL-4) and interleukin-12 (IL-12) in plasma were determined by ELISA in 60 HSP children (HSP group) and 21 healthy children (Control group). Peripheral blood mononuclear cells (PBMC) of 22 HSP patients and 21 healthy children were cultured in vitro and then were transformed into dendritic cells. The levels of IL-12 in the supernatant were detected by ELISA and the positive expression rate of CD1a(+) was detected by indirect immunofluorescence procedure.

RESULTS1) The levels of IFN-gamma and the ratio of IFN-gamma/IL-4 in plasma of the HSP group were lower than those of the Control group (IFN-gamma 30.59 +/- 11.27 pg/mL vs 43.38 +/- 19.19 pg/mL; IFN-gamma/IL-4 ratio 0.70 +/- 0.28 vs 1.33 +/- 0.57) (P < 0.01). The levels of IL-12 in the HSP group were also lower than those of the Control group (153.95 +/- 91.88 pg/mL vs 323.06 +/- 162.34 pg/mL; P < 0.01). In contrast, the levels of IL-4 were higher than those of the Control group (45.08 +/- 9.19 pg/mL vs 32.95 +/- 7.10 pg/mL; P < 0.01). The plasma levels of IL-12 positively correlated with the IFN-gamma levels (r=0.52, P < 0.01) and the ratio of IFN-gamma/IL-4 (r=0.43, P < 0.01) in the HSP group. 2) The IL-12 levels in the supernatant of the HSP group were lower than those of the Control group (357.06 +/- 153.56 pg/mL vs 489.80 +/- 213.45 pg/mL; P < 0.05), and had a positive correlation with the plasma IL-12 levels (r=0.74, P < 0.01). 3) The positive expression rate of CD1a(+) of the HSP group was lower than that of the Control group [(27.42 +/- 10.75)% vs (35.68 +/- 12.18)%; P < 0.05], and positively correlated with the IL-12 levels in the supernatants (r=0.57, P < 0.01) and in plasma (r=0.68, P < 0.01).

CONCLUSIONSThere was an imbalance of TH1/TH2 in HSP children. The decrease of TH1 function had a positive correlation with the low levels of IL-12 in plasma, while the latter correlated closely with decreased number and / or function of dendritic cells, suggesting that the decreased number and / or function of dendritic cells in peripheral blood resulted in the imbalance of TH1/TH2 indirectly.

Adolescent ; Antigens, CD1 ; analysis ; Cells, Cultured ; Child ; Child, Preschool ; Dendritic Cells ; immunology ; Female ; Humans ; Interferon-gamma ; blood ; Interleukin-12 ; biosynthesis ; blood ; Interleukin-4 ; blood ; Male ; Purpura, Schoenlein-Henoch ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology

BACKGROUNDThe heavy incidence and mortality of hemorrhagic fever with renal syndrome, as well as no specific drugs in curing the disease, clearly indicate the need for development of the more effective hantavirus vaccine. Refining the DNA vaccination strategy to elicit more clinically efficacious immune responses is now under intensive investigation. In the present study, we examined the effects of using an interleukin-12 expression plasmid as a genetic adjuvant to enhance the immune responses induced by a DNA vaccine based on the S gene encoding nucleocapsid protein against hantavirus.

METHODSBALB/c mice were immunized three times by intramuscular inoculations of DNA vaccine encoding of hantanvirus nucleocapsid protein alone or in combination with a plasmid expressing murine interleukin-12 (pcIL-12). Booster immunizations were employed 2 times at 2-week interval. To evaluate the humoral and cellular immune responses, antigen-specific lymphocyte proliferation and antibody production were assayed by MTT method and ELISA respectively. The level of interleukin-4 and interferon-gamma in the splenic lymphocytic cultured supernatant were detected with ELISA kit at day 5, 10, 17, 35 and 42 after primary immunization.

RESULTSAntigen-specific IgG antibodies was increased markedly at day 17 in the experiment groups and reached a plateau after day 35. As pcIL-12 co-injected, a significant inhibition of antigen-specific IgG levels was displayed over the period and the antibody mean titre was decreased to only about 1:50 at day 42 after primary immunization, significantly lower than the group immunized with pcDNA3.1 + S alone, in which the mean titre was about 1:70. Interferon-gamma was increased remarkably by the co-injection of pcIL-12 compared with the injection of pcDNA3.1 + S alone. However, the production of interleukin-4 was inhibited by pcIL-12 co-injection. Furthermore, pcIL-12 co-injection efficiently enhanced antigen-specific lymphocyte proliferation.

CONCLUSIONHumoral and cytokine responses elicited by pcDNA3.1 + S inoculation can be modulated by co-inoculation with pcIL-12 and efficiently induced Th1-dominant immune responses.

Animals ; Cytokines ; biosynthesis ; Genetic Therapy ; Hantavirus ; immunology ; Immunoglobulin G ; blood ; Immunophenotyping ; Interleukin-12 ; genetics ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred BALB C ; Nucleocapsid ; genetics ; immunology ; Vaccines, DNA ; immunology ; Viral Vaccines ; immunology

Immunization with dendritic cells (DCs) pulsed with tumor antigen can activate tumor-specific cytotoxic T lymphocytes (CTL), which is responsible for tumor protection and regression. In this study, we examined whether DCs pulsed with necrotic tumor lysates can efficiently prevent malignant melanoma tumor cell metastasis to the lung. DCs derived from mouse bone marrow were found to produce remarkably elevated levels of IL-12 after being pulsed with the tumor lysates. Moreover, immunization with these DCs induced CTL activation and protected mice from metastasis development by intravenously inoculated tumor cells. In addition, these DCs activated NK cells in vitro in a contact-dependent manner, and induced NK activities in vivo. Furthermore, NK cell depletion before DC vaccination significantly reduced the tumor-specific CTL activity, IFN-g production, and IFN-gamma- inducible gene expression, and eventually interfered with the antitumor effect of tumor-pulsed DCs. Finally, similar findings with respect to NK cell dependency were obtained in the C57BL/ 6J-bg/bg mice, which have severe deficiency in cytolytic activity of NK cells. These data suggest that the antitumor effect elicited by DC vaccination, at least in a B16 melanoma model, requires the participation of both cytolytic NK and CD8+ T cells. The findings of this study would provide important data for the effective design of DC vaccines for cancer immunotherapy.
Animals ; Antigen Presentation/immunology ; CD8-Positive T-Lymphocytes/immunology ; Cancer Vaccines/*therapeutic use ; Cell Line, Tumor ; Cytokines/biosynthesis/immunology ; Dendritic Cells/immunology/*transplantation ; Female ; Interferon Type II/biosynthesis/immunology ; Interleukin-12/biosynthesis/immunology ; Killer Cells, Natural/*immunology ; Lung Neoplasms/immunology/prevention & control/secondary ; Lymphocyte Activation/immunology ; Lymphocyte Depletion ; Melanoma, Experimental/immunology/secondary/*therapy ; Mice ; Mice, Inbred C57BL ; Monocyte Chemoattractant Proteins/biosynthesis/immunology ; Research Support, Non-U.S. Gov't ; T-Lymphocytes, Cytotoxic/immunology

Animals ; Coculture Techniques ; Colonic Neoplasms ; pathology ; therapy ; Cytokines ; pharmacology ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; Female ; Immunophenotyping ; Immunotherapy, Adoptive ; Interferon-gamma ; biosynthesis ; Interleukin-12 ; biosynthesis ; Killer Cells, Natural ; immunology ; Lung Neoplasms ; prevention & control ; secondary ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C