Genetic Predisposition to Disease ; Humans ; Interleukin-12 Subunit p40 ; physiology ; Receptors, Interferon ; physiology ; Receptors, Interleukin-12 ; physiology ; Toll-Like Receptors ; physiology ; Tuberculosis ; genetics ; immunology

Cytokines ; physiology ; Humans ; Interferon-gamma ; physiology ; Interleukin-1 ; physiology ; Interleukin-10 ; physiology ; Interleukin-12 ; physiology ; Interleukin-6 ; physiology ; Interleukin-8 ; physiology ; Rotavirus Infections ; immunology ; Tumor Necrosis Factor-alpha ; physiology

Cell Survival ; drug effects ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; physiology ; Humans ; Immunophenotyping ; Interleukin-10 ; pharmacology ; Interleukin-12 ; genetics ; secretion ; Interleukin-12 Subunit p35 ; Interleukin-12 Subunit p40 ; Protein Subunits ; genetics ; secretion ; RNA, Messenger ; analysis

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

In this study, we evaluated the biological properties of human mesenchymal stem cells transfected (hMSC) with a plasmid vector expressing human cytokine interleukin-12 (IL-12). Surface markers were analysed by immunophenotyping using flow cytometry. Differentiation capability was evaluated towards adipogenesis and osteogenesis. We demonstrated that successfully transfected hMSC retained their surface immunophenotypes and differentiation potential into adipocytes and osteocytes. These results indicate that hMSC may be a suitable vehicle for gene transduction.
Antigens, Surface/metabolism ; Biological Markers/metabolism ; Bone Marrow Cells/cytology ; Bone Marrow Cells/metabolism ; Cell Differentiation/physiology ; Cells, Cultured ; Flow Cytometry ; Immunophenotyping ; Interleukin-12/genetics ; Interleukin-12/metabolism ; Mesenchymal Stem Cells/*cytology ; Mesenchymal Stem Cells/metabolism ; Transfection

OBJECTIVEGram-negative bacteria-induced multiple organ failure/dysfunction syndrome (MOF/MODS) is one of the leading causes of death through the world. The member of immunoglobulin family programmed death-1 (PD-1) is a negative immune regulator. This study investigated the protective effect of PD-1 as well as the underlying mechanism in LPS-induced endotoxemia.

METHODSTen PD-1(+/+) and ten PD-1 knockout (PD-1(-/-)) mice were injected peritoneally with LPS (10 mg/kg), and the survival was observed within 72 hrs after LPS injection. The other 40 PD-1(+/+) and 40 PD-1(-/-) mice were injected peritoneally with LPS (5 mg/kg). Blood samples were collected before injection and 1.5, 3 and 6 hrs after LPS injection (n=10 each time point). Serum levels of various inflammatory mediators were measured using ELISA.

RESULTSThe survival rate in PD-1(-/-) mice was noticeably lower than that in PD-1(+/+) mice after 10 mg/kg LPS injection. Serum levels of inflammatory mediators TNF-α, IL-1β, IL-12 and IL-17 in PD-1/mice were higher than those in PD-1(+/+) mice after 5 mg/kg LPS injection.

CONCLUSIONSPD-1 can protect mice from LPS-induced endotoxemia probably through its regulation on inflammatory mediator production.

Animals ; Antigens, Surface ; physiology ; Apoptosis Regulatory Proteins ; physiology ; Endotoxemia ; prevention & control ; Female ; Interleukin-12 ; blood ; Interleukin-17 ; blood ; Interleukin-1beta ; blood ; Lipopolysaccharides ; toxicity ; Mice ; Programmed Cell Death 1 Receptor ; Tumor Necrosis Factor-alpha ; blood

OBJECTIVETo evaluate the therapeutic effect and the expression level of a tumor-specific replication-competent adenovirus and a replication-defective adenovirus expression mouse recombinant IL-12 (mIL-12) gene on hepatocellular carcinoma (HCC) in vitro.

METHODSThe cytotoxicity of replication-competent adenovirus with E1B-55 000 attenuated CNHK200-mIL12 and ONYX-015 (dl1520), and replication-defective adenovirus Adv-mIL12 were evaluated by MTT and replication assay in two HCC cell lines (HepG2 and Hep3B) and human normal hepatocyte line (LO2). Western blot and ELISA were used to determine the expression level of mIL-12.

RESULTSCNHK200-mIL12 replicated in HepG2 and Hep3B with an increase of 3,160-fold and 630-fold respectively in 96 h post-infection. CNHK200-mIL12 could kill HepG2 and Hep3B cells at a very low MOI (Multiplicity of Infection) and in short time course (HepG2:MOI = 0.2, on day 4; Hep3B:MOI = 0.005, on day 2), while it had no significant effect on LO2. Furthermore, the expressing level of mIL-12 in CNHK200-mIL12 treated HCC cell lines was much higher than that in Adv-mIL12 treated one (HepG2 101-fold, Hep3B 20-fold respectively).

CONCLUSIONReplication-competent adenovirus is more effective than replication-defective adenovirus in both cytotoxicity and efficiency of gene transfer in HCC, and holds great promise in the area of HCC therapy.

Adenoviridae ; genetics ; physiology ; Carcinoma, Hepatocellular ; pathology ; therapy ; Cell Line, Tumor ; Cell Survival ; Genetic Therapy ; Genetic Vectors ; Humans ; Interleukin-12 ; biosynthesis ; genetics ; Liver Neoplasms ; pathology ; therapy ; Virus Replication

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

The purpose of this study was to investigate clinical and immunological responses to Demodex on the ocular surface. Thirteen eyes in 10 patients with Demodex blepharitis and chronic ocular surface disorders were included in this study and treated by lid scrubbing with tea tree oil for the eradication of Demodex. We evaluated ocular surface manifestations and Demodex counts, and analyzed IL-1beta, IL-5, IL-7, IL-12, IL-13, IL-17, granulocyte colony-stimulating factor, and macrophage inflammatory protein-1beta in tear samples before and after the treatment. All patients exhibited ocular surface manifestations including corneal nodular opacity, peripheral corneal vascularization, refractory corneal erosion and infiltration, or chronic conjunctival inflammatory signs before treatment. After treatment, Demodex was nearly eradicated, tear concentrations of IL-1beta and IL-17 were significantly reduced and substantial clinical improvement was observed in all patients. In conclusion, we believe that Demodex plays an aggravating role in inflammatory ocular surface disorders.
Acari/drug effects/physiology ; Adolescent ; Adult ; Aged ; Animals ; Blepharitis/drug therapy/*immunology/parasitology ; Chemokine CCL4/analysis ; Female ; Granulocyte Colony-Stimulating Factor/analysis ; Humans ; Interleukin-12/analysis ; Interleukin-13/analysis ; Interleukin-17/analysis ; Interleukin-1beta/analysis ; Interleukin-5/analysis ; Interleukin-7/analysis ; Male ; Middle Aged ; Tea Tree Oil/therapeutic use ; Tears/metabolism