OBJECTIVETo explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) of vascular endothelial cells.

METHODSTryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-alpha in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively.

RESULTSIL-6 and TNF-alpha mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-alpha protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group.

CONCLUSIONReduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-alpha in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.

Animals ; Cell Line ; Culture Media, Conditioned ; Down-Regulation ; genetics ; Endothelial Cells ; enzymology ; secretion ; Gene Expression Regulation, Enzymologic ; Interleukin-6 ; genetics ; secretion ; Mice ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Transgenes ; Tryptases ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; secretion

OBJECTIVETo study whether the protective effects of glutamine against intestinal injury are associated with the regulation of intestinal secretion of NF-κB and TNF-α by glutamine.

METHODSTwenty-four 10-day-old Wistar rat pups were randomly intraperitoneally injected with normal sodium of 1 mL/kg (control group; n=8), LPS of 5 mg/kg (endotoxemia group; n=8) or LPS of 5 mg/kg plus glutamine of 10 mL/kg (glutamine group; n=8). The rats were sacrificed 3 hrs after injection. A segment of distal ileum was dissected. The pathologic changes of the small intestine were observed under an optical microscope (hematoxylin-eosin staining). The expression of intestinal NF-κB mRNA and protein were detected by RT-PCR and Western blot respectively. The TNF-α level in intestinal tissues was measured using ELISA.

RESULTSThe structure of the small intestine in the control group remained normal. The inflammation cells infiltration and the edema of interstitial substance and epithelials were observed in the endotoxemia and glutamine groups. The pathologic changes in the glutamine group were significantly alleviated when compared with the endotoxemia group. The expression of NF-κB mRNA and protein in the glutamine group was significantly lower than that in the endotoxemia group (P<0.01). The secretion of TNF-&alpha; in the glutamine group was significantly lower than that in the endotoxemia group (P<0.01).

CONCLUSIONSThe protective effects of glutamine against intestinal injury may be associated with the down-regulation of intestinal NF-κB mRNA and protein expression and the reduction of the secretion of TNF-&alpha;.

Animals ; Endotoxemia ; metabolism ; Female ; Glutamine ; pharmacology ; Intestines ; drug effects ; metabolism ; Male ; NF-kappa B ; analysis ; genetics ; secretion ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes.

METHODS3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA.

RESULTSNYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group.

CONCLUSIONSNYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.

3T3-L1 Cells ; Adipocytes ; drug effects ; secretion ; Adiponectin ; secretion ; Animals ; Carrier Proteins ; genetics ; physiology ; Glucose ; metabolism ; Glucose Transporter Type 4 ; analysis ; metabolism ; Insulin ; pharmacology ; Interleukin-6 ; secretion ; Mice ; Resistin ; analysis ; Transfection ; Tumor Necrosis Factor-alpha ; secretion

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.
Animals ; Enterotoxins ; genetics ; immunology ; Female ; Interferon-gamma ; secretion ; Interleukin-2 ; secretion ; Lymphocyte Activation ; Mice ; Mice, Inbred BALB C ; Mutant Proteins ; genetics ; immunology ; Receptors, Antigen, T-Cell ; immunology ; Superantigens ; genetics ; immunology ; Tumor Necrosis Factor-alpha ; secretion

The human telomerase reverse transcriptase (hTERT) promoter can be used for the tumor-specific expression of transgenes in order to induce selective cancer cell death. The hTERT core promoter is active in cancer cells but not in normal cells. To examine whether the combination of TNF-related apoptosis inducing ligand (TRAIL) treatment and cancer cell-selective expression of the TRAIL-death receptor could induce cell death in TRAIL-resistant cancer cells, we generated a death receptor-4 (DR4)-expressing adenovirus (Ad-hTERT-DR4), in which the expression of DR4 is driven by the hTERT promoter. Upon infection, DR4 expression was slightly increased in cancer cell lines, and cell death was observed in TRAIL-resistant cancer cell lines but not in normal human cells when DR4 infection was combined with TRAIL treatment. We also generated an adenovirus that expresses a secretable isoleucine zipper (ILZ)-fused, extracellular portion of TRAIL (Ad-ILZ-TRAIL). In cells infected with Ad-ILZ-TRAIL, TRAIL was expressed, secreted, oligomerized and biologically active in the induction of apoptosis in TRAIL-sensitive cancer cells. When Ad-hTERT-DR4 infected TRAIL-resistant HCE4 cells and Ad-ILZ-TRAIL infected TRAIL-resistant HCE7 cells were co-cultured, cell deaths were evident 24 h after co-culture. Taken together, our results reveal that the combination of TRAIL and cancer cell-specific expression of DR4 has the potential to overcome the resistance of cancer cells to TRAIL without inducing significant cell death in normal cells.
Tumor Necrosis Factor-alpha/genetics/*pharmacology/secretion ; Telomerase/genetics ; TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor/genetics/*metabolism ; Promoter Regions (Genetics) ; Neoplasms/genetics/metabolism/pathology ; Membrane Glycoproteins/genetics/*pharmacology/secretion ; Humans ; Drug Resistance, Neoplasm ; DNA-Binding Proteins/genetics ; Cell Line ; Apoptosis Regulatory Proteins/genetics/*pharmacology/secretion ; Apoptosis/*drug effects ; Antineoplastic Agents/*pharmacology ; Adenoviridae/genetics

BACKGROUNDIslet transplantation represents an ideal therapeutic approach for treatment of type 1 diabetes but islet function and regeneration may be influenced by necrosis or apoptosis induced by oxidative stress and other insults. Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme into biliverdin, releasing free iron and carbon monoxide. It has also been reported to be an antioxidant enzyme which can improve the function of grafted islets by cytoprotection via free radical scavenging and apoptosis prevention. In the present study, we investigated whether transduction of HO-1 genes into human islets with an adenovirus vector has cytoprotective action on islets cultured in vitro and discuss this method of gene therapy for clinical islet transplantation.

METHODSCadaveric pancreatic islets were isolated and purified in vitro. Transduction efficiency of islets was determined by infecting islets with adenovirus vector containing the enhanced green fluorescent protein gene (Ad-EGFP) at multiplicities of infection (MOI) of 2, 5, 10, or 20. Newly isolated islets were divided into three groups: EGFP group, islets transduced with Ad-EGFP using MOI = 20; HO-1 group, transduced with adenovirus vectors containing the human HO-1 gene using MOI = 20; and control group, mock transduced islets. Insulin release after glucose stimulation of the cell lines was determined by a radioimmunoassay kit and the stimulation index was calculated. Flow cytometry was used to detect apoptotic cells in the HO-1 group and in the control group after induction by recombinant human tumor necrosis factor-alpha (rTNFalpha) and cycloheximide (CHX) for 48 hours.

RESULTSAdenovirus vectors have a high efficiency of gene transduction into adult islet cells. Transduction of islets with the Ad-EGFP was most successful at MOI 20, at which MOI fluorescence was very intense on day 7 after transduction and EGFP was expressed in cultured islet cells for more than four weeks in vitro. The insulin release in the control group was (182.36 +/- 58.96) mIU/L after stimulation by high glucose media (16.7 mmol/L), while insulin release from the HO-1 group and the EGFP group were (270.09 +/- 89.37) mIU/L and (175.95 +/- 75.05) mIU/L respectively. Compared to the control group and the EGFP group, insulin release in the HO-1 group increased significantly (P < 0.05). After treatment with rTNFalpha and CHX the apoptotic ratio of islet cells was (63.09 +/- 10.86)% in the HO-1 group, significantly lower than (90.86 +/- 11.25)% in the control group (P < 0.05).

CONCLUSIONSTransduction of human islets with Ad-HO-1 can protect against TNF-alpha and CHX mediated cytotoxicity. The HO-1 gene also appears to facilitate insulin release from human islets. Transduction of donor islets with the adenovirus vector containing an HO-1 gene might have potential value in clinical islet transplantation.

Adenoviridae ; genetics ; Apoptosis ; drug effects ; Cycloheximide ; pharmacology ; Cytoprotection ; Genetic Therapy ; Heme Oxygenase-1 ; genetics ; physiology ; Humans ; Insulin ; secretion ; Islets of Langerhans ; physiology ; Transduction, Genetic ; Tumor Necrosis Factor-alpha ; pharmacology

To compare the anti-tumor effects of transmembrane TNF-alpha (TM-TNF) and secreted TNF-alpha (S-TNF) in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-alpha (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mutant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-alpha gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-alpha or its mutants and effectively kill H22 in vitro. The transfected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5:1 or 1:1 (effector/target cells, E/T) after the third day of H22 challenge, respectively. At the E/T = 5:1, the NIH3T3/TM-TNFm induced the highest tumor regression, while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T = 1:1 in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.
3T3 Cells ; Adenoviruses, Human ; genetics ; Animals ; Cytotoxicity, Immunologic ; immunology ; Fibroblasts ; cytology ; immunology ; Liver Neoplasms, Experimental ; immunology ; pathology ; Membrane Proteins ; secretion ; Mice ; Mutation ; Transfection ; Tumor Cells, Cultured ; Tumor Necrosis Factor-alpha ; genetics ; immunology ; secretion

Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; genetics ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Humans ; Mucin 5AC ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Respiratory System ; cytology ; Tumor Necrosis Factor-alpha ; pharmacology ; Up-Regulation ; drug effects

OBJECTIVETo investigate the effect of ADMA on macrophage migration inhibitory factor (MIF) expression and tumor necrosis factor-&alpha; (TNF-&alpha;) and IL-8 secretion in THP-1 monocyte-derived macrophages. METHIDS: THP-1 monocytes were induced to differentiate into macrophages by a 24-h incubation with 160 nmol/L PMA. The THP-1 monocyte-derived macrophages were exposed to different concentrations of ADMA for 24 h, and the changes in MIF mRNA and protein expressions were analyzed with RT-PCR and Western blotting, respectively. Enzyme-linked immunosorbent assay was used to detect the levels of TNF-&alpha; and IL-8 in the supernatant of THP-1-derived macrophages following ADMA treatments.

RESULTSADMA obviously up-regulated MIF mRNA and protein expressions in THP-1-derived macrophages in a concentration- dependent manner. Exposure of the cells to 15 µmol/L ADMA for 24 h showed the most potent effect in up-regulating MIF mRNA and protein expressions. ADMA treatment also resulted in a dose-dependent increase of the levels of TNF-&alpha; and IL-8 in the culture supernatant of the macrophages, and the peak levels occurred following the treatment with 15 µmol/L ADMA.

CONCLUSIONADMA can up-regulate MIF expression and induce TNF-&alpha; and IL-8 secretion in THP-1 monocyte-derived macrophages.

Arginine ; analogs & derivatives ; pharmacology ; Cell Differentiation ; Cell Line ; Humans ; Interleukin-8 ; secretion ; Intramolecular Oxidoreductases ; genetics ; metabolism ; Macrophage Migration-Inhibitory Factors ; genetics ; metabolism ; Macrophages ; cytology ; metabolism ; Monocytes ; cytology ; Phenanthrenes ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; secretion

OBJECTIVETo investigate the effects of dendritic cells (DCs) transfected with survivin gene, and to observe the effective and specific anti-tumor immunological effect induced by modified DC in vitro.

METHODSSurvivin gene was transfected to DCs with liposomes. Survivin expression could be detected both in DCs cells and in cell culture with method of Western blot. Cytokines as well as cellular surface molecule such as IL-12, TNF-alpha, CD1 alpha, CD83, MHCII, CD80 and CD86 were detected. The competence of inducing human specific cytotoxic T lymphocyte (CTLs) was also detected with MTT.

RESULTSSurvivin expression could be detected both in DCs which were transfected with survivin cDNA and in cell culture superior. The IL-12 and TNF-alpha level was (265.2 +/- 32.7), (437.1 +/- 83.5) pg/ml, and much higher in transgened DC cells than blank DC cells (P < 0.05). CD1 alpha, CD83, MHCII, CD80 and CD86 was high expressed in survivin-DC cells, however, it was low expressed in blank DC cells. The lyse rate to gastric cancer cell, colon cancer cell and bile duct cancer cell was 65%, 77%, and 85% respectively, and these were much higher than those of blank DC cells.

CONCLUSIONSDCs transfected with survivin gene could induce specific cytotoxic T lymphocytes and strikingly raised DC cell's antigen present function, and have specific CTL killing activity.

Antigens, CD ; metabolism ; Dendritic Cells ; immunology ; Gastrointestinal Neoplasms ; therapy ; Humans ; Immunotherapy, Active ; In Vitro Techniques ; Inhibitor of Apoptosis Proteins ; Interleukin-12 ; secretion ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; secretion