OBJECTIVEHuman soluble tumor necrosis factor receptor (sTNFR) can interfere with the biological functions of interleukin-1, which may be appropriate to the treatment of periodontitis. The eukaryote expression vector of the human sTNFR gene must be constructed prior to conducting transgene therapy of periodontitis.

METHODSBoth sTNFR gene and plasmid pcDNA 3.1 (+) DNA were digested with Kpn I and Xho I. After purification, the two fragments were ligated by TakaRa DNA Ligation Kit (Ver 2.0). This recombinant DNA was then transformed into E. Coli Competent Cells JM109 and positive clones were selected on the LB agarose plate containing ampicillin (80 microg/ul).

RESULTSSix single clones were indentified by double digestion with kpn I and xho I and two fragments with the size of 5.4 kb and 1.0 kb were produced as expected.

CONCLUSIONThe sTNFR gene was successfully inserted into the eukaryote expression vector plasmid pcDNA 3.1 (+) by recombination technology in vitro.

Escherichia coli ; Eukaryota ; Eukaryotic Cells ; Genetic Vectors ; Humans ; Plasmids ; Receptors, Tumor Necrosis Factor ; Receptors, Tumor Necrosis Factor, Type I ; Tumor Necrosis Factor-alpha

PURPOSE: This study was performed to evaluate anti-apoptotic mechanism of Sulfated Glycoprotein-2 (SGP-2) in tumor necrosis factor-alpha (TNF-alpha) induced apoptosis of prostatic cancer cell. MATERIALS AND METHODS: To suppress the activity of natively secreted SGP-2 by prostatic cancer cells, varying amount of the blocking antibody to SGP-2 (anti-SGP-2 antibody, 1.25~2.0microgram/ml) was applied. Then TNF-alpha or agonistic antibody to type I TNF-alpha receptor (agonistic-TNFR1 antibody) were applied for the induction of apop tosis. The changes of cell number and cytotoxicity were assessed by trypan blue dye exclusion assay. The exact amount of early and late apoptosis were analyzed and compared by flowcytometric analysis. RESULTS: By blocking SGP-2 with anti-SGP-2 antibody (2.0microgram/ml), apoptosis was signi ficantly increased in both TNF-alpha (96.8 +/- 1.4%) and agonistic-TNFR1 antibody treated PC3 cells (95.2 +/- 0.9%) compared to control group (41.4 +/- 4.7%), especially after 36 hours incubation (p<0.05). The more suppressed the activity of SGP-2 by anti-SGP-2 antibody, significantly the more apoptosis was happened (p<0.05). After 24 hours incubation, the increase of apoptosis by the suppression of SGP-2 was significantly greater in TNF-alpha treated PC3 cells (45.6 +/- 27.6%) than in agonistic- TNFR1 antibody treated ones (28.6 +/- 4.7%). However after 36 hours the difference in degree of apop tosis between two groups disappeared. CONCLUSIONS: We found that SGP-2 blocked the apoptosis which was induced by agonistic-TNFR1 antibody as like TNF-alpha induced one. These results implies the possi bility of the direct action of SGP-2 on type I TNF-alpha receptor. However the difference between two groups in the degree of apoptosis after 24 hours incubation period sug gest the more efficient blocking effect of SGP-2 on agonistic-TNFR1 induced apoptosis than on TNF-alpha induced one. For clarification of these difference, further research about other influencing factors such as type II TNF-alpha receptor activation is essential.
Apoptosis* ; Cell Count ; Clusterin* ; Prostatic Neoplasms* ; Receptors, Tumor Necrosis Factor, Type I ; Trypan Blue ; Tumor Necrosis Factor-alpha

PURPOSE: In prostate cancer, the anti-apoptotic mechanism of sulfated glycoprotein-2 (clusterin) against tumor necrosis factor-alpha (TNF-alpha) receptors and the action of type 2 TNF-alpha receptor (TNFR2) were investigated. MATERIALS AND METHODS: TNF-alpha, agonistic-TNF type 1 receptor (TNFR1) antibody, agonistic-TNF-R2 antibody and their combination were treated in PC3 cell line with or without anti-clusterin. Cytotoxicity was assessed by trypan blue dye exclusion assay. By using flowcytometric analysis, the exact amount of apoptosis and their changes were assessed. RESULTS: Apoptosis was significantly increased in both agonistic-TNFR1 antibody and TNF-alpha treated cases after blocking the activity of clusterin. The more the anti-clusterin antibody added, the more the apoptosis occurred. The increase of total apoptosis was greater in TNF-alpha treated cells than in agonistic-TNFR1 antibody treated ones. However, there was no increase of apoptosis in agonistic-TNFR2 antibody and TNF-alpha with agonistic-TNFR2 antibody treated cases, respectively. CONCLUSIONS: Clusterin prevents TNF-alpha induced apoptosis by affecting TNFR1. The difference in degree of apoptosis between agonistic-TNFR1 antibody treated cells and TNF-alpha treated ones suggests the possibility of the action of TNFR2. It may be associated with affinity of TNF-alpha to the tumor cell surface.
Apoptosis ; Cell Line ; Clusterin ; Diminazene ; Prostate ; Prostatic Neoplasms ; Receptors, Tumor Necrosis Factor ; Receptors, Tumor Necrosis Factor, Type I ; Receptors, Tumor Necrosis Factor, Type II ; Trypan Blue ; Tumor Necrosis Factor-alpha

The aim of this study was to detect the expression and cell cycle specificity of Fas, TNFRI and TNFRII in human peripheral blood lymphocytes (PBL), and to study the potential role of Fas, TNFRIand TNFRII in cell cycle specific apoptosis. The improved double-parameter flow cytometry was used to detect the expressions of Fas, TNFRI and TNFRII and cell cycle specificity in PBL which were incubated for 24 hours in the presence or absence of phytohaematoagglutinin (PHA) respectively. Apoptosis induced by IgM type anti-Fas and TNF-alpha was detected by API method. The results showed that compared with PBL treated in the absence of PHA in G(0) phase, the ratio of Fas, TNFRI and TNFRII expressions in PHA-stimulated PBL entering cell cycle increased (35.55 +/- 6.63)%, (30.63 +/- 2.66)%, (26.62 +/- 5.14)% respectively (P < 0.01), and mainly appeared at G(1)-phase; no apoptosis was induced by anti-Fas and TNF-alpha in G(0)-phase PBL cultured in the absence of PHA. On the contrary, the apoptosis was induced by anti-Fas and TNF-alpha in PBL which entered cell cycle after stimulation with PHA and mainly initiated at G(1)-Phase. It is concluded that there is evident dose-effect relationship between apoptotic receptor and receptor-mediated apoptosis. Moreover, the cell cycle specificity of receptor-mediated apoptosis is correlated with the cell cycle specific expressions of apoptotic receptor. The induction of apoptosis by apoptotic factors (anti-Fas and TNF-alpha) depends on whether cell entering cell cycle or not.
Apoptosis ; Cell Cycle ; Humans ; Lymphocytes ; cytology ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Receptors, Tumor Necrosis Factor, Type II ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo observe the effects of intra-articular ozone injection at different concentrations on the contents of tumor necrosis factor-α (TNF-α), TNF receptor I (TNFR I), and TNFR II in the serum and synovium of rats with rheumatoid arthritis (RA) and explore the therapeutic mechanism of ozone in RA treatment.

METHODSForty-eight Wistar rats were randomized into 8 groups, including 5 ozone groups receiving intra-articular injection of 10, 20, 30, 40 or 50 µg/ml ozone, a blank control group, an oxygen group and a RA model group. All the rats, except for those in the blank control group, were subjected to hypodermic injection of bovine collagen II and complete Freunds adjuvant to induce RA. Ozone treatment was administered once weekly for 3 weeks starting at 21 days after the modeling. The swelling and thickness of the hind paws were observed, and the serum and synovial contents of TNF-α, TNFR I, and TNFR II were detected.

RESULTSAt the end of treatment, the paw thickness was reduced significantly in rats with 40 µg/ml ozone injection compared with that in the model RA group (P<0.01). The serum contents of TNF-α, TNFR I and TNFR II showed no significant difference between the RA model group, oxygen group and the ozone groups, but their synovial contents showed significant reductions in rats with 40 and 50 µg/ml ozone injection (P<0.01); the synovial TNFR I was significantly higher in 40 µg/ml ozone group than in the model group (P<0.05).

CONCLUSIONIntra-articular injection of 40 µg/ml ozone can attenuate synovitis in rats with RA, the mechanism of which may involve the inhibition of TNF-α and TNFR II activity and enhancement of TNFR I activity in the synovium.

Animals ; Arthritis, Rheumatoid ; metabolism ; therapy ; Injections, Intra-Articular ; Male ; Ozone ; administration & dosage ; therapeutic use ; Rats ; Rats, Wistar ; Receptors, Tumor Necrosis Factor, Type I ; metabolism ; Receptors, Tumor Necrosis Factor, Type II ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

PURPOSE: Numerous reports have suggested that apoptosis may play an important role in postresuscitation syndrome. The aim of this study is to assess the levels of molecules that are associated with apoptosis in the serum of patients who underwent successful resuscitation after cardiac arrest. METHODS: The serum levels of cytochrome c, tumor necrosis factor type 1 receptor (TNF-R1) and Fas ligand in 11 patients were measured at 0, 4, 12, 24, 48 and 72 hours after successful resuscitation. The primary endpoint consisted of survival to hospital discharge. Ten healthy volunteers were also evaluated as a control group. RESULTS: Patients with successful resuscitation had increased levels of cytochrome c and TNF-R1 at 0, 4, 12, 24 and 48 hours after return of spontaneous circulation (ROSC), as compared with those levels of the healthy volunteers (p<0.05). Higher levels of TNF-R1 at 12, 24 and 48 hours after ROSC were found in the non-survivors as compared to those levels of the survivors (p=0.01, 0.03, 0.02). The Fas ligand level at ROSC was also higher in the patients with successful resuscitation (p=0.00). However, the Fas ligand levels at 24, 48 and 72 hours after ROSC were lower in the patients with successful resuscitation than those levels in the healthy volunteers. CONCLUSION: These results suggest that apoptosis belonging to the TNF-R1 and cytochrome c pathways may be involved in the pathogenesis of postresuscitation syndrome. The serum levels of the death-receptor TNF-R1 could serve to quantitate the severity of injury and to prognosticate the survival outcomes.
Apoptosis ; Cardiopulmonary Resuscitation ; Cytochromes ; Cytochromes c ; Fas Ligand Protein ; Heart Arrest ; Humans ; Receptors, Tumor Necrosis Factor, Type I ; Resuscitation ; Survivors ; Tumor Necrosis Factor-alpha

Apoptosis ; drug effects ; HeLa Cells ; Hepatocytes ; cytology ; drug effects ; Humans ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology

Objective: To detect the expression levels of M1-type polarization and autophagy-related indicators in the liver of trichloroethylene (TCE) -sensitized mice, and to explore the role of liver tumor necrosis factor-α (TNF-α) and tumor necrosis factor receptor 1 (TNFR1) in regulating M1-type Kupffer cells autophagy in liver injury in TCE-sensitized mice. Methods: In November 2019, according to simple random grouping, 45 SPF grade BALB/c female mice (6-8 weeks old) were divided into 4 groups: blank control group (<i>ni>=5) , solvent control group (<i>ni>=5) , TCE treatment group (<i>ni>=18) , TCE+R7050 (inhibitor) treatment group (<i>ni>=17) . Transdermally sensitized mice, 24 h after the last challenge, the mice were divided into TCE sensitized group and TCE non-sensitized group according to the skin reaction score. The livers of mice were harvested, and the pathological changes of the livers were observed under light and electron microscopes. Western blotting was used to detect the expressions of TNF-α, TNFR1 and autophagy-related indexes. The expression of inducible nitric oxide synthase (iNOS) , a marker of M1-type Kupffer cells, was detected by immunohistochemistry, and the occurrence of autophagy in M1-type Kupffer cells was detected by immunofluorescence double-labeling method. Results: The sensitization rate of TCE treatment group was 38.9% (7/18) , and TCE+R7050 treatment group was 35.3% (6/17) , with no significant difference between the two groups (<i>Pi>=1.000) . Compared with the blank control group, mice in the TCE sensitized group had abnormal liver ocytes, obvious liver injury, reduced mitochondria and broken endoplasmic reticulum. Western blotting results showed that the expressions of TNF-α and TNFR1 protein in the liver of the mice in the TCE sensitized group increased, the expression of iNOS protein in M1-type Kupffer cells increased, and the expressions of autophagic microtubule-associated protein 1 light-chain 3 (LC3B) and Beclin1 protein were decreased (<i>Pi><0.05) . The results of immunohistochemistry showed that iNOS was not significantly expressed in the blank control group and solvent control group, and a small amount of expression was found in the TCE non-sensitized group, the positive staining area was obvious in TCE sensitized group, and the expression of iNOS was significantly increased (<i>Pi><0.05) . Immunofluorescence results showed that the iNOS protein levels in the blank control group, solvent control group and TCE non-sensitized group were lower, and only partially colocalized with P62; the colocalization of iNOS with P62 in the TCE sensitized group was significantly increased. Conclusion: TNF-α/TNFR1 signaling pathway may promote liver injury in TCE-sensitized mice by inhibiting autophagy of M1-type Kupffer cells.
Animals ; Autophagy ; Female ; Kupffer Cells ; Liver ; Mice ; Mice, Inbred BALB C ; Receptors, Tumor Necrosis Factor, Type I ; Solvents ; Trichloroethylene/toxicity* ; Tumor Necrosis Factor-alpha

tumor tissues harvested at pre-treatment and TKI-resistant post-treatment periods. In addition, a public microarray dataset from patient-derived xenograft model for TKI-treated ccRCC (GSE76068) was retrieved. Commonly altered pathways between the datasets were investigated by Ingenuity Pathway Analysis using commonly regulated differently expressed genes (DEGs). The significance of candidate DEG on intrinsic TKI resistance was assessed through immunohistochemistry in a separate cohort of 101 TKI-treated ccRCC cases.RESULTS: TNFRSF1A gene expression and tumor necrosis factor (TNF)-α pathway were upregulated in ccRCCs with acquired TKI resistance in both microarray datasets. Also, high expression (> 10% of labeled tumor cells) of TNF receptor 1 (TNFR1), the protein product of TNFRSF1A gene, was correlated with sarcomatoid dedifferentiation and was an independent predictive factor of clinically unfavorable response and shorter survivals in separated TKI-treated ccRCC cohort.CONCLUSION: TNF-α signaling may play a role in TKI resistance, and TNFR1 expression may serve as a predictive biomarker for clinically unfavorable TKI responses in ccRCC.]]>
Biomarkers ; Carcinoma, Renal Cell ; Cohort Studies ; Dataset ; Drug Resistance ; Gene Expression ; Gene Expression Profiling ; Heterografts ; Humans ; Immunohistochemistry ; Protein-Tyrosine Kinases ; Receptors, Tumor Necrosis Factor ; Receptors, Tumor Necrosis Factor, Type I ; Tumor Necrosis Factor-alpha

INTRODUCTIONRheumatoid arthritis (RA) is a chronic, deforming arthritis that can lead to disabilities and poor quality of life. Cytokines are protein mediators of inflammation and are produced as a result of the activation of various cellular reactions. They are the final mediators and/or regulators of the inflammatory process.

MATERIALS AND METHODSThe sera from 64 RA patients were assayed for both Th-1 and Th-2 related cytokines and soluble TNF-alpha receptors (IFN-gamma, TGF-beta, TNF-alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, sTNF-R1 and sTNFR2) using ELISA.

RESULTSThe pro-inflammatory cytokines (IL-1, IL-6, IL-8, IL-18 and TNF- alpha) were significantly elevated in RA patients, while TGF-beta, an immunomodulatory cytokine, was elevated in control individuals. When the RA patients were categorised as active or inactive based on DAS scores, similar cytokines profiles were observed in both RA sub-groups. However, assays of sTNF-R1 and sTNFR-2 were noted to be significantly elevated in inactive RA patients when compared to active patients.

CONCLUSIONOur findings indicate that local production of cytokine inhibitors is capable of diminishing disease activity and cytokine activity.

Adult ; Aged ; Arthritis, Rheumatoid ; blood ; pathology ; Cell Differentiation ; Cytokines ; blood ; Female ; Humans ; Male ; Middle Aged ; Receptors, Tumor Necrosis Factor, Type I ; chemistry ; Receptors, Tumor Necrosis Factor, Type II ; chemistry ; Transforming Growth Factor beta ; chemistry