HUMANGGP:017444 - FACTA Search

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Query: HUMANGGP:017444 (TNF)
61,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe the production and purification of recombinant equine tumor necrosis factor alpha (rETNF alpha), generation and characterization of murine monoclonal antibodies (Mabs) and rabbit polyclonal antibodies (Pabs) against ETNF alpha, and development of a sensitive enzyme-linked immunosorbent assay (ELISA). Genomic-derived DNA sequences encoding mature ETNF alpha were reconstructed by the polymerase chain reaction (PCR) and oligonucleotide-directed mutagenesis and were cloned into the vector pFLAG-1 for expression in Escherichia coli. rETNF alpha was purified by anti-FLAG immunoaffinity chromatography and then used as immunogen for production of murine Mabs and rabbit Pabs. Three Mabs (6H4, 9B10, and 12F6) were obtained from one fusion. All three Mabs recognized rETNF alpha on western blots. Mabs 6H4 and 9B10 recognized similar epitopes on rENTF alpha and neutralized both rETNF alpha and native ETNF alpha (nETNF alpha) in a WEHI cell cytotoxicity assay. A sensitive ELISA was developed using Mab 6H4 and biotin-labeled rabbit Pabs. The ELISA was shown to detect levels of ENTF alpha as low as 100 pg/ml and was used to demonstrate the induction of ETNF alpha in horses with experimental endotoxemia. The rETNF alpha, antibodies, and ELISA developed in this report should be useful tools for studies of TNF-mediated diseases in horses.
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PMID:Equine tumor necrosis factor alpha: cloning and expression in Escherichia coli, generation of monoclonal antibodies, and development of a sensitive enzyme-linked immunosorbent assay. 128 21

The numerous biologic activities of TNF appear mediated by two types of specific cell surface receptors of 55 to 60 kDa (TR55) and 75 to 80 kDa (TR75) molecular mass, respectively. The role of TR55 in the activation of the nuclear transcription factor kappa B (NF-kappa B) was investigated using an antagonistic, mAb, H398, specific for the human TR55. The human leukemic T cell line, Jurkat, which expresses both types of receptors at comparable levels, was used to test for NF-kappa B activation by electrophoretic mobility shift assays using as a probe an oligonucleotide encompassing the two tandemly arranged kappa B sites of the HIV-1 LTR enhancer. mAb H398 is shown to efficiently block not only TNF- but also lymphotoxin-mediated activation of NF-kappa B. Furthermore mAb H398 also impeded TNF- or lymphotoxin-mediated activation of chloramphenicol acetyl transferase gene expression from the HIV-1-LTR as determined by transient transfection assays. These findings indicate that both, induction of NF-kappa B binding to DNA, and transcriptional activity can be efficiently inhibited by selective blockade of TR55. Finally it is shown, that human TR55 confers NF-kappa B inducibility when expressed in the mouse pre-B cell line 70Z/3, which does not respond to TNF in its parental state. Together, the results of this study indicate that TR55 is both necessary and sufficient for mediating TNF activation of NF-kappa B.
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PMID:Inhibition of tumor necrosis factor (TNF)-mediated NF-kappa B activation by selective blockade of the human 55-kDa TNF receptor. 131 30

Immunogenic tumor variants were previously derived after transplantation in vivo into nude mice of NIH/3T3-transformed cell lines. Nude-passaged cell lines were rejected by immunocompetent H-2q NIH mice, were recognized by specific CTL clones, and expressed new retroviral Ag. The aim of the present work was to investigate whether somatically acquired proviral sequences were present in the genome of nude-passaged cells and to test directly for a causative relationship between murine leukemia virus (MuLV) expression and immunogenicity. Southern blot analysis of PstI-digested DNA indicated that in contrast to the parental NIH/3T3 transformed cell lines (pT, T12N/5a, NS-1) all the nude-passaged immunogenic variants (pT-nude, T12N/5a-nude, NS-1-nude) contained newly acquired ecotropic-related proviruses. Immediately after in vitro establishment, these tumors displayed multiple integration sites as assessed by analysis of 3' proviral-cellular junctions. Long term in vitro culture of one of the cell lines (pT-nude) resulted in a cell line (pT-nude/vitro) that was clonal or oligo-clonal with respect to viral integration. Northern blot analysis established that the new proviruses were actively transcribed in all the immunogenic variants. To assess whether the somatically acquired ecotropic proviral sequences encode for target structures recognized by specific CTL, obtained after immunization of NIH mice with pT-nude, the parental cell line pT was transfected with plasmids containing the entire AKV MuLV genome, the cloned AKV gag or env genes. Screening of transfectants for their ability to stimulate the production of TNF by anti-pT-nude effectors indicated that cells transfected with the entire ecotropic virus or with MuLV-env gene products could be recognized by an NIH anti-pT-nude CTL line and NIH anti-pT-nude Kq-restricted CTL clones as well as the immunizing target pT-nude.
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PMID:Involvement of somatically acquired ecotropic viruses in the immunogenicity of nude-transplanted NIH/3T3 transformed cell lines. 131 2

While investigating the modulation of the growth and function of the FRTL-5 rat thyroid cell line by recombinant human tumor necrosis factor-alpha (TNF alpha), we noticed that pronounced changes in several response parameters occurred with increasing passage number. For young cells (passage less than 20), TNF alpha by itself slightly increased [3H]thymidine incorporation and DNA content, and had a minimal effect on basal 125I uptake. When combined with TSH, TNF alpha had no influence on TSH-stimulated [3H]thymidine incorporation, but significantly inhibited TSH-stimulated 125I uptake. Compared with young cells, aged cells (passage greater than 40), in contrast, developed a high sensitivity to TNF alpha. TNF alpha markedly stimulated [3H]thymidine incorporation into DNA, inhibited TSH-stimulated 125I uptake per micrograms DNA, but dramatically decreased the total DNA content and cell number. TSH augmented the TNF alpha effect in aged cells, resulting in a further reduction of DNA content. Aphidicolin, a specific inhibitor of DNA polymerase-alpha which is associated with DNA replication, dramatically inhibited TNF alpha-induced [3H]thymidine incorporation in both young and aged cells; this suggested that the effect of TNF alpha on FRTL-5 cell growth is related to DNA replication, rather than DNA repair. 51Cr release from FRTL-5 cells, a measure of cytotoxicity, increased 2-fold over baseline in aged cells at a dose of 400 ng/ml TNF alpha and decreased to 70% of baseline in young cells at this same dose. The protein kinase-A (PKA) and protein kinase-C (PKC) signal transduction mechanisms of TNF alpha in aged cells (passage greater than 40) were also studied. TNF alpha increased cAMP and also increased relative PKA and PKC activity in 1-40 min. Phorbol myristate acetate (PMA), an activator of PKC, increased [3H]thymidine incorporation and DNA content. PMA did not affect the TNF alpha-induced increase in [3H]thymidine incorporation or its reduction of DNA content. When the cells were pretreated with a high concentration of PMA (1 microM/24 h) to down-regulate PKC, the TNF alpha dose-dependent increase in [3H]thymidine incorporation and decrease in DNA content were only slightly inhibited, suggesting that the main effects of TNF alpha are independent of PKC. We conclude that the sensitivity of FRTL-5 cells to the cytotoxic effect of TNF alpha increases with aging.
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PMID:Aging of FRTL-5 rat thyroid cells causes sensitivity to cytotoxicity induced by tumor necrosis factor-alpha. 132 86

Tumor necrosis factor alpha (TNF alpha) is likely to exert a major influence in the pathogenesis of glomerulopathies. Besides its proinflammatory properties. TNF alpha interacts with cell growth and synthesis of components of the fibrinolytic system. In this study, we report the effects of recombinant human TNF alpha on the synthesis of tissue-type plasminogen activator (t-PA) and its inhibitor (PAI-1) by human mesangial cells in culture. We first demonstrate that TNF alpha binds specifically to a single class of high affinity receptors (Kd 5.10(-11) M; 1500 receptors/cell). TNF alpha has an antimitogenic effect on human mesangial cells since it decreased DNA synthesis, measured by 3H-thymidine incorporation, in a dose-dependent manner. Release of cytosolic LDH and incorporated 51Cr was not increased by 100 ng/ml TNF alpha as compared with control, indicating that this monokine is not cytotoxic for cultured human mesangial cells. Zymographic analysis and reverse fibrin autography disclosed a 120 kD t-PA-PAI-1 complex and a 50 kD free form of PAI-1 in the supernatants of both unstimulated and TNF-stimulated cells; PAI-1 was released in excess and free t-PA was not observed. TNF alpha (0 to 100 ng/ml) had no effect on t-PA synthesis, but enhanced PAI-1 release in a time- and dose-dependent manner (97% increase of PAI-1 synthesis after a 24 hour incubation). This effect was abolished by cycloheximide, suggesting that protein synthesis was required. Northern blot analysis showed that TNF alpha increased the steady-state PAI-1 mRNA levels in a time-dependent manner, with a maximal effect at two hours.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tumor necrosis factor alpha increases antifibrinolytic activity of cultured human mesangial cells. 132 51

Distinctions between tumor necrosis factor, TNF-alpha, and lymphotoxin, TNF-beta, have previously been based on the differences between their protein sequences, biological activity, and molecular regulation. In the past year, elucidation of the molecular nature of the two molecules and the interactions with common receptors has emphasized their similarities, although profound differences continue to emerge with regard to their mode of production, transcription rates, mRNA half-lives, and the importance of various DNA regulatory sequences. A role for both TNF-alpha and TNF-beta has recently been suggested with regard to disease, particularly multiple sclerosis. The past year has also seen the description of an extensive microsatellite polymorphic system which should provide a more definitive understanding of the association of the TNF locus with disease.
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PMID:Tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta). 132 40

The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.
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PMID:Transcriptional regulation of the thrombomodulin gene. 133 Oct 78

Agents that can arrest cellular proliferation are now providing insights into mechanisms of growth factor action and how this action may be controlled. It is shown here that the macrophage activating agents tumor necrosis factor-alpha (TNF alpha), interferon-gamma (IFN gamma), and lipopolysaccharide (LPS) can maximally inhibit colony stimulating factor-1 (CSF-1)-induced, murine bone marrow-derived macrophage (BMM) DNA synthesis even when added 8-12 h after the growth factor, a period coinciding with the G1/S-phase border of the BMM cell cycle. This inhibition was independent of autocrine PGE2 production or increased cAMP levels. In order to compare the mode of action of these agents, their effects on a number of other BMM responses in the absence or presence of CSF-1 were examined. All three agents stimulated BMM protein synthesis; TNF alpha and LPS, but not IFN gamma, stimulated BMM Na+/H+ exchange and Na+,K(+)-ATPase activities, as well as c-fos mRNA levels. IFN gamma did not inhibit the CSF-1-induced Na+,K(+)-ATPase activity. TNF alpha and LPS inhibited both CSF-1-stimulated urokinase-type plasminogen activator (u-PA) mRNA levels and u-PA activity in BMM, whereas IFN gamma lowered only the u-PA activity. In contrast, LPS and IFN gamma, but not TNF alpha, inhibited CSF-1-induced BMM c-myc mRNA levels, the lack of effect of TNF alpha dissociating the inhibition of DNA synthesis and decreased c-myc mRNA expression for this cytokine. These results indicate that certain biochemical responses are common to both growth factors and inhibitors of BMM DNA synthesis and that TNF alpha, IFN gamma, and LPS, even though they all have a common action in suppressing DNA synthesis, activate multiple signaling pathways in BMM, only some of which overlap or converge.
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PMID:Biochemical events accompanying macrophage activation and the inhibition of colony-stimulating factor-1-induced macrophage proliferation by tumor necrosis factor-alpha, interferon-gamma, and lipopolysaccharide. 133 37

We detected the HBV DNA in peripheral blood monocytes of 70 patients with chronic hepatitis B and 20 healthy controls by polymerase chain reaction (PCR) and Southern transfer hybridization technique, the HLA-DR expression on monocytic membrane and tumor necrosis factor-alpha (TNF alpha) content and interleukin 1 (IL 1) activity in their monocytic culture supernatant were also measured respectively by indirect immunofluorescence technique, ELISA, and thymocytic multiplication assay. HBV DNA sequences were detected in monocytes of 42(60%) of the 70 patients. The percentage of monocyte-expressing HLADR and the TNF alpha content in monocyte culture supernatant were significantly elevated in patients with HBV DNA-positive monocytes compared with those patients with HBV-DNA-negative monocytes. The percentage of monocyte-expressing HLA-DA and the level of TNF alpha produced by monocytes correlated markedly with the intensity of HBV DNA-positive signal in monocytes. These data suggested that HBV infection in monocytes might cause a series of functional changes in these cells.
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PMID:[Hepatitis B virus infection in monocytes and their functional changes in patients with chronic hepatitis B]. 133

Recombinant TNF as a single agent for human cancer appears to be of limited value. However, rTNF has synergistic anticancer effects when combined with chemotherapeutic drugs targeted at DNA topoisomerase II. This effect of rTNF has been observed in several in vitro and in vivo tumor models, both in animal and human studies. The mechanism of this interaction appears to involve lesions to the DNA of tumor cells mediated by inhibition of DNA topoisomerase II. The combinations of rTNF plus doxorubicin and rTNF plus etoposide administered systemically are currently under evaluation by clinical trials in patients with advanced cancers. Determination of the efficacy of such combination therapy must await the completion of phase I and II trails. Other routes of administration that might increase the local concentration of rTNF and could be combined with topoisomerase II-targeted drugs include intravesical administration and the use of tumor-infiltrating lymphocytes.
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PMID:Tumor necrosis factor and chemotherapeutic drugs targeted at DNA topoisomerase II for the treatment of genitourinary malignancies. 134 88

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