Expression and purification of woodchuck tumour necrosis factor alpha

The production of recombinant woodchuck cytokines is an essential prerequisite to study the immune response to hepadnavirus infection in the woodchuck model. Woodchuck tumour necrosis factor-alpha (TNF-alpha) was expressed in mammalian cells and in Escherichia coli. A test system for the biological activity of woodchuck TNF-alpha was established on basis of its cytotoxic effect to the murine fibrosarcoma cell line L929. Recombinant TNF-alpha was purified and used for the production of neutralizing antisera.     

Mechanism of tumour necrosis factor alpha mediated eosinophil survival

Prolonged eosinophil survival is an essential step in the late and chronic phases of allergic inflammation and is regulated by the eosinophil survival cytokines. Our work has demonstrated that tumour necrosis factor (TNF)-alpha enhances survival (Trypan blue exclusion test) of human peripheral blood eosinophils from mildly allergic patients in a dose-dependent manner. The survival activity of TNF-alpha was inhibited by anti-TNF-RI, anti-TNF-RII antagonist antibodies and anti-granulocyte-monocyte colony-stimulating factor (GM-CSF) neutralizing antibodies but not by anti-interleukin (IL)-3 or anti-IL-5 antibodies. Furthermore, TNF-alpha-induced GM-CSF release from eosinophils. Anti-TNF-alpha antibodies also inhibited GM-CSF release from eosinophils induced by rat mast cell sonicate, which enhances eosinophil survival. To define the signal transduction pathway involved in GM-CSF production, eosinophils were incubated either with various mitogen-activated protein kinases (MAPK) inhibitors (MEK, JNK, P38), or Cyclosporin A (calcineurin inhibitor), or MG-132 (proteasome inhibitor). Only the proteasome inhibitor significantly decreased both TNF-alpha-enhanced eosinophil survival (from 38.1+/-4.1% to 13.3+/-1.4%) and GM-CSF release (from 6.2+/-0.7 pg/ml to 0.3+/-0.1 pg/ml). TNF-alpha also induced nuclear factor-kappaB (NF-kappaB) translocation to the nucleus, an essential step in GM-CSF mRNA production. All these findings provide evidence that NF-kappaB is involved in TNF-alpha-enhanced eosinophil survival through the regulation of GM-CSF production by eosinophils.     

Induction of tumour necrosis factor alpha in experimental animals treated with hepatotoxicants.

Administrations of hepatotoxicants namely carbon tetrachloride (CCl4:0.4 ml in 1.2 ml of liquid paraffin) and ANIT (1 ml of 1.5% solution in liquid paraffin) in Charles foster rats (force fed) and D-galactosamine (8 mg in water per swiss albino mouse, ip) induce the release of TNF-alpha in case of CCl4 and D-galactosamine. High TNF-alpha level was observed up to 48 hr in CCl4 and up to 24 hr in D-galactosamine treated animals. Elevated levels of biochemical like ALP and SGPT are also recorded. TNF-alpha level can be measured of tissue damage and prognosis in case of hepatitis.     

Association between circulating microRNA-126 expression level and tumour necrosis factor alpha in healthy smokers

Introduction: Smoking contributes to the death of a million people worldwide each year. Smokers experience an alteration in tumour necrosis factor-alpha (TNF-α), and the risk of expected lung cancer. The study aimed at investigating the expression levels of mir-126 and mir-124, as well as TNF-α as possible biomarkers of expected smoking-related diseases.

Methods: Twenty-five male smokers’ age and sex-matched with 25 non-smokers were recruited for the present study. Plasma expression levels of mir-126 and mir-124 were evaluated using quantitative real-time PCR. Lipid profile, TNF-α, interleukin-6 and C-reactive protein were assessed in plasma of each participant.

Results: Plasma miR-126 was statistically down-regulated in smokers relative to non-smokers; however, mir-124 did not show any significant changes between groups. Among the measured parameters, mir-126 and tumour necrosis factor alpha (TNF-α) displayed a good discrimination and sensitivity between smokers and non-smokers (AUC = 0.809 (95% CI: 0.668–0.95; p?<?0.001) and 0.742(95% CI: 0.584–0.9; p?<?0.01), respectively. Also, the combined evaluation of miR-126 and TNF-α levels showed high discrimination (AUC= 0.889 (95% CI: 0.779–1.00; p?<?0.0001), sensitivity = 85%, and specificity = 80% in the diagnosis of smokers with non-smokers.

Conclusions: MiR-126 and TNF-α are potential biomarkers of smoking-related diseases and are important in assessing the expected tobacco-related harm.     

Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes

Type 2 diabetes is associated with increased circulating concentrations of markers of the acute-phase response and interleukin-6 (IL-6). An augmented acute-phase response may be a mechanism which explains many of the clinical and biochemical features of type 2 diabetes and its complications. We sought to confirm that circulating concentrations of the cytokine acute-phase mediators IL-6 and tumour necrosis factor alpha [TNFalpha] are elevated in type 2 diabetes, and investigated blood as a source of cytokines in type 2 diabetes. Blood samples from 20 type 2 diabetic and 17 age-matched healthy subjects were incubated in vitro for 24 hr with and without lipopolysaccharide (LPS) stimulation and secreted cytokines measured. Plasma IL-6 and TNFalpha were significantly increased in type 2 diabetes compared to normal subjects. However, basal production of IL-6 and TNFalpha in cultured diabetic blood was markedly depressed in comparison with non-diabetic samples. IL-6 and TNFalpha production was increased in blood in response to LPS, reaching similar levels in diabetic and non-diabetic subjects, though IL-6 was slightly but significantly higher in controls. We conclude that circulating levels of IL-6 and TNFalpha are increased in type 2 diabetes but there is downregulation of basal cytokine production in blood cells in type 2 diabetes. Blood has the capacity to produce cytokines in diabetes which contribute to the augmented acute-phase response, but the main source of the increased plasma IL-6 and TNFalpha concentrations may be from non-circulating cells.     

The neutrophil migration induced by tumour necrosis factor alpha in mice is unaffected by glucocorticoids

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFalpha), IL-8, C5a and leukotriene B(4) (LTB(4)). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulated in vitro with LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFalpha. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFalpha was also unaffected by pretreatment of the mice with dexamethasone. Moreover, neutrophil migration induced by intraperitoneal injection of LPS was completely blocked by pretreatment of the mice with a monoclonal antibody against murine TNFalpha. In conclusion, our results support the hypothesis that, in contrast to the role of TNF in rats (where it indirectly induces neutrophil migration), in mice, it may be an important mediator in the recruitment of neutrophils to inflammatory sites.     

Inhibitory effect of esculentoside A on tumour necrosis factor alpha production by human monocytes

Esculentoside A (EsA) is a saponin isolated from the roots of Phytolacca esculenta. Previous experiments have shown that it has strong anti-inflammatory effects. Tumour necrosis factor (TNF) is a very important inflammatory mediator. It is known that there are two types of TNF-TNFalpha is from macrophages/monocytes and TNFbeta is from activated lymphocytes. In order to study the mechanism of the anti-inflammatory effect of EsA, it was determined whether TNFalpha production from human peripheral monocytes was altered by EsA under lipopolysaccharide (LPS)-stimulated conditions. EsA was found to decrease TNFalpha production in a dose-dependent manner at concentrations higher than 1 mumol/l EsA. Recent studies have shown that EsA has a curative effect on chocolate cyst and other inflammatory diseases. Our previous studies have shown that EsA could reduce the release of platelet activating factor (PAF) from rat macrophages, and inhibit interleukin-1 and interleukin-6 production from routine macrophages. The reducing effects of EsA on the release of TNFalpha, IL-1, IL-6 and PAF may explain its anti-inflammatory effect.     

Molecular steps of tumor necrosis factor receptor-mediated apoptosis

Until recently it was believed that TNF-induced apoptosis is mediated exclusively by TNF-RI because TNF-RII lacks death domain. However, it has been demonstrated that TNF-RII enhances TNF-RI-mediated apoptosis. In this review, I have discussed the evidence and mechanisms by which TNF-RII regulates TNF-alpha-induced apoptosis. A role of RIP is emphasized and novel mechanisms of FLIP-mediated inhibition of apoptosis are discussed. In addition, various mechanisms of TNF-induced activation of mitochondrial pathway of apoptosis have been reviewed.     

Clinical applications of tumour necrosis factor

Tumour necrosis factor (TNF) is a polypeptide hormone produced in vivo by activated macrophages and lymphocytes. TNF has diverse effects in vivo and has a physiological role as an immune modulator, as a mediator of the immune response, both through activation of neutrophils and eosinophils, and also affects the vascular endothelium. TNF also has antiviral activity and causes alterations in lipid metabolism. In disease states excessive production of TNF may have adverse affects. TNF has been implicated as a mediator of endotoxic shock, inflammatory joint disease, immune deficiency states, allograft rejection, and in the cachexia associated with malignant disease and some parasitic infections. When used in pharmacological doses, TNF is cytotoxic to many malignant cells in vitro and in vivo. The mechanisms underlying cytotoxicity are not fully elucidated but involve both a direct toxic effect to the cell and an indirect effect on tumour vasculature. Cytotoxicity is not universal and TNF may act as a differentiating agent or growth factor for some haematological cell types. So far the clinical application of TNF has been as a treatment for cancer in Phase I and II trials in patients with advanced disease and its efficacy here is still unproven. TNF may have potential for clinical application in combination therapy for cancer. There is experimental evidence for its interaction with other biological agents and cytotoxic drugs. The use of specific antibodies to inhibit production of TNF, or other agents to antagonise the toxic effects of TNF may have clinical relevance in counteracting septic shock and the clinical manifestations of TNF in inflammatory and neoplastic disease.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.     

Endotoxaemia, production of tumour necrosis factor alpha and polymorphonuclear neutrophil activation following strenuous exercise in humans

To examine whether endotoxaemia accompanying long-term, strenuous physical exercise is involved in exercise-induced increase in plasma tumour necrosis factor alpha (TNF-alpha) concentration and polymorphonuclear neutrophil (PMN) activation, 14 male recreational athletes [mean age 28 (SEM 1) years] were studied. Exercise consisted of a 1.5-km river swim, a 40-km bicycle race, and a 10-km road race. Mean time to complete the race was 149.8 (SEM 4.8) min. The plasma concentrations of granulocyte myeloperoxidase (MPO) and TNF-alpha were significantly higher than baseline values immediately and 1 h after exercise (P<0.001). Both variables returned to pre-race levels the day after exercise. Marked, transient decreases in plasma concentrations of anti-lipopolysaccharide (LPS) immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies directed against a panel of selected smooth gram-negative LPS were observed after the race, reaching in most cases minimal values in the blood sample drawn immediately following the completion of the triathlon. There was no significant correlation between the magnitude of PMN activation, as assessed by the increase in plasma concentrations of MPO, and the humoral markers of endotoxaemia and TNF-alpha. An inverse, highly significant relationship between the increase in plasma TNF-alpha concentrations and the changes in circulating anti-LPS IgM antibodies concentrations was observed (r = -0.7; P<0.01). These findings suggest that exercise-induced endotoxaemia was involved in the release of TNF-alpha, that the magnitude of the TNF-alpha response to exercise was down-regulated by anti-LPS antibodies of the IgM class, and that the production of TNF-alpha and endotoxaemia did not seem to play a role in the activation of circulating PMN in the exercising subjects.     

Inhibition of fibronectin-activated migration of microvascular endothelial cells by interleukin-1alpha, tumour necrosis factor alpha and interferon gamma

The effect of interferon gamma (IFN) and the inflammatory cytokines tumour necrosis factor alpha (TNF) and interleukin 1alpha (IL-1) on micro- and macrovascular endothelial cell (EC) proliferation and migration was analysed. Whereas both micro- and macrovascular EC were growth-inhibited in response to the aforementioned cytokines, only microvascular EC were sensitive to TNF, IL-1 and IFN as inhibitors of fibronectin-activated cell migration. In addition, because microvascular EC play a crucial role in angiogenesis, and the formation of new capillaries depends upon the presence of angiogenic polypeptides, we evaluated the synthesis of fibroblast growth factor (FGF) type 1 and 2, Vascular Endothelial Growth Factor (VEGF) and Hepatocyte Growth Factor (HGF) in our system. Both micro- and macrovascular EC produce large amounts of FGF-2, which is mainly localized in the nucleus, and almost undetectable levels of FGF-1. In addition, the two cell types synthesize notable levels of VEGF and no HGF. Whether these findings are relevant to the different in vivo functions of EC residing different districts remains the focus of additional studies.