Ulstrastructural localization of tumour necrosis factor-alpha

Summary The application of an antibody against tumour necrosis factor-alpha (TNF) to thin sections of plastic-embedded mouse tissue has identified sites of TNF activity in normal and endotoxin-treated C₃N/HeN mice. Prior to endotoxin treatment, TNF was observed in the secretory granules of the antibacterial Paneth cell and one type of crypt endocrine cell. Four hours after endotoxin treatment, these two types of intestinal cell were found to have degranulated. In addition, endotoxin treatment resulted in the appearance of TNF in the secretory granules of all eosinophils, neutrophils and monocytes in the bone marrow, spleen, lung and the proximal intestine. TNF was also observed in the internal elastic lamina (IEL) of arterioles. These results suggest that the process of TNF induction specifically targets the immune system and the vasculature. An invasive stimulus, such as circulating endotoxin, can provoke the immune cells to be armed with TNF. That same stimulus may cause arteriole smooth muscle cells to secrete TNF. TNF secretion in the presence of arteriole smooth muscle cells may play a role in the adjustment of arteriole tone. In the venules, TNF may be responsible for platelet and neutrophil accumulation which leads to embolism formation.     

Trypsin induces tumour necrosis factor-alpha secretion from a human leukemic mast cell line

Trypsin activating both proteinase-activated receptor (PAR) 2 and PAR4 plays an important role in inflammation. We have investigated the potential of trypsin to induce TNF-alpha secretion from the human leukemic mast cell line (HMC-1). HMC-1 cells co-express both PAR2 and PAR4, and their agonist trypsin signals to HMC-1 cells. Trypsin (100 nm), SLIGKV-NH(2) (100 microm, corresponding to the PAR2 tethered ligand), or GYPGQV-NH(2) (100 microm, corresponding to the PAR4 tethered ligand) induced tumour necrosis factor (TNF)-alpha secretion from HMC-1 cells. TNF-alpha secretion by trypsin was significantly blocked by pretreatment with 50 microm PD098059, MEK-1 inhibitor. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) in HMC-1 cells without any detectable activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinase homologue. These results show that trypsin may induce TNF-alpha secretion following activation of ERK via both PAR2 and PAR4 on HMC-1 cells.     

Effects of tumour necrosis factor-alpha on BrdU incorporation in cultured human enterocytes

Bromodeoxyuridine incorporation is a useful method for studying the pattern of DNA synthesis in proliferating cells. The distribution pattern of incorporated BrdU in villus enterocytes of duodenal explants was analysed after exposure to TNFalpha in organ culture. TNFalpha caused a consistent, low level uptake of BrdU in the portion of the nucleus close to the nuclear membrane, this pattern was absent from the control cultures. As these epithelial cells are terminally arrested in G(0), the BrdU incorporation was thought not to be due to S phase DNA synthesis, but rather a response to the cytotoxic influence of TNFalpha. Microtitre plate proliferation assays of cell density and DNA synthesis were devised to study the effects of TNFalpha on confluent monolayers of the human foetal jejunal cell line I407 and the mouse fibrosarcoma cell line L929. Both cell lines showed a similar response to TNFalpha. Exposure to TNFalpha alone did not reduce cell numbers but did cause a significant increase in DNA synthesis (p < 0.05). When cycloheximtde was added in tandem with TNFalpha there was a significant reduction in cell number (p < 0.001) and level of DNA synthesis (p < 0.01) indicative of cell death. The DNA of cells exposed to TNFalpha and cycloheximide was fragmented when viewed on an electrophoresis gel. The results show that BrdU incorporation might be a good indicator of damage to the DNA of cells after cytotoxic insult. TNFalpha may be responsible for villus enterocyte damage in enteropathies such as coeliac disease and GVHR of the small bowel.     

Development of a sandwich ELISA for the quantification of enterovirus 71

Since 2008, enterovirus 71 (EV71) has been responsible for high-mortality seasonal epidemics of hand, foot and mouth disease in China. Currently many groups in the world are in the process of developing EV71 vaccines to combat this deadly disease. We have developed three EV71-specific monoclonal antibodies, and in this study we report the establishment of a fast and cost-effective sandwich ELISA kit for measurement of virus concentration in EV71 vaccines using a pair of mouse anti-EV71 monoclonal antibodies. The system is specific for EV71 virus, with no cross-reactivity to coxsackievirus A16, H1N1, rabies, and hepatitis A. Using a reference EV71 vaccine standard, the sensitivity of the assay kit was determined to be 0.82 U/ml, with a linear range between 3.75 and 120 U/ml.     

Development and validation of a sensitive ELISA for quantification of secretory IgA in rat saliva and faeces.

Non-invasive measures of immunological markers are an attractive means of stress assessment in laboratory animals. Salivary IgA has been used successfully as a stress marker in the human, and several reports indicate the potential of secretory IgA as a non-invasive measure of stress in animals. The present paper describes the development of an ELISA using commercially available components for the quantification of rat IgA and validation of this assay for the quantification of rat secretory IgA in saliva and faeces. The concentration of IgA in rat saliva varied significantly between duplicate samples obtained from individual rats, and the viscosity and small total volume of rat saliva gave unsatisfactory results for IgA. Faecal IgA was present in high concentrations, and duplicate samples varied by only 2-3%. However, faecal IgA seemed less stable than IgA in other biological compartments, and this finding must be taken into consideration when using quantitative measurements of IgA as a marker of mucous humoral immune status.     

Mechanisms of the induction of apoptosis in human hepatoma cells by tumour necrosis factor-alpha.

Tumour necrosis factor alpha (TNF-alpha) at 20 ng/ml induced apoptosis in human hepatoma cells in vitro. The effect of TNF-alpha-induced apoptosis was exacerbated by the hypoxanthine-xanthine oxidase (HX/XO) system and cycloheximide (CHX), but alleviated by superoxide dismutase (SOD), suggesting that TNF-alpha-induced apoptosis may be due to oxidative stress, and independent of protein synthesis. TNF-alpha elevated free Ca(2+)concentration, triggered lipid peroxidation and decreased the expression of bcl-2 protein. The findings suggest that TNF-alpha-induced apoptosis may be involved in stimulating Ca(2+)-dependent endonuclease activity and increasing membrane lipid peroxidation. Bcl-2 may play a pivotal role in serving as a Ca(2+)regulator or antioxidant, preventing lipid peroxidation in the process.     

An alternative approach to the detection of lignin: a note on the application of ELISA using polyclonal antibodies

A specific detection method for lignin has been developed using polyclonal antibodies raised against BSA coupled lignin. This method was found to be highly sensitive and linear in enzyme linked immunosorbant assay (ELISA) within the coating lignin concentration range of 0.01 7g/ml to 1 7g/ml. The interference in the detection due to the presence of lignin breakdown products and other coexisting biopolymers could be eliminated at an antiserum dilution of 1:25600.     

Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA.

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in many aspects of acute phase and immune responses. Species specificity in the biological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and in vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents. In this paper, we describe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris. Recombinant TNF-alpha was produced intracellularly in a soluble form, cells were lysed and the protein purified by ammonium sulphate precipitation, Sephadex G75 fractionation and finally, ion-exchange chromatography. The purified recombinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which is within 1 Da of the value predicted by the sequence data, taking into account N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101. Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, than the human standard preparation. Polyclonal antibodies were raised against purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA). The ELISA was sensitive to 10 pg.ml- 1 rat TNF-alpha and was specific for TNF-alpha, showing no cross-reactivity with rat IL-1alpha, rat IL-1beta, rat IL-1Ra or rat IL-6. The ELISA was used to measure TNF-alpha in the plasma of rats injected with bacterial endotoxin and in cultures of rat white blood cells. The ELISA was shown to be a robust method suitable for use in assaying samples generated in both in vivo or in vitro experiments.     

Paradoxical effects of tumour necrosis factor-alpha in adjuvant-induced arthritis

Anti-tumour necrosis factor (TNF)alpha therapy is highly effective in rheumatoid arthritis and it is surprising, therefore, that a recent study showed that intraperitoneal administration of recombinant TNFalpha reduced the severity of adjuvant-induced arthritis and decreased IFNgamma expression in cultured draining lymph node cells. Furthermore, in untreated arthritic rats, maximal TNFalpha expression in draining lymph node cells coincided with spontaneous disease remission, suggesting a role for endogenous TNFalpha in recovery from arthritis. If confirmed in further studies, these findings suggest that, in addition to its well-established pro-inflammatory properties, TNFalpha may also play a disease-limiting role in this model of rheumatoid arthritis by suppressing effector T cell responses.     

Signalling to translational activation of tumour necrosis factor-alpha expression in human THP-1 cells

Monocytic THP-1 cells expressed tumour necrosis factor-alpha (TNF-alpha) mRNA, but hardly any detectable TNF-alpha protein and a partially activated MAP kinase ERK-2 in the unstimulated state. Stimulation with phorbol ester led to expression of TNF-alpha protein without significant changes in mRNA, a response that was sensitive to the MEK-1/2 inhibitors PD98059 and U0126. A calcium signal also led to expression of TNF-alpha protein, but now accompanied by a rapid increase in mRNA. A synergistic effect between phorbol ester and calcium ionophore was evident at the level of TNF-alpha protein, but not its mRNA. Stimulation with anisomycin led to a TNF-alpha expression that was sensitive to the p38 inhibitor SB203580. Actinomycin D lowered TNF-alpha mRNA in a similar way as PD98059 but was less inhibitory on PMA- or anisomycin-induced formation of TNF-alpha, thus confirming that these agents acted by causing translational derepression. Thus, in THP-1 cells MAP kinase pathways involving MEK-1/2 and possibly ERK-2 as well as the human p38 analogue were essential for basal TNF-alpha mRNA expression and translational activation.     

Mitogenic action of tumour necrosis factor-alpha and interleukin-8 on explants of human duodenal mucosa

Our aim is to examine whether tumour necrosis factor-alpha (TNF-alpha) and interleukin affect the mitotic activity in explants of human duodenal mucosa and to estimate the release of cytokines from explants incubated with TNF-alpha. Biopsy specimens of normal duodenal mucosa were taken from 19 subjects that underwent upper endoscopy for investigation of dyspeptic symptoms or chronic gastrointestinal bleeding. The specimens were processed following guidelines for organ culture technique. Paired biopsy specimens from 12 subjects were cultured for 23 h to achieve steady state and thereafter the explants were incubated 25 h with 10(-13)-10(-9) M of TNF-alpha or IL-8. Mitoses were arrested in the metaphase by adding vincristine sulphate for the last three hours. The explants were then fixed and processed for microdissection. Fifteen crypts were microdissected and the total number of metaphases was determined using the whole crypt as reference volume. The number of metaphases per crypt was also estimated in explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies. Additional duodenal explants from seven subjects were incubated with 10(-10) M TNF-alpha for 25 h. Thereafter the release of IL-1-beta, IL-6, IL-8 and interferon gamma (IFN-gamma) into the culture medium was measured by enzyme immunoassay and expressed as pg/mg protein. TNF-alpha and IL-8 significantly increased the number of metaphases/crypts (P<0.0001). The addition of anti-IL-8 slightly reduced the number of metaphases/crypt compared to the values observed in the explants incubated with 10(-10) M TNF-alpha alone (P<0.0001). The number of metaphases/crypt in the explants incubated with 10(-10) M TNF-alpha in the presence of anti-IL-8 antibodies was, however, markedly and significantly higher than that of the controls (P<0.000). TNF-alpha induced the release of IL-8 (P<0.01) and IL-6 (P<0.05) from the duodenal explants. TNF-alpha and IL-8 are potent mitogens to human small intestinal crypts. The mitogenic action of TNF-alpha is primarily a direct effect of the cytokine and only to a minor extent mediated by a secondary production of IL-8 in the duodenal explant. Our findings indicate that TNF-alpha and IL-8 may participate in the regulation of cell proliferation in the human small intestinal epithelium.     

The effect of tumour necrosis factor-alpha (TNF-alpha) muteins on human neutrophils in vitro

Tumour necrosis factor-alpha (TNF-alpha) has been implicated as an important inflammatory mediator. In vitro, TNF-alpha is reported to activate human polymorphonuclear neutrophils (PMN), inducing responses such as phagocytic activity, degranulation and oxidative metabolism. Biological responses to TNF-alpha are initiated by its binding to specific cell surface receptors, and various studies have shown that the major TNF receptor species on PMN is the 75 kDa receptor. To verify the suggestion that the receptor binding domain includes the region close to the N-terminus of the TNF-alpha molecule, four TNF-alpha derivatives termed muteins were constructed, using a synthetic cDNA fragment substituting the N-terminal 3-7 selected hydrophilic or hydrophobic amino acids in the original TNF-alpha genomic DNA. Binding of muteins to PMN was assessed using monoclonal antibodies recognizing either the 55 kDa (p55) or the 75 kDa (p75) TNF receptor subtypes. Blocking by muteins of anti-p75 antibody binding to PMN was as expected from their N-terminal amino acid composition and hydrophilic properties. Hydrophilic muteins competed well with anti-TNF receptor antibodies for binding to the p75 receptor. In contrast, hydrophobic muteins were unable to block anti-p75 binding. Similarly, degranulation, chemiluminescence or enhancement of the PMN response to specific stimuli by the muteins correlated with the hydrophilic properties of the muteins. The significance of these observations in relation to the molecular structure of TNF-alpha is discussed.     

Production and utilization of polyclonal antibodies against nisin in an ELISA and for immuno-location of nisin in producing and sensitive bacterial strains

Specific nisin polyclonal antibodies (PAb) were produced in rabbits using nisin Z produced by Lactococcus lactis subsp. lactis biovar diacetylactis UL 719. Antisera were obtained from white female New Zealand rabbits that were first immunized with a nisin Z-keyhole limpet haemocyanin conjugate and boosted with free nisin Z. Nisin-specific PAb were purified by affinity chromatography with a yield of 15 mg specific antinisin 100 ml-1 serum. The detection limit of the ELISA test for nisin Z was 0.75 ng ml-1 in buffer but was 1.7 and 3.5 ng ml-1 in milk and complex media broth spiked (5, 10, 20 microg ml-1) with nisin Z, respectively. In nisin Z-spiked samples, the average concentration was between 90 and 107% of actual added amount. In contrast, when the bioassay (microtitration method) was used, only 50-63% of nisin Z biological activity could be detected. In addition, the affinity-purified nisin PAb, antirabbit IgG gold conjugate and transmission electron microscopy were successfully used to locate nisin Z on producing cells and to observe its bactericidal effects against sensitive cells.     

Identification of the forms of human leukemia using polyclonal antibodies to erythroblast antigen

Using polyclonal antibodies to an interspecies antigen of erythroblasts (Ag-Eb) with a molecular weight 69 000 D this antigen was revealed by immunofluorescence on the cells of the peripheral blood of patients with erythroleukemias and, in several cases, in those with undifferentiated leukemias. The possibility was shown of using these antibodies as a diagnostic tool when studying erythroleukemias and acute undifferentiated leukemias.     

Apremilast,a novel PDE4 inhibitor,inhibits spontaneous production of tumour necrosis factor-alpha from human rheumatoid synovial cells and ameliorates experimental arthritis

Introduction  

Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis.     

Synergistic induction of apoptosis in human endothelial cells by tumour necrosis factor-alpha and transforming growth factor-beta

Serum deprivation stimulates endothelial apoptosis while albumin inhibits this and has been proposed as important in confining apoptotic remodelling to poorly perfused vessels. Tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta are also reported to induce endothelial apoptosis. To investigate the comparative roles of these stimuli, the effect of TNF-alpha and TGF-beta, alone or in combination, in the presence or absence of serum or albumin was studied. There was strong synergy between the cytokines in inducing human umbilical vein endothelial cell apoptosis, but only in the absence of serum. Synergy was destroyed by boiling cytokines and was not affected by polymyxin B. Dose response experiments revealed greater activity of TGF-beta(1) than TGF-beta(2). The synergy was protein synthesis dependent and apoptosis was confirmed by DNA gel electrophoresis, transmission electron microscopy and FACS analysis. Data suggests a role for synergistic activation of endothelial cell apoptosis by TNF-alpha and TGF-beta(1) but perhaps only in poorly perfused vessels deprived of serum factors.     

An ELISA method for the detection and quantification of human heparanase

Heparanase is a mammalian endo-beta-D-glucuronidase that cleaves heparan sulfate side chains at a limited number of sites. Heparanase enzymatic activity is thought to participate in degradation and remodeling of the extracellular matrix and to facilitate cell invasion associated with tumor metastasis, angiogenesis, and inflammation. Traditionally, heparanase activity was well correlated with the metastatic potential of a large number of tumor-derived cell types. More recently, heparanase upregulation was detected in an increasing number of primary human tumors, correlating, in some cases, with poor postoperative survival and increased tumor vascularity. The present study was undertaken to develop a highly sensitive ELISA suitable for the determination and quantification of human heparanase in tissue extracts and body fluids. The assay preferentially detects the 8+50 kDa active heparanase heterodimer vs. the latent 65 kDa proenzyme and correlates with immunoblot analysis of heparanase containing samples. It detects heparanase at concentrations as low as 200 pg/ml and is suitable for quantification of heparanase in tissue extracts and urine.     

Tumor necrosis factor-alpha dependent cytotoxicity of human skin mast cells is enhanced by anti-IgE antibodies

Mast cells dispersed from human skin and purified by density-gradient centrifugation were cytotoxic toward the mouse fibrosarcoma cell line WEHI-164. Skin mast cells were not cytotoxic toward the NK cell-sensitive cell line K562. Killing of WEHI-164 occurred over a prolonged (greater than 18 h) period of incubation with mast cells and was effectively inhibited by polyclonal antibodies and mAb against TNF-alpha suggesting that this cytokine plays an important role in mast cell-mediated cytotoxicity. Whereas lysates of rat peritoneal mast cells exhibited cytotoxicity toward WEHI-164, this was not found with lysates of unstimulated skin mast cells suggesting that TNF-alpha is not stored preformed in the latter. Killing of WEHI-164 cells by skin mast cells was enhanced by anti-IgE and there was a significant correlation between histamine release and cytotoxicity after activation with this stimulus. We conclude that human skin mast cells are a potential source of TNF-alpha and suggest that these cells, particularly after activation, might contribute to the synthesis of this multifunctional cytokine in inflammatory sites.     

Development of a double-antibody sandwich ELISA for sensitive detection of Yersinia pestis

We developed a biotin–streptavidin-based sandwich ELISA for the sensitive and specific detection of Yersinia pestis. In this assay, the F1 capsular protein and Y. pestis were captured by anti-F1 mouse monoclonal antibody followed by detection with biotinylated-anti-F1 rabbit polyclonal antibody and HRP-conjugated streptavidin. The developed F1 ELISA could detect not only the F1 protein up to 29 and 17 pg/ml but also Y. pestis up to 177.8 and 129.2 CFU/ml in PBS buffer and human serum, respectively. In addition, the F1 ELISA did not show any cross-reactivity with various proteins and bacterial strains.