Establishment of a cell-based assay for examining the expression of tumor necrosis factor alpha (TNF-α) gene

Tumor necrosis factor alpha (TNF-alpha) is a proinflammatory cytokine produced by activated macrophages and lymphocytes and involved in many inflammatory diseases. Preventing the production or action of TNF-alpha is a potent therapeutic strategy for these inflammatory diseases. Since there is a lack of rapid and effective assay for examining the expression TNF-alpha in macrophages, we attempt to establish a reporter system to assess TNF-alpha gene expression through measuring luciferase activity. In this study, mouse macrophage cell line RAW 264.7 was stably transfected with a luciferase reporter pGL3-TNFPro-UTR, which contains TNF-alpha promoter and 3'-untranslated region (3'-UTR). The TNF-alpha-luciferase reporter cell line is used for assessing the expression of TNF-alpha gene induced by LPS in the presence or absence of chemicals that inhibit the biosynthesis of TNF-alpha such as dexamethasone and emodin, and also for measuring change of expression of TNF-alpha gene under downregulation of the expression of steroid receptor coactivator-3, a modulator for TNF-alpha. The luciferase activity correlated well with the ELISA results for TNF-alpha production, therefore, the TNF-alpha-luciferase reporter cell line is a sensitive, effective tool for studying the expression of TNF-alpha gene.     

Glial tumor necrosis factor alpha (TNFα) generates metaplastic inhibition of spinal learning

Injury-induced overexpression of tumor necrosis factor alpha (TNFα) in the spinal cord can induce chronic neuroinflammation and excitotoxicity that ultimately undermines functional recovery. Here we investigate how TNFα might also act to upset spinal function by modulating spinal plasticity. Using a model of instrumental learning in the injured spinal cord, we have previously shown that peripheral intermittent stimulation can produce a plastic change in spinal plasticity (metaplasticity), resulting in the prolonged inhibition of spinal learning. We hypothesized that spinal metaplasticity may be mediated by TNFα. We found that intermittent stimulation increased protein levels in the spinal cord. Using intrathecal pharmacological manipulations, we showed TNFα to be both necessary and sufficient for the long-term inhibition of a spinal instrumental learning task. These effects were found to be dependent on glial production of TNFα and involved downstream alterations in calcium-permeable AMPA receptors. These findings suggest a crucial role for glial TNFα in undermining spinal learning, and demonstrate the therapeutic potential of inhibiting TNFα activity to rescue and restore adaptive spinal plasticity to the injured spinal cord. TNFα modulation represents a novel therapeutic target for improving rehabilitation after spinal cord injury.     

Is tumor necrosis factor alpha a trigger for the initiation of endometrial prostaglandin F(2alpha) release at luteolysis in cattle?

To determine the physiological significance of tumor necrosis factor alpha (TNFalpha) in the regulation of luteolytic prostaglandin (PG) F(2alpha) release by the bovine endometrium, the effect of TNF-alpha on PGF(2alpha) output by the endometrial tissues in vitro was investigated and compared with the effect of oxytocin (OT). Furthermore, the presence of specific receptors for TNFalpha in the bovine endometrium during the estrous cycle was determined. Endometrial slices (20-30 mg) taken from six stages of the estrous cycle (estrus: Day 0; early I: Days 2-3; early II: Days 5-6; mid-: Days 8-12; late: Days 15-17; and follicular: Days 19-21), as determined by macroscopic examination of the ovaries and uterus, were exposed to TNFalpha (0.06-6 nM) and/or OT (100 nM). OT stimulated PGF(2alpha) output at the follicular stage and at estrus (P < 0.001), but not at the late luteal stage. On the other hand, the stimulatory effects of TNFalpha on PGF(2alpha) output were observed not only at the follicular stage but also at the late luteal stage (P < 0.001). When the endometrial tissues at late luteal stage were simultaneously exposed to TNFalpha (0.6 nM) and OT (100 nM), the stimulatory effect on PGF(2alpha) output was higher than the effect of TNFalpha or OT alone (P < 0.05). Specific binding of TNFalpha to the bovine endometrial membranes was observed throughout the estrous cycle. The concentration of TNF-alpha receptor at the early I luteal stage was less than the concentrations at other luteal stages (P < 0.01). The dissociation constant (K(d)) values of the endometrial membranes were constant during the estrous cycle. The overall results lead us to hypothesize that TNFalpha may be a trigger for the output of PGF(2alpha) by the endometrium at the initiation of luteolysis in cattle.     

Effector mechanism of human monocyte-mediated cytotoxicity: role of a new tumor cytotoxic factor distinct from interleukin 1 and tumor necrosis factorα

Human blood monocytes activated to the tumoricidal state were previously found to release a factor(s) responsible for tumor cell killing. The activity of the tumor cytotoxic factor(s) (TCF) was determined by release assay of radioactivity from human A375 melanoma cells. On fractionation of the supernatant of activated monocytes by Ultrogel AcA34 and TSK-G3000SW gel chromatographies two major peaks of the material with TCF activity with MWs of 30,000 and 15,000, called TCF-I and TCF-11, respectively were obtained. TCF-II could be neutralized by polyclonal anti-IL-1 antiserum, but anti-IL-1 antiserum did not neutralize either factor. TCF-I was separated by ampholine column electrofocusing into three major fractions with TCF activity at pI 5, 6 and 6.8, named TCF-1, TCF-1 and TCF-1, respectively. The cytotoxic and IL-1 activities of TCF-1 were neutralized by anti-IL-1 serum, whereas those of TCF-1 and TCF-1 were not completely neutralized by anti-IL-1 or anti-IL-1 antiserum. On DEAE ion-exchange chromatography (TSK DEAE 5PW) TCF-I gave two peaks with TCF activity (TCF-I1 and TCF-I2). TCF-I1 was slightly neutralized by anti-TNF antibody, but TCF-I2 was not affected by antisera against IL-1 and IL-1, or anti-TNF antibody, thus ruling out the possibility that tumor necrosis factor (TNF) might be involved in tumor cell killing mediated by TCF-I2. These results indicate that human monocyte-mediated cytotoxicity against human A375 melanoma cells is mediated in part by a tumor cytotoxic factor (TCF; MW, 30,000; pI 6), differing from IL-1 and TNF.     

The nucleotide sequence of a wheat γ-gliadin genomic clone

γ-Gliadins are a subclass of the storage proteins from wheat. We have isolated from a wheat genomic library a clone encoding a γ-gliadin gene. The amino acid sequence derived from the gene sequence reveals a mature protein (308 amino acids, excluding a 19 amino acid signal sequence) with a characteristic repeated structure of the consensus heptapeptide PQQPFPQ corresponding to the high glutamine (35.4%) and proline (17.5%) content. The gene does not contain an intron, and possesses a typical eukaryotic promoter.     

Characterization of golimumab,a human monoclonal antibody specific for human tumor necrosis factor α

We prepared and characterized golimumab, a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p = 0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2-fold; p = 0.017) and adalimumab (3.3-fold; p = 0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8°C vs. 69.5°C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.Key words: TNF, golimumab, neutralization, affinity, bioassay, arthritis, stability, solubility     

Characterization of golimumab,a human monoclonal antibody specific for human tumor necrosis factor α

We prepared and characterized golimumab (CNTO148), a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p=0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2 fold; p=0.017) and adalimumab (3.3-fold; p=0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8 °C vs. 69.5 °C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.     

Effect of one night of sleep loss on changes in tumor necrosis factor alpha (TNF-α) levels in healthy men

Total sleep deprivation in humans is associated with increased daytime sleepiness, decreased performance, elevations in inflammatory cytokines, and hormonal/metabolic disturbances.To assess the effects of 40 h of total sleep deprivation (TSD) under constant and well controlled conditions, on plasma levels of TNF-α and its receptor (TNFR1), interleukin-6 (IL-6), cortisol and C-reactive protein (CRP), sleepiness and performance, 12 healthy men (29 ± 3 years) participated in a 5-days sleep deprivation experiment (two control nights followed by a night of sleep loss and one recovery night). Between 0800 and 2300 (i.e. between 25 and 40 h of sleep deprivation), a serial of blood sampling, multiple sleep latency, subjective levels of sleepiness and reaction time tests were completed before (day 2: D2) and after (day 4: D4) one night of sleep loss. We showed that an acute sleep deprivation (i.e. after 34 and 37 h of sleep deprivation) induced a significant increase in TNF-α (P < 0.01), but there were no significant changes in TNFR1, IL-6, cortisol and CRP. In conclusion, our study in which constant and controlled experimental conditions were realized with healthy subjects and in absence of psychological or physical stressors, an acute total sleep deprivation (from 34 h) was sufficient to induce secretion of pro-inflammatory cytokine such as TNF-α, a marker more described in chronic sleep restriction or deprivation and as mediators of excessive sleepiness in humans in pathological conditions.     

Mycoplasma capricolum membranes induce tumor necrosis factor α by a mechanism different from that of lipopolysaccharide

Summary Heat-inactivated (60°C, 45 min)Mycoplasma capricolum strain JR cells activate murine macrophages to secrete high levels of tumór necrosis factor (TNF) and to lyse tumor target cells efficiently. Fractionation of the intactM. capricolum cells, obtained from cells harvested at the exponential phase of growth, shows that their capacity to induce TNF secretion by macrophage resides exclusively in the membrane fraction. The macrophage-mediated cytolysis following activation byM. capricolum membranes was significantly inhibited by specific anti-recombinant murine TNF antibodies.M. capricolum membranes are a potent inducer of TNF as the commonly used bacterial lipopolysaccharide, indicated by their doseresponse curve for macrophage activation. Our study further showed thatM. capricolum membranes and lipopolysaccharide synergize to augment TNF secretion by C57BL/6-derived macrophages markedly. Moreover, lipopolysaccharide-unresponsive C3H/HeJ-derived macrophages, were pronouncedly activated byM. capricolum membranes, which do not contain lipopolysaccharide. These findings suggest that the mechanism by whichM. capricolum membranes activate macrophages differs from that of lipopolysaccharide. Results of preliminary experiments show that human monocytes as well secrete TNF following activation byM. capricolum membranes. Thus, in contrast with the prohibitive toxicity of lipopolysaccharide to animals and humans,M. capricolum membranes, which contain no lipopolysaccharide and are nontoxic in nature, may be of therapeutic value in the treatment of cancer.This study was supported by the Ernst David Bergmann Fund of the Hebrew University, Jerusalem, the Concern II Foundation for Cancer Research, Los Angeles, and the Society of Research Associates of the Lautenberg Center     

Intermolecular binding between TIFA-FHA and TIFA-pT mediates tumor necrosis factor alpha stimulation and NF-κB activation

The forkhead-associated (FHA) domain recognizes phosphothreonine (pT) with high specificity and functional diversity. TIFA (TRAF-interacting protein with an FHA domain) is the smallest FHA-containing human protein. Its overexpression was previously suggested to provoke NF-κB activation, yet its exact roles in this signaling pathway and the underlying molecular mechanism remain unclear. Here we identify a novel threonine phosphorylation site on TIFA and show that this phosphorylated threonine (pT) binds with the FHA domain of TIFA, leading to TIFA oligomerization and TIFA-mediated NF-κB activation. Detailed analysis indicated that unphosphorylated TIFA exists as an intrinsic dimer and that the FHA-pT9 binding occurs between different dimers of TIFA. In addition, silencing of endogenous TIFA resulted in attenuation of tumor necrosis factor alpha (TNF-α)-mediated downstream signaling. We therefore propose that the TIFA FHA-pT9 binding provides a previously unidentified link between TNF-α stimulation and NF-κB activation. The intermolecular FHA-pT9 binding between dimers also represents a new mechanism for the FHA domain.     

Purification of recombinant proteins with an example of tumor necrosis factor thymosin-α1

Hybrid protein, cancer necrosis factor thymosin-α1 (TNF-T), when synthesizing in strain-producer of Escherichia coli SG200-50 with plasmid pThy315, was a part of “inclusion bodies” mostly in the form of a high-molecular complex with other proteins due to the S-S bonds formation. An approach of purification of TNF-T has been proposed, which is based on the destruction of the complex in the presence of sodium dodecylsulfate (DDS-Na) and dithiotreitol (DDT) followed by gel-filtration on Sephadex G-100 and renaturation by ultrafiltration on hollow fibers. The method allows the isolation of electrophoretically homogeneous TNF-T containing no DDS-Na and having high cytotoxic activity against cancer cells of mouse adenocarcinome L-929. The yield of TNF-T achieved 80% relative its content in biomass and 30% relative the total protein.     

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic α-amylase

Porcine pancreatic α-amylase (1,4-α-d-glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Relationship of tumor necrosis factor α expression with activation of sphingomyelinase and lipid peroxidation after removal of cholestatic factor

The goal of this work was to study the expression of tumor necrosis factor α (TNFα), sphingomyelin cycle activation, and lipid peroxidation (LPO) processes after the removal of a cholestatic factor in the liver subjected to different durations of cholestasis. Restored bile flow after a 9-day hepatic cholestasis normalized sphingomyelinase (SMase) activity and levels of TNFα and LPO products. The removal of a cholestatic factor after a 12-day cholestasis did not normalize the studied parameters: SMase activity and the levels of TNFα and LPO products remained much higher compared to control. A significant positive correlation between TNFα expression, SMase activity, and LPO rate has been revealed. The obtained data indicate that hepatocyte apoptosis after bile outflow restoration in late cholestasis can be due to the activation of the sphingomyelin cycle, LPO, and TNFα expression. The synergistic interaction can sharply increase the proapoptotic capacity of each of these factors since TNFα activates SMase and LPO, SMase activity depends on the LPO rate, while ceramide, an SMase-produced secondary messenger of apoptosis, can induce oxidative stress.     

Interleukin-4 plus tumor necrosis factor α augments the antigenicity of melanoma cells

Immune cytokines are important regulators of the immune response to neoplastic cells. We previously reported that interleukin 4 (IL-4) and either tumor necrosis factor α (TNF) or interferon γ (IFN) synergistically inhibit melanoma cell growth and induce cell differentiation. In the present study we used various combinations of IL-4, IFN and TNF to enhance the antigenicity of melanoma cells. IL-4 plus TNF significantly increased the ability of melanoma cells to stimulate cytotoxic T cells (CTL) and act as targets of these CTL; IL-4 plus IFN was somewhat less effective, while TNF plus IFN was not as effective. IL-4 plus TNF also increased the expression of HLA class I and HLA-DR antigens on melanoma cells. The CTL lines examined in this study were CD3⁺CD4⁺ and oligoclonal. These preclinical results suggest that the immune response to melanoma whole-cell vaccines might be enhanced by pretreating vaccine cells with IL-4 plus TNF.     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.