Inhibition of tumor necrosis factor alpha signaling by anti-tumor necrosis factor alpha antibodies and pentoxifylline is unable to prevent fumonisin hepatotoxicity in mice.

Fumonisin B1 (FB1), a mycotoxin from Fusarium verticillioides, disrupts sphingolipid metabolism by inhibiting ceramide synthase leading to modulation of cytokines including tumor necrosis factor (TNF) alpha. Current study investigated the effect of interrupting TNFalpha signaling, known to be involved in FB1 hepatotoxicity. Male C57BL/6N mice were injected intravenously once with anti-TNFalpha antibodies or treated with pentoxifylline at 150 mg/kg intraperitoneally twice a day for 5 days to inhibit TNFalpha production before and during subcutaneous injection of 2.25mg FB1/kg daily for 5 days; mice were sampled one day after the last treatment. Results showed that both anti-TNFalpha antibodies and pentoxifylline did not prevent FB1 hepatotoxicity; the latter was somewhat augmented, indicated by increases in circulating alanine aminotransferase and aspartate aminotransferase, and incidence of apoptotic hepatocytes. Anti-TNFalpha antibodies did not alter FB1-induced accumulation of free sphingoid bases or expression of TNFalpha in liver following the FB1 treatment. Pentoxifylline significantly reduced accumulation of free sphinganine and expression of TNFalpha. Neither anti-TNFalpha antibodies nor pentoxifylline altered FB1-induced expression of interleukin-12, interferongamma, lymphotoxinbeta, and c-myc. Expression of c-myc, an inducer of cell death, increased after interference with TNFalpha signaling. These findings suggest a dual role of TNFalpha signaling activation in FB1 hepatotoxicity.     

人肿瘤坏死因子受体75在人肿瘤坏死因子α介导的细胞毒效应中的双重功能

目的：研究人肿瘤坏死因子受体75（hTR75）在hTNFα所引发的细胞毒效应中的功能。方法：用电脉冲将hTR75基因表达载体转入只有内源性hTR55表达的HEp-2细胞中构建了两种TNF受体同时表达的HEp-2-A75细胞株。通过定点突变构建及在大肠杆菌中高效表达,分离纯化出两种分别对hTR55以及hTR75受体具有选择结合活性的hTNFα突变体。野生型hTNFα及其这两种突变体对HEp-2以及HEp-2-A75细胞的细胞毒效应采用比色法测定并进行了比较。结果：RT－PCR以及间接ELISA的结果表明hTR75在RNA转录以及蛋白质翻译水平均获得了表达,其表达量由Scatchard分析确定。HEp-2细胞中hTR75的存在可以明显增强HEp-2细胞对hTNFα的敏感性。结论：hTR75受体在介导hTNFα的细胞毒活性中具有双重功能,即hTR75不但自身可介导部份hTNFα所引发的细胞毒效应,它对hTR55所介导的细胞毒效应还具有相当的协同作用。     

Role of tumor necrosis factor receptor 1 (p55) in hepatocyte proliferation during acetaminophen-induced toxicity in mice

Hepatocyte proliferation represents an important part of tissue repair. In these studies, TNF receptor 1 (TNFR1) knockout mice were used to analyze the role of TNF-alpha in hepatocyte proliferation during acetaminophen-induced hepatotoxicity. Treatment of wild-type (WT) mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was associated with proliferation of hepatocytes surrounding the damaged areas, which was evident at 24 h. The cell cycle regulatory proteins, cyclin D1 and cyclin A, were also up regulated in hepatocytes. In contrast, in TNFR1-/- mice, which exhibit exaggerated acetaminophen hepatotoxicity, hepatocyte proliferation, and expression of cyclin D1 and cyclin A, as well as the cyclin dependent kinases, Cdk4 and Cdk2, were reduced. The cyclin-dependent kinase inhibitor p21 was also induced in the liver following acetaminophen administration. This was greater in TNFR1-/- mice compared to WT mice. To investigate mechanisms mediating the reduced hepatic proliferative response of TNFR1-/- mice, we analyzed phosphatidyl inositol-3-kinase (PI-3K) signaling. In both WT and TNFR1-/- mice, acetaminophen caused a rapid increase in total PI-3K within 3 h. Acetaminophen also increased phosphorylated PI-3K, but this was delayed 6-12 h in TNFR1-/- mice. Expression of Akt, a downstream target of PI-3K, was increased in both WT and TNFR1-/- mice in response to acetaminophen. However, the increase was greater in WT mice. Acetaminophen-induced expression of phosphorylated STAT3, a key regulator of cytokine-induced hepatocyte proliferation, was also delayed in TNFR1-/- mice relative to WT. These data suggest that TNF-alpha signaling through TNFR1 is important in regulating hepatocyte proliferation following acetaminophen-induced tissue injury. Delayed cytokine signaling may account for reduced hepatocyte proliferation and contribute to exaggerated acetaminophen-induced hepatotoxicity in TNFR1-/- mice.     

Pharmacokinetics of tumor necrosis factor alpha after intravenous administration in rats. Dose dependence and influence of tumor necrosis factor beta

2, 10, 20, 100 and 500 micrograms/kg/0.5 h of recombinant human tumor necrosis factor alpha (TNF-alpha) were administered to rats by intravenous infusion over 30 min. At high doses (greater than 10 micrograms/kg/0.5 h) the plasma half-life (t1/2, about 0.5 h) of TNF-alpha was practically dose independent. In contrast at the lowest dose (2 micrograms/kg/0.5 h) the elimination of TNF-alpha was markedly faster: A plasma half-life of only 5-8 min was determined. However, when in addition to 2 micrograms/kg/0.5 h of TNF-alpha a high dose of recombinant human tumor necrosis factor beta (TNF-beta) was infused the plasma half-life was enhanced up to values at high doses (greater than 10 micrograms/kg/0.5 h). Thus at the lowest dose the speed of elimination of TNF-alpha was enhanced which could be prevented by TNF-beta.     

新型重组人肿瘤坏死因子工程菌的发酵工艺研究

目的研究并建立新型重组人肿瘤坏死因子 (nrhTNF)的发酵工艺。方法通过探讨nrhTNF工程菌在试管和锥形摇瓶中的生长和表达规律 ,筛选并确定其最适宿主菌、最佳培养基及最佳诱导表达条件 ,然后在 5L自控发酵罐中进行分批补料培养。结果nrhTNF工程菌在 5L发酵罐中培养至终密度A60 0nm30左右时 ,目的蛋白的表达最高 ,保持在菌体总蛋白的 5 0 %附近 ,发酵时间为 12～ 13h。结论确定了周期短、产率高且稳定可靠的发酵工艺 ,为nrhTNF的工业化生产奠定了基础。     

Mitigation of tumor necrosis factor alpha cytotoxicity by aurintricarboxylic acid in human peripheral B lymphocytes

The aims of this study were to ascertain whether aurintricarboxylic acid (ATA), an endonuclease inhibitor, known to interfere, with the actions of cytokines such as interferons, is able to antagonize the toxic effects produced by tumor necrosis factor alpha (TNF-alpha) in human healthy peripheral B lymphocytes and try to elucidate the molecular machinery through which this possible antagonism takes place. Results evidenced that the balance of survival signals of human B lymphocytes in the presence of TNF-alpha was altered by the interaction of TNF-alpha with a salicylate compound, ATA. Apoptosis effected by TNF-alpha alone was suppressed in the presence of ATA, and this effect appeared essentially characterized by: (i) phosphorylation of phosphatidylinositol-3 kinase (PI-3K), influencing in turn protein kinase B/Akt (Akt) and Bad phosphorylation; (ii) nuclear translocation of the nuclear factor kappa B (NF-kappaB) and (iii) nuclear translocation of protein kinase C zed (PKCzeta). Reversal of TNF-alpha/ATA effects occurred in the presence of the PI-3K specific inhibitors wortmannin or LY294002 in the culture medium and was coincident with inhibition of the translocation of PKCzeta in the nucleus, while NF-kappaB was less affected. These results indicate, therefore, that PI-3K-mediated activation and nuclear transfer of PKCzeta might be essential steps of ATA antagonism against TNF-alpha, suggesting that possible ATA pharmacological applications might be taken into account for staving off systemic or local toxic effects produced by TNF-alpha.     

In vivo evidence for protease-catalysed mechanism providing bioactive tumor necrosis factor alpha

Mice pretreated by intravenous injection of 42 mg/kg of the serine protease inhibitor alpha 1-antitrypsin prior to a hepatotoxic dose of D-galactosamine/lipopolysaccharide (GalN/LPS) were fully protected against hepatitis. Pretreatment with alpha 1-antitrypsin with doses up to 300 mg/kg at different times failed to protect galactosamine sensitized animals against tumor necrosis factor alpha (TNF alpha)-induced hepatitis. No bioactive TNF alpha was detectable in serum of mice protected against GalN/LPS-induced hepatitis by pretreatment with alpha 1-antitrypsin. In contrast, abundant amounts of TNF were found in sera of GalN/LPS-treated control animals. It is concluded that a serine protease sensitive to alpha 1-antitrypsin provides bioactive TNF alpha by proteolytic cleavage of a TNF alpha precursor.     

Paradoxical role of tumor necrosis factor alpha in fumonisin-induced hepatotoxicity in mice

The use of tamoxifen (TAM) has been questioned on the chemotherapy and chemoprevention of breast cancer due to several estrogen receptor-independent cytotoxic effects. As an alternative, its more active metabolite 4-hydroxytamoxifen (OHTAM) has been proposed with presumed lower side effects. In this work, the potential OHTAM toxicity on rat liver mitochondrial bioenergetics in relation to the multiple deleterious effects of TAM was evaluated. OHTAM, at concentrations lower than those putatively reached in tissues following the administration of TAM, does not induce significant perturbations on the respiratory control ratio (RCR), ADP/O, transmembrane potential (DeltaPsi), phosphorylative capacity and membrane integrity of mitochondria. However, at high concentrations, OHTAM depresses the DeltaPsi, RCR and ADP/O, affecting the phosphorylation efficiency, as also inferred from the DeltaPsi fluctuations and pH changes associated with ADP phosphorylation. Moreover, OHTAM, at concentrations that stimulate the rate of state 4 respiration in parallel to the decrease in the DeltaPsi and phosphorylation rate, causes mitochondrial swelling and stimulates both ATPase and citrate synthase activities. However, the OHTAM-observed effects, at high concentrations, are not significant relatively to the damaging effects promoted by TAM and suggest alterations to mitochondrial functions due to proton leak across the mitochondrial inner membrane.     

Partial biochemical characterization of spiroplasma membrane component inducing tumor necrosis factor alpha

We have recently found that membranes of Spiroplasma spp. strain MQ-1 (hereafter referred to as MQ-1) induce both tumor necrosis factor alpha (TNF alpha) secretion by bone marrow macrophages and blast transformation of lymphocytes via a mechanism different from that operated by bacterial lipopolysaccharide (LPS). This report presents evidence indicating that the MQ-1-derived membrane component(s) which activates bone marrow macrophages to secrete TNF alpha is, at least in part, protein. This conclusion is supported by our findings that TNF alpha secretion was reduced following exposure of MQ-1 membranes to elevated temperatures, extreme acidic pH treatment and incubation with protease K or pronase. Furthermore, following lipid extraction of MQ-1 membranes, most of both induction of TNF alpha secretion and blast transformation activities appeared in the 'protein' fraction. When membranes were chromatographed on a phenyl-Sepharose column, two major peaks were obtained, one containing most of the TNF alpha induction activity and the other the mitogenic activity. Neither peak coeluted with the peak of bulk membrane lipids. The possibility that the spiroplasma membrane component inducing TNF alpha secretion is acylated protein is discussed.     

Tunicamycin potently inhibits tumor necrosis factor-induced hepatocyte apoptosis

The protein glycosylation inhibitor tunicamycin protected male BALB/c mice from tumor necrosis factor α-induced liver failure. Tunicamycin also inhibited tumor necrosis factor-induced cell death in primary hepatocyte cultures with a median inhibitory concentration of 8 nM, but not in the tumor cell line WEHI 164 clone 13. Hepatocyte death in our culture system was characterized by DNA fragmentation and apoptotic changes. These two characteristic signs of programmed cell death were also inhibited by tunicamycin treatment. These data suggest that protein glycosylation is an early and causal event of tumor necrosis factor (TNF)-induced parenchymal cell death in the liver.     

聚乙二醇化重组人肿瘤坏死因子-α:制备和抗肿瘤活性

AIM: To assess the merits of polyethylene glycol-modified recombinant human tumor necrosis factor alpha (PEG-rHuTNF-alpha). METHODS: The rHuTNF-alpha was modified with N-succinimidyl succinate monomethoxy polyethylene glycol (SS-PEG) of three different molecular weights. The PEG-rHuTNF-alpha was separated into fractions of various molecular weights by gel filtration chromatography. In vitro activities of various fractions were determined with L929 cell assay and in vivo anti-tumor potencies of main fractions were studied with respect to necrosis of S-180 solid tumor. RESULTS: The rHuTNF-alpha could be modified using SS-PEG under mild conditions. The main fraction of PEG5000-rHuTNF-alpha contained four PEG molecules, and PEG12000-rHuTNF-alpha and PEG20000-rHuTNF-alpha contained two PEG molecules, respectively. There was a higher activity when rHuTNF-alpha was coupled to less numbers of the same molecular weight PEG molecules. When PEG-rHuTNF-alpha was of the same molecular weight, rHuTNF-alpha modified with bigger molecular weight PEG molecules had a higher activity. PEG-rHuTNF-alpha was resistant to proteolysis, and over 70 % activity remained after 8 h, but the activity of rHuTNF-alpha was time-dependently diminished by incubation with bovine trypsin. PEG5000-rHuTNF-alpha (1500 IU per mouse) had a similar anti-tumor potency compared with rHuTNF-alpha (3000 IU per mouse). PEG12000-rHuT NF-alpha (1500 IU per mouse) had an increased anti-tumor potency compared with rHuTNF-alpha (3000 IU per mouse). In particular, PEG20000-rHuTNF-alpha at a dose of 1500 IU per mouse had a higher anti-tumor potency than rHuTNF-alpha at a dose of 6000 IU per mouse. CONCLUSION: PEG-modified rHuTNF-alpha could be more suitable for therapeutic use     

Effects of tumor necrosis factor alpha on replication of varicella-zoster virus.

Replication of varicella-zoster virus (VZV) and expression of VZV nuclear antigen are inhibited in human embryonic lung fibroblast (HEL) cells pretreated with recombinant tumor necrosis factor (TNF) alpha for 24 h. This antiviral activity is completely blocked by the addition of monoclonal antibodies against TNF. TNF acts synergistically with interferons alpha and gamma. When TNF is added to HEL cells after VZV adsorption, virus replication is still inhibited. When VZV-infected HEL cells are co-cultured with HEL cells which have been pretreated with TNF or grown in the presence of TNF, spread of VZV from VZV-infected HEL cells to uninfected cells is unaffected. No interferon is detected in the supernatants or cell lysates of HEL cells cultured with TNF and antibodies to alpha-, beta- and gamma-interferon have no effect on the antiviral action of TNF.     

TNF-α、HGF在急性胰腺炎中的变化及意义

目的：探讨急性胰腺炎（AP）患者血清肿瘤坏死因子α（TNF-α）、肝细胞生长因子（HGF）的水平变化，分析其变化规律及对病情判断的价值。方法：应用酶联免疫吸附法（ELISA）测定39例AP及20例对照组血清TNF-α和HGF水平。结果：AP患者血清TNF-α和HGF水平明显高于对照组（P〈0．01），在发病第一天，重症AP患者TNF-α水平明显高于轻症AP患者，而HGF则轻症AP患者较高，差异有显著性（P〈0．01）。结论：TNF-α和HGF参与了AP的发生、发展过程，可作为AP早期诊断的参考指标。     

Immunomodulating effects of flavonoids on acute and chronic inflammatory responses caused by tumor necrosis factor alpha

Flavonoids have beneficial activities which modulate oxidative stress, allergy, tumor growth and viral infection, and which stimulate apoptosis of tumor cells. In addition to these activities, dietary flavonoids are able to regulate acute and chronic inflammatory responses. Here we describe new aspects of regulatory mechanisms by which flavonoids suppress production of tumor necrosis factor-alpha (TNF-alpha) by macrophages, microglial cells and mast cells stimulated with lipopolysaccharide (LPS) and others via toll-like receptors (TLRs), and TNF-alpha-mediated acute and chronic inflammatory responses. Treatment with flavonoids such as luteolin, apigenin, quercetin, genistein, (-)-epigallocatechin gallate, and anthocyanidin resulted in significant downregulation of LPS-elicited TNF-alpha and nitric oxide (NO) production and diminished lethal shock. In chronic diseases, pathogenesis of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis which is triggered by TNF-alpha, was improved by the oral administration of flavonoids after the onset of CIA. Here, we discuss that inhibitory effects of flavonoids on acute and chronic inflammation are due to regulation of signaling pathways, including the nuclear factor kappaB (NF-kappaB) activation and mitogen-activated protein (MAP) kinase family phosphorylation. FcetaRI expression by NF-kappaB activation was also reduced by flavonoids; while accumulation of lipid rafts, which is the critical step for signaling, was blocked by flavonoids. The intake of dietary flavonoids reduces acute and chronic inflammation due to blocking receptor accumulation and signaling cascades, and would assist individuals at high-risk from life-style related diseases.     

Effect of atorvastatin on adhesive phenotype of human endothelial cells activated by tumor necrosis factor alpha

We studied the effect of atorvastatin on the adhesive phenotype of human endothelial cells (HUVEC) stimulated by tumor necrosis factor (TNF)-alpha. Surface expression of adhesion molecules on HUVEC was examined by flow cytometry and confocal microscopy, and adhesion of monocytes (human THP-1 cell line) was measured in vitro under flow conditions. In TNF-alpha-activated HUVEC, atorvastatin significantly enhanced surface expression of vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule (ICAM)-1, E-selectin, and fractalkine, when compared with TNF-alpha stimulation alone. This enhancement was reversed by mevalonate or geranylgeranyl pyrophosphate (GGPP) and was mimicked by an inhibitor of geranylgeranylation. The enhancing effect of atorvastatin was restricted to TNF-alpha-inducible adhesion molecule and was the reflect of an increased protein synthesis (mRNA and protein) and not of a reduced shedding. Confocal microscopy examination showed that atorvastatin also altered the surface distribution of adhesion molecules. Adhesion of human THP-1 cells on TNF-alpha-activated HUVEC was significantly reduced by atorvastatin (-42% at 1 microM). Mevalonate or GGPP restored the TNF-alpha-induced adhesive potential. These results show that atorvastatin, by inhibiting prenylation of G proteins, enhances the TNF-alpha-induced expression of adhesion molecules at the endothelial cell surface and also alters their surface distribution which may account for the reduced binding of monocytes.     

新型重组人肿瘤坏死因子-α的研究进展

提高肿瘤坏死因子 α(TNF α)的抗肿瘤活性 ,降低其毒副作用 ,是将人TNF α应用于临床的关键。此文综述了近年来对TNF α进行结构基因改造、构建融合蛋白基因用以制备高效、毒副作用小的新型抗肿瘤药物的研究进展。     

Protection of lethal toxicity of endotoxin by Salvia miltiorrhiza BUNGE is via reduction in tumor necrosis factor alpha release and liver injury

Lipopolysaccharide (LPS) has been implicated as one of the major cause of Gram-negative bacteria-induced sepsis that are life-threatening syndromes occurring in intensive care unit patients. Many natural products derived from medicinal plants may contain therapeutic values on protecting endotoxemia-induced sepsis by virtue their ability to modulate multiple pro-inflammatory cytokines. In the present study, we show that Salvia miltiorrhiza (SM) BUNGE or Danshen, used in treatment of various systemic and surgical infections in the hospitals of China, was able to block the lethal toxicity of LPS in mice via suppression of TNF-alpha release and protection on liver injury. The ability of SM to suppress LPS-induced TNF-alpha release is further confirmed by in vitro experiments conducted on human peripheral blood leukocytes (PBL) and the RAW 264.7 macrophage cell line. Immunophenotyping by flow cytometry shows improved T-helper cell (CD4) and T-suppressor cells (CD8) ratio in SM-treated PBL and splenocytes of LPS-challenged mice. The drop in plasma glutamate-pyruvate transaminase (GPT) induced by LPS provides evidence that SM can protect hepatic damage. The present study explains some known biological activities of SM, and supports the clinical application of SM in the prevention of inflammatory diseases induced by Gram-negative bacteria.