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Tumor necrosis factor (TNF) is an inflammatory cytokine that is upregulated in a number of cardiomyopathies. Adverse cardiac remodeling and dilation result from degradation of the extracellular matrix by matrix metalloproteinases (MMPs). We investigated whether TNF can directly trigger expression and activation of MMPs in cardiac cells. We compared MMP expression profile and activities between primary cultures of mouse neonatal cardiomyocytes and cardiofibroblasts and in cellular and extracellular compartments. In response to recombinant TNF (rTNF, 20 ng/ml), cardiomyocytes exhibited faster and more pronounced superoxide production compared with cardiofibroblasts, concomitant with increased expression of several MMPs. MMP9 levels increased more rapidly and about twofold more in cardiomyocytes than in cardiofibroblasts. TNF did not induce MMP2 expression. Expression of collagenases (MMP8, MMP12, MMP13, and MMP14) increased significantly, while total collagenase activity increased to a greater degree in conditioned medium of cardiomyocytes than in cardiofibroblasts, rTNF-mediated MMP expression and activation were dependent on superoxide production and were blocked by apocynin, an NADPH oxidase inhibitor. We identified phosphatidylinositol 3-kinase (PI3K)[gamma] as a key factor in TNF-mediated events since TNF-induced superoxide production, MMP expression, and activity were significantly suppressed in cardiomyocytes and cardiofibroblasts deficient in PI3K[gamma]. We further demonstrated that the TNF-superoxide-MMP axis of events is in fact activated in heart disease in vivo. Wild-type and [TNF.sup.-/-] mice subjected to cardiac pressure overload revealed that TNF deficiency resulted in reduced superoxide levels, collagenase activities, PI3K activity, and fibrosis leading to attenuated cardiac dilation and dysfunction. Our study demonstrates that TNF triggers expression and activation of MMPs faster and stronger in cardiomyocytes than in cardiofibroblasts in a superoxide-dependent manner and via activation of PI3K[gamma], thereby contributing to adverse myocardial remodeling in disease. reactive oxygen species; NADPH oxidase; pressure overload doi:10.1152/ajpcell.00351.2009.

Neutrophils (PMNs) are a vital part of host defense and are the principal leukocyte in innate immunity. Interleukin (IL)-18 is a proinflammatory cytokine with roles in both innate and adaptive immunity. We hypothesize that PMNs contain preformed IL-18, which is released in response to specific inflammatory stimuli. Isolated PMNs were stimulated with a battery of chemoattractants (5 min to 24 h), and IL-18 release was measured. PMNs were also separated into subcellular fractions and immunoblotted with antibodies against IL-18 or were fixed and probed with antibodies to IL-18 as well as to the contents of granules, intracellular organelles, and filamentous actin (F-actin), incubated with fluorescent secondary antibodies, and examined by digital microscopy. Quiescent PMNs contained IL-18 in the cytoplasm, associated with F-actin, as determined by positive fluorescence resonance energy transfer (FRET+). In turn, TNF-[alpha] stimulation disrupted the association of IL-18 with F-actin, induced a FRET+ interaction of IL-18 with lipid rafts, and elicited IL-18 release. Manipulation of F-actin status confirmed the relationship between IL-18 and F-actin in resting PMNs. Consequently, incubation with monomeric IL-18 binding protein inhibited TNF-[alpha]-mediated priming of the PMN oxidase. We conclude that human PMNs contain IL-18 associated with F-actin in the cytoplasm and TNF-[alpha] stimulation causes dissociation of IL-18 from F-actin, association with lipid rafts, and extracellular release. Extracellular IL-18 participates in TNF-[alpha] priming of the PMN oxidase as demonstrated by inhibition with the IL-18 binding protein. F-actin; fluorescent resonance energy transfer; lipid rafts doi:10.1152/ajpcell.00011.2009.

Tumor necrosis factor-[alpha] (TNF-[alpha]) upregulates the expression of monocyte chemoattractant protein-1 (MCP-1) and adhesion molecules in type 2 diabetes. We hypothesized that TNF-[alpha] and MCP- 1 may interact to contribute to the evolution of vascular inflammation and endothelial dysfunction in coronary arterioles in type 2 diabetes. To test this hypothesis, we administered anti-MCP-1 to block MCP-1 signaling in genetically modified mice with type 2 diabetes ([Lepr.sub.db]) and in heterozygote (m [Lepr.sup.db]) lean control. Anti-MCP-1 partially restored vasodilation to the endothelium-dependent vasodilator acetylcholine in isolated, cannulated, and pressurized coronary arterioles in [Lepr.sup.db] mice but did not affect vasodilation in m [Lepr.sup.db] mice. Anti-MCP-1 attenuated super-oxide production and the protein expression of nitrotyrosine, which is an indicator of peroxynitrite production, in isolated coronary arterioles of [Lepr.sup.db] mice. Immunostaining results showed that the expression of MCP-1 and vascular cellular adhesion molecule-1 is colocalized with endothelial cells and macrophages. Anti-TNF-[alpha] or anti-MCP- 1 markedly reduced macrophage infiltration and the number of MCP-1-positive endothelium in [Lepr.sup.db] mice. The neutralization of TNF-[alpha] or anti-MCP-1 reduced the expression of adhesion molecules, suggesting that proinflammatory cytokines interact to amplify the signaling process that leads to vascular dysfunction. These findings demonstrate that the endothelial dysfunction occurring in type 2 diabetes is the result of the effects of the inflammatory cytokine TNF-[alpha] and TNF-[alpha]-related signaling, including the expression of MCP-1 and adhesion molecules, which further exacerbates vessel inflammation and oxidative stress. coronary microcirculation; cytokines; inflammation; vasodilation doi: 10.1152/ajpheart.00396.2009

Here we report that T-cell lymphomas characterized by the expression of anaplastic lymphoma kinase (ALK+ TCL) fail to express the TNF[alpha] and frequently display DNA methylation of the TNF[alpha] gene promoter. While only a subset of the ALK+ TCL-derived cell lines showed a high degree of the promoter methylation, all 6 showed low to nondetectable expression of the TNF[alpha] mRNA, and none expressed the TNF[alpha] protein. All 14 ALK+ TCL tissue samples examined displayed some degree of the TNF[alpha] promoter methylation, which was the most prominent in the distal portion of the the promoter. Treatment with a DNA methyltransferase inhibitor, 5'-aza-2'-deoxy-cytidine (5-ADC), reversed the promoter methylation and led to the expression of TNF[alpha] mRNA and protein. Furthermore, in vitro DNA methylation of the promoter impaired its transcriptional activity in the luciferase reporter assay. This impairment was seen even if only either distal or proximal portion were methylated, with methylation of the former exerting a more profound inhibitory effect. Notably, the ALK+ TCL cell lines uniformly expressed the type 1 TNF[alpha] receptor (TNF-R1) protein known to transduce the TNF[alpha]-induced pro-apoptotic signals. Moreover, exogeneous TNF[alpha] inhibited growth of the ALK+ TCL cell lines in a dose-dependent manner and induced activation of the members of the cell apoptotic pathway: Caspase 8 and caspase 3. These findings provide additional rationale for the therapeutic inhibition of DNA methyltransferases in ALK+ TCL. They also suggest that treatment with TNF[alpha] may be highly effective in this type of lymphoma.

The incidence of tuberculosis is increased during treatment of autoimmune diseases with anti-TNF antibodies. This is a significant clinical complication, but also provides a unique model to study immune mechanisms in human tuberculosis. Given the key role for cell-mediated immunity in host defense against Mycobacterium tuberculosis, we hypothesized that anti-TNF treatment impairs T cell--directed antimicrobial activity. Anti-TNF therapy reduced the expression in lymphocytes of perforin and granulysin, 2 components of the T cell--mediated antimicrobial response to intracellular pathogens. Specifically, M. tuberculosis--reactive [CD8.sup.+][CCR7.sup.-][CD45RA.sup.+] effector memory T cells ([T.sub.EMRA] cells) expressed the highest levels of granulysin, lysed M. tuberculosis, and infected macrophages and mediated an antimicrobial activity against intracellular M. tuberculosis. Furthermore, [T.sub.EMRA] cells expressed cell surface TNF and bound the anti-TNF therapeutic infliximab in vitro, making them susceptible to complement-mediated lysis. Immune therapy with anti-TNF was associated with reduced numbers of [CD8.sup.+] [T.sub.EMRA] cells and decreased antimicrobial activity against M. tuberculosis, which could be rescued by the addition of [CD8.sup.+] [T.sub.EMRA] cells. These results suggest that anti-TNF therapy triggers a reduction of [CD8.sup.+] [T.sub.EMRA] cells with antimicrobial activity against M. tuberculosis, providing insight into the mechanism whereby key effector T cell subsets contribute to host defense against tuberculosis., Introduction The treatment of patients with monoclonal anti-TNF antibodies (infliximab) has led to a 4-fold increased incidence of tuberculosis (1), representing a major complication of anti-TNF therapy. Despite this potential [...]

ABSTRACT Diabetes is a common endocrine disorder, primarily characterised by elevated plasma glucose levels. The disease affects all age groups worldwide. Most patients suffer from Type 2 diabetes, which is [...]

We have previously documented that human mesangial cell (HMC)-derived TNF-[alpha] is an important mediator involved in the glomerulo-tubular communication in the development of interstitial damage in IgA nephropathy (IgAN). With the strategic position of podocytes, we further examined the role of mesangial cells in the activation of podocytes in IgAN. There was no binding of IgA from patients with IgAN to podocytes. Podocytes cultured with IgA from patients with IgAN did not induce the release of growth factors or cytokines. Furthermore, podocytes did not express mRNA of known IgA receptors. In contrast, IgA-conditioned medium (IgA-HMC medium) prepared by culturing HMC with IgA from patients with IgAN for 48 h significantly increased the gene expression and protein synthesis of TNF-[alpha] by podocytes with a 17-fold concentration above that of IgA-HMC medium. The upregulation of TNF-[alpha] expression by podocyte was only abolished by a neutralizing antibody against TNF-[alpha] but not by other antibodies. Exogenous TNF-[alpha] upregulated the synthesis of TNF-[alpha] by podocytes in an autocrine fashion. IgA-HMC medium prepared with IgA from patients with IgAN also significantly upregulated the expression of both TNF-[alpha] receptor 1 and 2 in podocytes. Our in vitro finding suggests podocytes may play a contributory role in the development of interstitial damage in IgAN by amplifying the activation of tubular epithelial cells with enhanced TNF-[alpha] synthesis after inflammatory changes of HMC. tumor necrosis factor-[alpha]; mesangial cell; tubulointerstitial injury

Previously we have shown that both Rac 1 and c-Jun [NH.sub.2]-terminal kinase (JNK1/2) are key proapoptotic molecules in tumor necrosis factor TNF-[alpha]/cycloheximide (CHX)-induced apoptosis in intestinal epithelial cells, whereas the role of reactive oxygen species (ROS) in apoptosis is unclear. The present studies tested the hypothesis that Racl-mediated ROS production is involved in TNF-[alpha]-induced apoptosis. In this study, we showed that TNF-[alpha]/CHX-induced ROS production and hydrogen peroxide ([H.sub.2][O.sub.2])-induced oxidative stress increased apoptosis. Inhibition of Racl by a specific inhibitor NSC23766 prevented TNF-[alpha]-induced ROS production. The antioxidant, N-acetylcysteine (NAC), or rotenone (Rot), the mitochondrial electron transport chain inhibitor, attenuated mitochondrial ROS production and apoptosis. Rot also prevented JNK1/2 activation during apoptosis. Inhibition of Racl by expression of dominant negative Racl decreased TNF-[alpha]-induced mitochondrial ROS production. Moreover, TNF-[alpha]-induced cytosolic ROS production was inhibited by Racl inhibition, diphenyleneiodonium (DPI, an inhibitor of NADPH oxidase), and NAC. In addition, DPI inhibited TNF-[alpha]-induced apoptosis as judged by morphological changes, DNA fragmentation, and JNK1/2 activation. Mitochondrial membrane potential change is Racl or cytosolic ROS dependent. Lastly, all ROS inhibitors inhibited caspase-3 activity. Thus these results indicate that TNF-[alpha]-induced apoptosis requires Racl-dependent ROS production in intestinal epithelial cells. intestinal epithelial cells-6; N17Rac1; diphenyleneiodonium; JNK 1/2; oxidative stress

Tumors elicit antitumor immune responses, but over time they evolve and can escape immune control through various mechanisms, including the loss of the antigen to which the response is directed. The escape of antigen-loss variants (ALVs) is a major obstacle to T cell--based immunotherapy for cancer. However, cancers can be cured if both the number of CTLs and the expression of antigen are high enough to allow targeting of not only tumor cells, but also the tumor stroma. Here, we showed that IFN-[gamma] and TNF produced by CTLs were crucial for the elimination of established mouse tumors, including ALVs. In addition, both BM-and non-BM--derived stromal cells were required to express TNF receptors and IFN-[gamma] receptors for the elimination of ALVs. Although IFN-[gamma] and TNF were not required by CTLs for perforin-mediated killing of antigen-expressing tumor cells, the strong inference is that tumor antigen--specific CTLs must secrete IFN-[gamma] and TNF for destruction of tumor stroma. Therefore, bystander killing of ALVs may result from IFN-[gamma] and TNF acting on tumor stroma., Introduction Cancers express antigens that are targets for specific CTLs (1), (2); however, tumor evasion by different mechanisms remains a significant obstacle to effective adoptive T cell therapy. Cancer cells [...]

The inflammatory bowel disease ulcerative colitis (UC) frequently progresses to colon cancer. To understand the mechanisms by which UC patients develop colon carcinomas, we used a mouse model of the disease whereby administration of azoxymethane (AOM) followed by repeated dextran sulfate sodium (DSS) ingestion causes severe colonic inflammation and the subsequent development of multiple tumors. We found that treating WT mice with AOM and DSS increased TNF-[alpha] expression and the number of infiltrating leukocytes expressing its major receptor, p55 (TNF-Rp55), in the lamina propria and submucosal regions of the colon. This was followed by the development of multiple colonic tumors. Mice lacking TNF-Rp55 and treated with AOM and DSS showed reduced mucosal damage, reduced infiltration of macrophages and neutrophils, and attenuated subsequent tumor formation. WT mice transplanted with TNF-Rp55-deficient bone marrow also developed significantly fewer tumors after AOM and DSS treatment than either WT mice or TNF-Rp55-deficient mice transplanted with WT bone marrow. Furthermore, administration of etanercept, a specific antagonist of TNF-[alpha], to WT mice after treatment with AOM and DSS markedly reduced the number and size of tumors and reduced colonic infiltration by neutrophils and macrophages. These observations identify TNF-[alpha] as a crucial mediator of the initiation and progression of colitis-associated colon carcinogenesis and suggest that targeting TNF-[alpha] may be useful in treating colon cancer in individuals with UC., Introduction Ulcerative colitis (UC) is an inflammatory bowel disease characterized by pathological mucosal damage and ulceration, which can involve the rectum and extend proximally (1). The incidence of UC in [...]

Terrestrial solar UVB radiation ([approximately equal to]295-320 nm) readily induces cyclobutane pyrimidine dimers (CPDs) in human skin DNA that result in characteristic mutations associated with nonmelanoma skin cancer. The proinflammatory cytokine TNF[alpha] is important in mouse skin chemical carcinogenesis and is thought to also play a role in UVR-induced skin cancer by its immunomodulatory properties. There is some in vitro evidence that CPDs initiate the production of TNF[alpha], and we tested this hypothesis by comparing the wavelength dependence (action spectrum) for TNF[alpha] protein induction in human skin in vivo with our earlier in vivo action spectra for CPD induction in four different epidermal layers of human skin. Normal volunteers (n = 35) were irradiated with physiologically relevant doses of monochromatic UVB (290-320 nm), and TNF[alpha] concentration was assessed, by high-sensitivity ELISA, in exudates from skin suction blisters raised 8 h after irradiation. An action spectrum, constructed from the slopes of the close-response curves at the different wavelengths, showed maximal efficacy at 300 nm. An excellent match was observed for TNF[alpha] and the CPD action spectrum for cells in the lower basal epidermis. These data strongly suggest that UVB-induced photodamage to DNA in the epidermal basal layer is a major trigger for TNF[alpha] production. The TNF[alpha] may originate directly from the keratinocytes in this layer or inflammatory cells that are rapidly recruited into the upper dermis (e.g., neutrophils) as a consequence of DNA photodamage to basal-layer keratinocytes. cytokine | DNA photodamage | photoimmunosuppression | cyclobutane pyrimidine dimer

CD137 is expressed on activated T cells and ligands to this costimulatory molecule have clinical potential for amplifying CD8 T cell immunity to tumors and viruses, while suppressing CD4 autoimmune [...]

Production of TNF-[alpha] and IL-1 in infectious and autoimmune diseases is associated with fever, fatigue, and sleep disturbances, which are collectively referred to as sickness behavior syndrome. In mice TNF-[alpha] and IL-1 increase nonrapid eye movement sleep. Because clock genes regulate the circadian rhythm and thereby locomotor activity and may alter sleep architecture we assessed the influence of TNF-[alpha] on the circadian timing system. TNF-[alpha] is shown here to suppress the expression of the PAR bZip clock-controlled genes Dbp, Tef, and Hlf and of the period genes Per1, Per2, and Per3 in fibroblasts in vitro and in vivo in the liver of mice infused with the cytokine. The effect of TNF-[alpha] on clock genes is shared by IL-1[beta], but not by IFN-[alpha], and IL-6. Furthermore, TNF-[alpha] interferes with the expression of Dbp in the suprachiasmatic nucleus and causes prolonged rest periods in the dark when mice show spontaneous locomotor activity. Using clock reporter genes TNF-[alpha] is found here to inhibit CLOCK-BMAL1-induced activation of E-box regulatory elements-dependent clock gene promoters. We suggest that the increase of TNF-[alpha] and IL-1[beta], as seen in infectious and autoimmune diseases, impairs clock gene functions and causes fatigue. behavior | circadian rhythms | cytokines | innate immunity

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ahj.2007.02.031 Byline: Tiago V. Pereira (a)(b), Martina Rudnicki (c), Rendrik F. Franco (d), Alexandre C. Pereira (a), Jose E. Krieger (a) Abstract: Under the hypothesis that the G-308A polymorphism in the promoter region of the tumor necrosis factor [alpha] gene might increase tumor necrosis factor [alpha] expression, several investigations have been performed to examine the influence of the -308A allele on the risk of cardiovascular events. The results of these studies, however, have been conflicting. To provide a more robust estimate of the putative effect of the G-308A polymorphism on the risk of cerebrocardiovascular events, we did 2 meta-analyses that examined the role of the -308A variant in both ischemic heart disease (IHD) and ischemic stroke. Author Affiliation: (a) Heart Institute (InCor), Sao Paulo University Medical School, University of Sao Paulo, Sao Paulo, Brazil (b) Department of Biochemistry and Molecular Biology, Federal University of Sao Paulo, Sao Paulo, Brazil (c) Clinical and Toxicological Analysis Department, Faculty of Pharmaceutical Sciences, University of Sao Paulo, Sao Paulo, Brazil (d) Fleury Research Institute, Sao Paulo, Brazil Article History: Received 3 December 2006; Accepted 22 February 2007 Article Note: (footnote) This work was supported by Coordenacao de Aperfeicoamento Pessoal de Nivel Superior, Sao Paulo, Brazil.

BACKGROUND: Polymorphisms in the proinflammatory cytokine genes tumor necrosis factor-[alpha] (TNF) and lymphotoxin-[alpha] (LTA, also called TNF-[beta]) have been associated with asthma and atopy in some studies. Parental smoking is [...]

The immediate-early gene Nur77, which encodes an orphan nuclear receptor, is rapidly induced by various stress stimuli, including tumor necrosis factor (TNF). Nur77 has been implicated in mediating apoptosis, particularly in T cells and tumor cells. We report here that Nur77 can play a role in antagonizing apoptosis in TNF signaling. Nur77 expression is strongly induced by TNF. Interestingly, unlike most antiapoptotic molecules, this induced expression of Nur77 is largely independent of NF-[kappa]B. Ectopic expression of Nur77 can protect wild-type, [TRAF2.sup.-/-], and [ReIA.sup.-/-] cells from apoptosis induced by TNF, whereas expression of a dominant-negative form of Nur77 (DN-Nur77) accelerates TNF-mediated cell death in the mutant cells. In mouse embryonic fibroblasts, Nur77 remains in the nucleus in response to TNF and is not translocated to the mitochondria, where it was reported to mediate apoptosis. Our results suggest that Nur77 is a survival effector protein in the context of TNF-mediated signaling.

Transcription of the tumor necrosis factor (TNF) gene is rapidly and transiently induced by lipopolysaccharide in cells of monocyte/macrophage lineage. Previous studies have suggested that in the mouse, multiple NF-[kappa]B/Rel-binding sites contribute to the TNF transcriptional response to LPS. But the role of these regulatory elements in transcriptional activation of the TNF-[alpha] gene in human monocytes remains unclear. Previously, a transcription factor, termed lipopolysaccharide-induced TNF-[alpha] factor (LITAF), was found to regulate TNF-[alpha] gene expression. However, the specific protein domain(s) of human (h)LITAF that interact with the hTNF-[alpha] promoter had not been identified. In this study, we identify by footprinting a sequence motif, CTCCC (-515 to -511), within the TNF-[alpha] promoter that binds to hLITAF. We also identify the region of hLITAF (amino acids 165-180) that was named peptide B and specifically mediates binding to the hTNF-[alpha] promoter. When THP-1 cells were stimulated with this peptide B, it was sufficient to induce TNF-[alpha] secretion. Induction of TNF-[alpha] transcription by LPS or peptide B depended on the presence of the -515 to -511 promoter region, which was found to be essential for hLITAF binding. Together, these findings help to clarify the mechanism of hLITAF/hTNF-[alpha] interaction and the manner by which hLITAF contributes to hTNF-[alpha] regulation in an attempt to design new pharmacological interventions to address TNF-related diseases. lipopolysaccharide-induced tumor necrosis factor-[alpha] factor | electrophoretic mobility-shift assay | DNA-binding site

The present study investigated transcriptional inactivation of TNF-[alpha] gene by nuclear factor-binding oligonucleotides (ON) and their effects on pulmonary inflammatory responses in mice. PCR-based gene mutation and gel shift assays were used to identify specific cis-acting elements necessary for nuclear factor binding and transactivation of TNF-[alpha] gene by lipopolysaccharide (LPS). LPS inducibility of TNF-[alpha] was shown to require transcriptional activation by NF-[kappa]B at multiple binding sites, including the -850 ([kappa]1), -655 ([kappa]2), and -510 ([kappa]3) sites, whereas the -210 ([kappa]4) site had no effect. Maximum inducibility was associated with the activation of [kappa]3 site. The sequence-specific, double-stranded ON targeting this site was most effective in inhibiting TNF-[kappa] activity induced by LPS. The inhibitory effect of ON on TNF-[kappa] bioactivity was also investigated using a murine lung inflammation model. Pretreatment of mice with ON, but not its mutated sequence, inhibited LPS-induced inflammatory neutrophil influx and TNF-[kappa] production by lung cells. Effective inhibition by ON in this model was shown to require a liposomal agent for efficient cellular delivery of the ON. Together, our results indicate that transcriptional inactivation of TNF-[alpha] gene can be achieved by using ON that compete for nuclear factor binding to TNF-[kappa] gene promoter. This gene inhibition approach may be used as a research tool or as potential therapeutic modality for diseases with etiology dependent on aberrant gene expression. tumor necrosis factor-[alpha]

Mice lacking the stress-signaling kinase SEK1 die from embryonic day 10.5 (E10.5) to E12.5. Although a defect in liver formation is accompanied with the embryonic lethality of sek[1.sup.-/-] mice, the mechanism of the liver defect has remained unknown. In the present study, we first produced a monoclonal antibody specifically recognizing murine hepatoblasts for the analysis of liver development and further investigated genetic interaction of sek1 with tumor necrosis factor-[alpha] receptor 1 gene (tnfr1) and protooncogene c-jun, which are also responsible for liver formation and cell apoptosis. The defective liver formation in sek[1.sup.-/-] embryos was not protected by additional tnfr1 mutation, which rescues the embryonic lethality of mice lacking NF-[kappa]B signaling components. There was a progressive increase in the hepatoblast cell numbers of wild-type embryos from E10.5 to E12.5. Instead, impaired hepatoblast proliferation was observed in sek[1.sup.-/-] livers from E10.5, though fetal liver-specific gene expression was normal. The impaired phenotype in sek[1.sup.-/-] livers was more severe than in [c-jun.sup.-/-] embryos, and sek[1.sup.-/-] [c-jun.sup.-/-] embryos died more rapidly before E8.5. The hepatoblast proliferation required no hematopoiesis, since liver development was not impaired in AML[1.sup.-/-] mice that lack hematopoietic functions. Stimulation of stress-activated protein kinase/c-Jun N-terminal kinase by hepatocyte growth factor was attenuated in sek[1.sup.-/-] livers. Thus, SEK1 appears to play a crucial role in hepatoblast proliferation and survival in a manner apparently different from NF-[kappa]B or c-Jun. Key words: SEK1; NF-[kappa]B; c-Jun; HGF; hematopoiesis; hepatogenesis.

Endotoxin induces marked changes in lipid metabolism via its effects on cytokines. To evaluate the role of tumor necrosis factor-alpha (TNF) in mediating changes of lipid metabolism in ruminants, we performed a crossover saline-controlled study in Holstein heifers (n = 8; 394.0 kg average BW), investigating the metabolic effects of a single intravenous administration of recombinant bovine TNF (rbTNF, 5.0 [micro]g/kg). Blood samples were taken from a jugular vein at 0 (1100, just before injection), 0.5, 6, 12, and 24 h after each treatment. Dry matter intake in the heifers was not affected by single administration of the rbTNF. The rbTNF produced early as well as later hypertriglyceridemia (P < 0.05) in dairy heifers. The rbTNF also induced an early and sustained rise (P < 0.05) in the plasma NEFA concentration. Plasma retinol concentration was decreased (P < 0.05) at 24 h after rbTNF injection, whereas the [alpha]-tocopherol concentration was not significantly affected by rbTNF treatment. At 0.5 and 24 h, there was an increase (P < 0.05) in the plasma concentration of the very-low-density lipoprotein (VLDL) fraction in rbTNF-treated heifers. Between 6 and 24 h after rbTNF treatment, concentration of the low-density lipoprotein fraction declined (P < 0.05) but the high-density lipoprotein fraction was not altered in the rbTNF-treated heifers. These results indicate that TNF produces a hypertriglyceridemic response associated with an increase of the VLDL fraction and a disturbance of retinol metabolism in dairy heifers. Key Words: Dairy Cattle, Lipoproteins, Retinol, Tumor Necrosis Factor, Vitamin E Acetate

Research has been conducted on the tumor necrosis factor-alpha converting enzyme (TACE). Results indicate that TACE recombinant and native forms possess a synthetic substrate which corresponds to the tumor necrosis factor-alpha cleavage site.

Byline: Michael Heesen (1), Brunhilde Blomeke (2), Bernhard Schluter (3), Nicole Heussen (4), Rolf Rossaint (1), Dagmar Kunz (5) Keywords: CD14 antigen Genetics: gene polymorphism Molecular biology: real-time PCR Fluorescence-activated cell sorting Cytokines: tumor necrosis factor-[alpha] Abstract: Objective: CD14 is a receptor for endotoxin and binds components of Gram-positive and Gram-negative bacteria. CD14-bearing monocytes respond to stimulation with the increased synthesis and release of cytokines. The recently described --260 C[right arrow]T promoter polymorphism of the CD14 gene has been found to be related to a risk of myocardial infarction. This study evaluated the role of this polymorphism in the expression of monocyte and soluble CD14. Moreover, the effect of the CD14 --260 genotypes for the ex vivo TNF-[alpha] response to endotoxin was analyzed in whole blood. Patients and participants: Ninety-five healthy blood donors were studied. Measurements and results: CD14 --260 genotyping was performed by means of a real-time PCR with fluorescence labeled hybridization probes. CD14 expression on human monocytes (mCD14) was assessed by fluorescence-activated cell sorting analysis with anti-CD14 monoclonal antibodies. Plasma levels of soluble CD14 (sCD14) were measured by ELISA. The TNF-[alpha] synthesis was determined by chemiluminescence in whole blood after endotoxin stimulation. There were no differences in mCD14 density, sCD14 levels, or the tumor necrosis factor-[alpha] concentrations between individuals with the three different CD14 --260 genotypes CC, CT, and TT. Conclusions: The CD14 --260 polymorphism does not affect the CD14 expression of unstimulated circulating monocytes or soluble CD14 plasma levels. Author Affiliation: (1) Department of Anesthesiology and Intensive Care Medicine, University Hospital of Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany (2) Department of Dermatology, University Hospital of Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany (3) Department of Clinical Chemistry, University Hospital of Munster, Albert-Schweitzer-Strasse 33, 48149 Munster, Germany (4) Department of Statistics and Biometry, University Hospital of Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany (5) Department of Clinical Chemistry and Pathobiochemistry, University Hospital of Aachen, Pauwelsstrasse 30, 52057 Aachen, Germany Article History: Received Date: 08/12/2000 Accepted Date: 21/08/2001 Article note: Final revision received: 21 August 2001 Electronic Publication

Byline: J.H. Weitkamp (1), F. Stuber (2), P. Bartmann (1) Keywords: Key words Tumor necrosis factor; Gene polymorphism; Neonate; Sepsis; Infection Abstract: In adult postoperative intensive care patients, the biallelic Ncol polymorphism within the tumor necrosis factor (TNF) locus has been shown to be a genomic marker for individuals with increased TNF-[alpha] response and poor prognosis in severe sepsis. We characterized the genomic distribution and allele frequency of the Ncol polymorphism in 23 preterm and term neonatal intensive care unit (NICU) patients with culture-proven sepsis and compared it with clinical and laboratory characteristics to assess its prognostic value for disease progression. Genotype analysis demonstrated the following absolute (relative) frequencies: 7 (0.31) infants were homozygous for the allele TNFB2 (Group A). 12 (0.52) infants were heterozygous (TNFB1/TNFB2) and four (0.17) infants homozygous for the allele TNFB1 (Group B). There was no significant difference compared to adult intensive care patients with severe sepsis (p = 0.31). The median gestational age of all infants (13 female and ten male) as well as for either group was 28 weeks (range 23--37) with a median birth weight of 845 g (range 560--2,720). The study population included a total of 16 very low birth weight (VLBW) infants, four in Group A and 12 in Group B. However, there was no significant difference for gestational age and birth weight in both groups (p = 0.82 and 0.71, respectively). Laboratory parameters as maximum and minimum leukocyte and thrombocyte counts, maximum immature to total neutrophil ratios (ITR), maximum C-reactive protein (CRP) levels, days of CRP levels &gt 5 mg/l and total days of antibiotic treatment, were not statistically different in both groups. In total, three infants (13%) died in consequence of their underlying disease. Two infants belonged to Group A and one to Group B. The statistical analysis of outcome variables (mortality, neurological impairment, failure to thrive) was not possible, because the study population was small and the reasons for poor outcome and death in these high-risk patients had to be considered multifactorial. In conclusion, in this pilot study the biallelic Ncol polymorphism within the TNF locus was not a prognostic marker for disease progression in high-risk NICU-admitted term and preterm infants with culture-proven sepsis. In order to detect differences in outcome similar to adult postsurgical patients with severe sepsis, an unfeasibly high number of NICU patients with culture-proven sepsis would need to be included for a similar study. Author Affiliation: (1) Div. of Neonatology, Center for Pediatrics, Bonn University, Adenauerallee 119, D-53113 Bonn, Germany Phone: +49-228-287-3408, Fax: +49-228-287-3314, DE (2) Hospital for Anesthiology and Intensive Care Medicine, Bonn University, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany, DE Article note: Received: September 28, 1999 * Revision accepted: January 25, 2000

Results reveal that splicing tumor necrosis factor-alpha gene, containing a cis-acting sequence (UTR), requires an RNA-activated protein kinase (PKR). Data further demonstrate that deletion of UTR sequence frees splicing from the requirement of PKR indicating interaction of these two factors in the regulation and expression of the gene.

Tumor necrosis factor (TNF) biology and the major histocompatibility complex in humans are discussed relative to linkage, with the TNF (ital) gene within the MHC gene cluster, that can make interpretation of studies in animal models difficult. TNF seems to have a significant role in promotion of inflammation, likely in a mode identified from TNF (ital) gene-targeted mice.

Bone resorption and remodeling is a very precisely controlled body process that requires osteoclasts to function as a part of the process. Mice that lack the TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in tooth eruption and bone remodeling, the result of impaired osteoclast activity. TRAF6 has been shown to be crucial in IL-1 and CD40 signaling and also in lipopolysaccharide (LPS) signaling, which would not be expected. It is essential for survival perinatally and postnatally, also unexpected.