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Although inflammation is known to influence bone turnover and bone mineral density (BMD), less is known about role of soluble tumor necrosis factor alpha receptor 1 (sTNFα-R1) in changes in bone turnover and BMD in the year after hip fracture. We studied 245 persons (117 men and 128 women) from the Baltimore Hip Studies. Bone turnover markers of resorption (carboxy-terminal type I collagen cross-links [CTX-I]) and formation (amino-terminal propeptide type I collagen [P1NP]), BMD of the contralateral hip, and sTNFα-R1 were measured within 15 days of hospitalization and 2, 6, and 12 months later. Latent class growth modeling was used to determine sTNFα-R1 trajectories. Weighted generalized estimating equations were used to examine the association of sTNFα-R1 trajectories with serum levels of CTX-I and P1NP and BMD; standardized beta coefficients (βˆ) are reported. Higher baseline sTNFα-R1 was significantly associated with a greater rate of CTX-I change (βˆ = 0.26, p = 0.004). Four distinct sTNFα-R1 trajectories were identified. The two groups with higher sTNFα-R1 levels during the year following fracture had faster increasing levels of CTX-I compared to the group with lowest sTNFα-R1 levels (men: group 3: βˆ = 0.76, p = 0.02; group 4: βˆ = 1.4, p

BACKGROUND End-stage liver disease caused by non-alcoholic steatohepatitis (NASH) is the second leading indication for liver transplantation. To date, only moderately effective pharmacotherapies exist to treat NASH. Understanding the pathogenesis of NASH is therefore crucial for the development of new therapies. The inflammatory cytokine tumor necrosis factor alpha (TNF-α) is important for the progression of liver disease. TNF signaling via TNF receptor 1 (TNFR1) has been hypothesized to be important for the development of NASH and hepatocellular carcinoma in whole-body knockout animal models. AIM To investigate the role of TNFR1 signaling in hepatocytes for steatohepatitis development in a mouse model of diet-induced NASH. METHODS NASH was induced by a western-style fast-food diet in mice deficient for TNFR1 in hepatocytes (TNFR1ΔHEP) and their wild-type littermates (TNFR1fl/fl). Glucose tolerance was assessed after 18 wk and insulin resistance after 19 wk of feeding. After 20 wk mice were assessed for features of NASH and the metabolic syndrome such as liver weight, liver steatosis, liver fibrosis and markers of liver inflammation. RESULTS Obesity, liver injury, inflammation, steatosis and fibrosis was not different between TNFR1ΔHEP and TNFR1fl/fl mice. However, Tnfr1 deficiency in hepatocytes protected against glucose intolerance and insulin resistance. CONCLUSION Our results indicate that deficiency of TNFR1 signaling in hepatocytes does not protect from diet-induced NASH. However, improved insulin resistance in this model strengthens the role of the liver in glucose homeostasis.

Introduction: Density and co-expression of tumor necrosis factor (TNF) receptors may vary among cell populations. However, the role and potential of these changes remain unclear. This study aimed to determine the density of expression and co-expression of TNFR1/2 and the dose-dependent effect of soluble TNF on these parameters. Methods: Epithelial-like (HEp-2, K-562, MCF-7, ZR-75/1) and lymphoblast-like (MOLT-4, HL-60, Raji, RPMI-8226, IM-9) cell lines were characterized for co-expression of TNFR1/2 using a modified flow cytometry protocol. The dose-dependent effects of rhTNF on TNF receptor expression in these lines were studied. Results: This study reports a protocol for the simultaneous quantitative evaluation of the of TNF receptor number and co-expression of membrane-bound TNFR1/2. Cells within one tumor cell line were found to differ regarding their expression of type 1 and 2 TNFα receptors; simultaneously, cells with all 4 variants of co-expression may be present in culture. Conclusion: We demonstrated a dose-dependent effect of TNF on changes in the expression of TNFR1/2 by the percentage of positive cells and by the number of receptors, which may be used to control TNF-mediated processes in target cells.

Tumor necrosis factor -alpha (TNFa) is strongly associated with fatty liver disease (i.e, hepatosteatosis). Cytokine production has been thought of as a consequence of hepatic lipid accumulation which becomes a critical factor in the development of chronic liver pathologies as well as insulin resistance. The purpose of this study was to test the hypothesis that TNFa directly regulates lipid metabolism in liver in the mutant peroxisome-proliferator activated receptor-alpha (PPARa-/-) mouse model with robust hepatic lipid accumulation. At 10 weeks of age, TNFa and TNF receptor 1 expression are increased in livers of PPARa-/- mice compared to wild type. PPARa-/- mice were then crossed with mice lacking the receptor for TNFa receptor 1 (TNFR1-/-). Wild type, PPARa-/-, TNFR1-/-, PPARa-/- x TNFR1-/- mice were housed on ad-libitum standard chow diet for up to 40 weeks. Increases in hepatic lipid and liver injury and metabolic disruption associated with PPARa ablation were largely blunted when PPARa-/- mice were crossed with TNFR1-/- mice. These data support the hypothesis that TNFR1 signaling is critical for accumulation of lipid in liver. Therapies that reduce pro-inflammatory responses, namely TNFa, could have important clinical implications to reduce hepatosteatosis and progression of severe liver disease.

Background and aimsTumor necrosis factor receptor type 1 (TNFR1) is associated with kidney disease and mortality risk in various populations. Whether or not kidney function mediates mortality risk is unknown. We evaluated associations of TNFR1 levels with measures of kidney function, cardiovascular events, and mortality in a population of veterans with stable ischemic heart disease.MethodsTNFR1 was measured from baseline serum samples in the Heart and Soul Study; elevated levels were defined by the highest quartile (Q4, >3.4ng/ml). We evaluated associations of high TNFR1 with baseline estimated glomerular filtration rate (eGFR) and urine albumin to creatinine ratio (ACR) and with longitudinal changes in eGFR (rapid loss), as well as with incident myocardial infarction (MI), heart failure hospitalizations (HF), and mortality over a median follow-up time of 8.9 years. Covariates included demographics and comorbid conditions.ResultsAmong 985 participants who had TNFR1 measurements, median TNFR1 was 2.33ng/ml (IQR 1.8-3.1). Relative to Q1, Q4 had higher risk of eGFR

Background Major depressive disorder (MDD) patients have shown elevated plasma levels of pro-inflammatory biomarkers compared to healthy controls. We hypothesized increased serum tumor necrosis factor-alpha receptor subtype 1 (TNF-α R1) is more associated with impaired brain function in patients with treatment-resistant depression (TRD) than those without TRD. Methods 34 MDD patients and 34 healthy subjects were recruited and we separated MDD patients to TRD group (n = 20) and non-TRD (n = 14) group. Pro-inflammatory cytokines were assessed by enzyme-linked immunosorbent assays. A standardized uptake values (SUV) of glucose metabolism measured by 18F-FDG positron-emission-tomography (PET) was applied to all subjects for subsequent region-of- interest analyses and whole-brain voxel-wise analyses. 18F-FDG-PET measures glucose uptake into astrocytes in response to glutamate release from neuronal cells, and was thus used as a proxy measure to quantify glutamatergic neurotransmission in the human brain. Results Post-hoc analysis revealed that TRD group had higher serum concentrations of TNF-α R1 compared to healthy control or non-TRD group. In the MDD group, higher serum concentrations of TNF-α R1 significantly correlated with decreased SUV in anterior cingulate cortex (ACC) and bilateral caudate nucleus. The ROI analysis further supported the negative correlations of plasma TNF-α R1 and SUV in the ACC and caudate nucleus. Such correlation is more consistent in TRD group than in non-TRD and HC groups. Limitation Glutamate neurotransmission and the effect of chronic stress on glutamate release in the brain were not measured directly. Conclusions Increased TNF-α R1 was associated with impaired glutamatergic neurotransmission of caudate nucleus and ACC in MDD patients, particularly in the TRD.

Although inflammation is known to influence bone turnover and bone mineral density (BMD), less is known about role of soluble tumor necrosis factor alpha receptor 1 (sTNFα-R1) in changes in bone turnover and BMD in the year after hip fracture. We studied 245 persons (117 men and 128 women) from the Baltimore Hip Studies. Bone turnover markers of resorption (carboxy-terminal type I collagen cross-links [CTX-I]) and formation (amino-terminal propeptide type I collagen [P1NP]), BMD of the contralateral hip, and sTNFα-R1 were measured within 15 days of hospitalization and 2, 6, and 12 months later. Latent class growth modeling was used to determine sTNFα-R1 trajectories. Weighted generalized estimating equations were used to examine the association of sTNFα-R1 trajectories with serum levels of CTX-I and P1NP and BMD; standardized beta coefficients (βˆ) are reported. Higher baseline sTNFα-R1 was significantly associated with a greater rate of CTX-I change (βˆ = 0.26, p = 0.004). Four distinct sTNFα-R1 trajectories were identified. The two groups with higher sTNFα-R1 levels during the year following fracture had faster increasing levels of CTX-I compared to the group with lowest sTNFα-R1 levels (men: group 3: βˆ = 0.76, p = 0.02; group 4: βˆ = 1.4, p 0.001; women: group 3; βˆ = 0.67, p = 0.02; group 4: βˆ = 1.3, p = 0.004). Men in the highest sTNFα-R1 group had a greater decline in BMD compared to the lowest sTNFα-R1 group (2-month βˆ = -0.01, p = 0.01; 6-month: βˆ = -0.09, p = 0.001; 12-months: βˆ = -0.1, p 0.001). An increasing rate of CTX-I was associated with a steeper decline in total hip BMD in those within higher sTNFα-R1 trajectory groups (p 0.001). CTX-I was significantly increased with sTNFα-R1 in both sexes. CTX-I and the highest sTNFα-R1 trajectory were significantly associated with declines in total hip BMD in men. Interventions that reduce systemic inflammation should be explored to reduce bone resorption and prevent a decline in BMD after hip fracture. © 2018 American Society for Bone and Mineral Research.

Liver disease is the main cause of morbidity and mortality worldwide. The spectrum of the disease ranged from fatty liver to hepatic inflammation, necrosis, progressive fibrosis, and hepatocellular carcinoma. We evaluated the serum levels of soluble tumor necrosis factor alpha receptor 1, total B-hexosaminidase and its isoenzymes Hex A and B activities, and nitric oxide in patients with liver diseases and their association with aminotransferase level. Seventy patients and 12 healthy subjects were recruited. Patients were divided into three groups: chronic hepatitis group (20 patients), liver cirrhosis group (30 patients), and malignant liver group (20 patients). Serum levels of soluble tumor necrosis factor alpha receptor 1, total B-hexosaminidase and its isoenzymes Hex A and B activities, and nitric oxide were measured. Serum levels of soluble tumor necrosis factor alpha receptor 1, total B-hexosaminidase activity, and nitric oxide were significantly higher in the liver disease patients. Serum levels of isoenzymes Hex A and B were significantly higher in malignant liver patients. Total B-hexosaminidase and its isoenzyme Hex A activity levels were significantly higher in positive HBsAg and positive anti-HCV patients. Serum levels of soluble tumor necrosis factor alpha receptor 1 were positively correlated with aminotransferase level. Taken together, these findings suggested that these biochemical indices might reflect ongoing disease activity and played an important role in the pathophysiology of liver diseases.

Purpose: The purpose of this study was to examine the influence of narrowband ultraviolet (NB-UVB) therapy on the serum concentration of soluble tumor necrosis factor receptor type 1 (sTNF-R1) in psoriasis patients. Methods: Twenty-seven patients received NB-UVB therapy three to five times a week for 4 weeks. The assessment of skin lesions using psoriasis area and severity index (PASI) and serum concentration of sTNF-R1 was performed at the baseline, at 2, 4 and 4 weeks after treatment cessation. The sera of healthy subject were used as controls. Results: The baseline PASI was 13.56±5.71, sTNF-R1 concentration was 1.89±0.43 ng/ml in patients and 1.48±0.30 ng/ml in controls (P

The immune/inflammatory signaling molecule tumor necrosis factor α (TNFα) is an important mediator of both constitutive and plastic signaling in the brain. In particular, TNFα is implicated in physiological processes, including fever, energy balance, and autonomic function, known to involve the hypothalamic paraventricular nucleus (PVN). Many critical actions of TNFα are transduced by the TNFα type 1 receptor (TNFR1), whose activation has been shown to potently modulate classical neural signaling. There is, however, little known about the cellular sites of action for TNFR1 in the PVN. In the present study, high-resolution electron microscopic immunocytochemistry was used to demonstrate the ultrastructural distribution of TNFR1 in the PVN. Labeling for TNFR1 was found in somata and dendrites, and to a lesser extent in axon terminals and glia in the PVN. In dendritic profiles, TNFR1 was mainly present in the cytoplasm, and in association with presumably functional sites on the plasma membrane. Dendritic profiles expressing TNFR1 were contacted by axon terminals, which formed non-synaptic appositions, as well as excitatory-type and inhibitory-type synaptic specializations. A smaller population of TNFR1-labeled axon terminals making non-synaptic appositions, and to a lesser extent synaptic contacts, with unlabeled dendrites was also identified. These findings indicate that TNFR1 is structurally positioned to modulate postsynaptic signaling in the PVN, suggesting a mechanism whereby TNFR1 activation contributes to cardiovascular and other autonomic functions.

The plasma level of the tumor necrosis factor-alpha receptor 2 (TNFR2) is associated with obesity phenotypes. However, the genetic polymorphisms for such an association have rarely been explored and are generally unknown. In this study, by employing a large sample of 1,873 subjects from 405 Caucasian nuclear families, we explored the association of 12 SNPs of the TNFR2 gene and obesity-related phenotypes, including body mass index (BMI), fat mass, and percentage fat mass (PFM). The within-family quantitative transmission disequilibrium test, which is robust to sample stratification, was implemented to evaluate the association of TNFR2 gene with obesity phenotypes. Evidence of association was obtained at SNP9 (rs5746059) with fat mass (P = 0.0002), BMI (P = 0.002), and PFM (P = 0.0006). The contribution of this polymorphism to the variation of fat mass and PFM was 6.24 and 7.82%, respectively. Individuals carrying allele A at the SNP9 site had a 4.6% higher fat mass and a 2.5% increased PFM compared to noncarriers. The results remained significant even after correction for multiple testing. Evidence of association between the TNFR2 gene and obesity phenotypes are also found in 700 independent Chinese Han and 1,000 random Caucasians samples. The results suggest that the TNFR2 gene polymorphisms contribute to the variation of obesity phenotypes.

Only recently, knockout mouse models were applied in studies on the pathogenesis of leptospirosis. Current data suggest an important role of innate immunity receptors and interferon gamma dependant cellular response on protection. It is not clear, however, whether T helper cell polarization influences on outcome of leptospiral infection. We report findings of experimental infection of C57BL/6 (interferon gamma or tumor necrosis factor alpha receptor deficient) and BALB/c (interleukin 4 deficient) mice infected by pathogenic Leptospira interrogans serovar Copenhageni. Specific cytokine gene deficiency had no impact on outcome since all animals survived. TNFR knockout mice, however, exhibited more severe residual renal inflammation during convalescence thus suggesting this cytokine is important in early control of infection, protecting kidneys from relevant pathology.

Tumor necrosis factor-α (TNF-α) is one of the main mediators of inflammatory response activated by fatty acids in obesity, and this signaling through TNF-α receptor (TNFR) is responsible for obesity-associated insulin resistance. Recently, TNF-α has shown to affect lipid metabolism including the regulation of lipase activity and bile acid synthesis. However, there is scanty in vivo evidence for the involvement of TNF-α in this process, and the mechanistic role of TNFR remains unclear. In this study, TNFR2 knockout mice (R2KO) and wild-type (WT) mice were fed commercial normal diet (ND) or high-fat diet (HFD) for 8 weeks. In R2KO/HFD mice, the increase in body weight and the accumulation of fat were significantly ameliorated compared with WT/HFD mice in association with the decrease in plasma total cholesterol (137.7 ± 3.1 vs. 98.6 ± 3.1 mg/dL, P

Tumor necrosis factor-alpha (TNF-alpha) is a highly pleiotropic cytokine produced mainly by activated macrophages. This cytokine has been found to mediate the growth of certain tumors, the replication of HIV-1, septic shock, cachexia, graft-versus-host disease, and autoimmune diseases. The binding of TNF-alpha to the p55 tumor necrosis factor receptor type I (TNFRI) is considered one of the initial steps responsible for the multiple physiologic effects mediated by TNF-alpha. The role of TNF-alpha as an inflammatory mediator through TNFRI makes both of these genes attractive targets for intervention in both acute and chronic inflammatory diseases. We have designed antisense oligodeoxynucleotides (ODNs) containing chemically modified purine and pyrimidine bases that specifically inhibit TNFRI expression and functions. These ODNs were designed to hybridize to the 3'-polyadenylation signal region of the TNFRI gene. In cell-based assays, gene-specific antisense inhibition occurred in a dose-dependent fashion at submicromolar concentrations in the presence of cellular uptake enhancing agents. Within ODN sets with a common pattern of stabilizing backbone substitution, the inhibition of the gene expression is found to be correlated with the affinity of the ODNs for their cognate mRNA target sites, providing direct evidence for an antisense mechanism of action. In addition, events triggered by the binding of TNF-alpha to TNFRI, such as the production of IL-6 and IL-8, were significantly reduced by treatment of cells with the anti-TNFRI ODN. Therefore, antisense ODNs can be used to control biologic processes mediated by TNF-alpha and may be useful as therapeutic agents to treat conditions resulting from overproduction of TNF-alpha.

Porphyromonas gingivalis aspiration pneumonia induces local and systemic cytokine responses, but the dynamic of the immune response following lung exposure to live P. gingivalis is poorly understood. Groups of 50 12-week-old male BALB/c mice were inoculated intratracheally with live P. gingivalis ATCC 33277 using low dose (2 x 10(5) colony-forming units [CFU]), high dose (2.9 x 10(9) CFU), or phosphate-buffered saline (PBS; sham-inoculated), and the 3 groups were sacrificed at 2, 6, 24, 72, 168 hours. Lung and serum samples were collected for tumor necrosis factor alpha (TNF-alpha), soluble TNF-alpha receptors (sTNFRs), interleukin (IL)-1beta, and IL-6 analysis and lung histology. Pneumonia, only observed in the high-dose group, was associated with an early increase in lung TNF-alpha, IL-1beta, and IL-6, whereas no significant changes were observed in lung sTNFRs. Serum sTNFRs were significantly increased in high-dose animals at all times. IL-1beta elevation occurred earlier in serum than in lungs. IL-1beta was also significantly elevated in serum from low-dose animals at 6 hours. Serum IL-6 and sTNFRs remained raised at 7 days, whereas all other measured cytokines returned to basal levels with resolution of pneumonia. Development of pneumonia is dependent on the P. gingivalis dose; however, part of the cytokine response is unique to the systemic compartment, even in animals that do not develop pneumonia.

ING proteins affect apoptosis, growth, and DNA repair by transducing stress signals such as DNA damage, binding histones, and subsequently regulating chromatin structure and p53 activity. p53 target genes, including the p21 cyclin-dependent kinase inhibitor and Bax, an inducer of apoptosis, are regulated by ING proteins. To identify additional targets downstream of p33ING1 and p32ING2, cDNA microarrays were performed on phenotypically normal human primary fibroblasts. The 0.36% of genes affected by ING proteins in primary fibroblasts were distinct from targets seen in established cells and included the HSP70 heat shock gene, whose promoter was specifically induced10-fold. ING1-induced expression of HSP70 shifted cells from survival to a death pathway in response to tumor necrosis factor alpha (TNF-alpha), and p33ING1b protein showed synergy with TNF-alpha in inducing apoptosis, which correlated with reduced NF-kappaB-dependent transcription. These findings are consistent with previous reports that HSP70 promotes TNF-alpha-mediated apoptosis by binding I-kappaBeta kinase gamma and impairing NF-kappaB survival signaling. Induction of HSP70 required the amino terminus of ING1b but not the plant homeodomain region that was recently identified as a histone binding domain. Regulation of HSP70 gene expression by the ING tumor suppressors provides a novel link between the INGs and the stress-regulated NF-kappaB survival pathway important in hypoxia and angiogenesis.

Recently, we demonstrated that mice deficient of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) were partly protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxicity. Here we extended the study and investigated TNF-alpha receptor 1 (−/−) (TNFR1) and TNF-alpha receptor 2 (−/−) (TNFR2) mice using a chronic MPTP dosing regimen (15 mg/kg MPTP on 8 consecutive days). One week after the last MPTP treatment, HPLC determination of striatal dopamine (DA) and immunostaining for the dopamine transporter (DAT) in the substantia nigra pars compacta (SNpc) was performed. MPTP treatment reduced striatal DA levels significantly; nigral DAT immunoreactivity was reduced to a lower extent. However, there was no difference in DA levels and the number of DAT positive neurons between TNFR1 (−/−), TNFR2 (−/−) and wild type mice after MPTP treatment. In contrast to TNF-alpha deficiency neither TNFR1 nor TNFR2 gene ablation showed protection against MPTP neurotoxicity, which argues for a protective mechanism of TNF-alpha not mediated by TNFR1 and TNFR2 signaling.

OBJECTIVE: Mice lacking the tumor necrosis factor-alpha receptor 2 (TNFR2) gene fed a high-fat diet gain less weight and display reduced leptin and insulin levels. In humans, plasma levels of the soluble fraction of TNFR2 (sTNFR2) circulate in proportion to the degree of insulin resistance. The purpose of this study was to evaluate a polymorphism in the 3' untranslated region of the TNFR2 gene on chromosome 1 in relation to BMI, leptin levels, and insulin resistance. RESEARCH DESIGN AND METHODS: Using single-strand conformation polymorphism, the polymorphism was analyzed in 107 nondiabetic subjects (60 women, 47 men) and in 110 consecutive patients with type 2 diabetes (79 women, 31 men). In a subset of 33 healthy subjects, insulin sensitivity (minimal model analysis) was also evaluated. RESULTS: Four alleles of the TNFR2 gene were identified (A1, A2, A3, and A4). BMI and serum leptin levels were significantly increased in young carriers of the A2 allele. Plasma sTNFR2 levels were similar among the different TNFR2 gene variants. However, in subjects who did not carry the A2 allele, in young subjects, and in women, plasma sTNFR2 levels were proportional to BMI and leptin levels. In the study sample, carriers of the A2 allele (n = 18) showed significantly increased BMI, fat mass, waist-to-hip ratio, serum total and VLDL triglyceride levels, and leptin levels and had a lower insulin sensitivity index than noncarriers of the A2 variant (n = 15). The frequency of the different alleles among diabetic subjects was similar to that in the control population. However, diet-treated diabetic subjects (n = 49) who were carriers of the A2 allele exhibited significantly higher BMI and leptin levels than diet-treated noncarriers of the A2 allele. CONCLUSIONS: The presence of the A2 allele in the TNFR2 gene may predispose subjects to obesity and higher leptin levels, which may in turn predispose them to insulin resistance or vice versa. The TNFR2 gene may be involved in weight-control mechanisms.

The eye has been described as an immunologically privileged site because it can accept grafts from allogeneic hosts. This immune privilege (29, 30) results from a combination of sequestration of autoantigen behind the blood-ocular barrier, induction of immune deviation phenomena (19, 20), presence of anti-inflammatory molecules in the eye (1, 7, 31–35, 39), and restriction of T-cell infiltration by the expression of Fas ligand (FasL) by ocular cells (8, 11–13). However, ocular viral infections induce an inflammatory response, including the recruitment and activation of T lymphocytes. This immune response, aimed at the clearance of the pathogen, may lead to either the protection or the destruction of the eye structure. This dual role is well illustrated by ocular diseases induced by infection with herpes simplex virus (HSV). Neutrophils and CD4+ T lymphocytes are key mediators of the immunopathological keratitis induced by corneal infection with HSV type 1 (HSV-1) (6, 36), whereas T lymphocytes protect the retina of the eye during acute retinal necrosis following HSV-1 injection (2). Thus, the mechanisms modulating an inflammatory reaction in the eye may depend on the site of viral replication, and they remain poorly defined. Rabies virus induces an ocular disease in mice and humans (14; S. Camelo et al., submitted for publication). After infection in the hind limbs, the neurotropic Challenge Virus Standard (CVS) strain of rabies virus travels along the spinal cord and spreads to the brain and then to the eye. Viral invasion through the optic nerve leads to infection of the retinal ganglion cell (RGC) from day 6 postinfection (p.i.), but not of the photoreceptors. Rabies virus also infects the trigeminal ganglia, resulting in infection of the cornea by 12 days p.i. in mice (Camelo et al., submitted) and also in humans (17, 40). Morphological study of humans (14) and terminal deoxynucleotidyltransferase-mediated dUTP-tetramethylrhodamine-conjugated nick end labeling (TUNEL) staining of mice (Camelo et al., submitted) indicate that rabies virus ocular infection induces little apoptosis of the RGC despite morphological alteration and apoptosis of uninfected photoreceptors. Tumor necrosis factor alpha (TNF-α) is involved in the immune response during CVS acute encephalitis (5). In this article, we address the role of the immune response and of TNF-α in the control of rabies virus infection in the eye. We used immunocytochemistry to describe the infiltration of inflammatory cells in the retinas of rabies virus-infected mice. Using mice lacking p55TNFR, the p55 TNF-α receptor (p55TNFR−/− mice), athymic nude mice, and wild-type BALB/c and C57BL/6 mice, we assessed the role of signaling through p55TNFR and of infiltrating T lymphocytes in the outcome of rabies virus ocular disease. Finally, the sensitivity of inflammatory cells to undergo apoptosis was assessed by a combination of immunocytochemistry and TUNEL assays on frozen eye sections. Susceptibility to rabies ocular pathology and cellular infiltration are under the control of p55TNFR. Rabies virus-induced ocular disease was more severe in nude mice than in BALB/c mice, and only rare T lymphocytes in the eyes of CVS-infected mice were apoptotic. Therefore, despite the privileged immune status of the eye, T lymphocytes are able to enter the eye and are required to limit rabies virus ocular disease. Rabies virus-mediated eye disease is a promising model for elucidation of the mechanisms regulating the antiviral response in an immunologically privileged site.

Background: The aim is to define the role of single nucleotide polymorphism on the progress of hepatitis B virus (HBV) infection. We evaluated polymorphisms of TNF- α -308, Vitamin D receptor Apa I and Taq I gene in patients with HBV infection. Methods: All subjects included were older than 18 years old. Sixty three patients had chronic HBV infection, 61 were HBsAg positive carriers and 59 were positive for anti-HBs and anti-HBc. Gene polymorphisms were evaluated by Amplification Refractory Mutation System PCR. For patients with chronic hepatitis, viral load, ALT levels, and histopathological evaluation of the liver were also compared. Results: Gender distribution was not different among groups; however, anti-HBs positive patients were significantly older than the other patients. ALT levels and viral load were significantly higher in chronic hepatitis group than the asymptomatic carriers group. Vitamin D receptor Apa I gene and Taq I gene and TNF- α -308 gene variant alleles were not different in all three groups. Variant alleles of three genes were not different in subgroups of chronic hepatitis patients formed according to ALT levels, viral load, histological activity index, and fibrosis score. Conclusions: Role of single nucleotide polymorphism in clinical status of various HBV infection states was not shown in this study. Considering the other studies performed with this aim, which strengthens the notion that ethnicity is an important factor, future studies with more patients from different ethnic groups may help to clear the role of polymorphisms in the clinical progress of HBV infection. doi: http://dx.doi.org/10.4021/gr544e

We examined the age-associated changes in tumor necrosis factor alpha (TNF alpha) receptor on peripheral blood monocytes and bone marrow macrophages obtained from slc:Wistar rats and slc:ICR mice. The TNF alpha receptor on monocytes and macrophages in both rats and mice showed high and low affinity sites. The Kd values of those sites gradually increased with age in both types of cells from both species. Binding capacities (receptor number) were low in 52-week-old animals. These results suggest that the TNF alpha receptor on monocytes and macrophages of these species might decrease with age in both quality and quantity.

Decreased severity of graft-versus-host disease after mismatched umbilical cord blood (UCB) transplantation may be attributed in part to the increased propensity to apoptosis of UCB T cells following activation. Interleukin (IL)-15, a pleiotropic cytokine that is essential for T-cell proliferation and survival, may serve as promising immunomodulative therapy post-CB transplantation for its anti-apoptotic effect. This study aimed to determine the kinetics of Fas or tumor necrosis factor-alpha receptor (TNFR) mediated caspase-3 expression and apoptosis of anti-CD3/anti-CD28 activated UCB T cells in the influence of IL-15. Activated caspase-3 expression was analyzed by Western blotting and the percentage of apoptotic cells was determined by annexin-V/propidium iodide (PI) flow cytometric staining. Significant expression of Fas and TNFR2 was detected on anti-CD3/anti-CD28 pre-activated UCB T cells. These cells were susceptible to anti-Fas but not TNF-alpha-induced apoptosis. Kinetic study shows that caspase-3 expression became evident at 6th-8th h following anti-Fas stimulation, while early apoptotic cells with annexin-V(+)/PI(-) expression appeared at 12th-16th h. IL-15, though successful in decreasing apoptosis in pre-activated UCB T cells, failed to completely prevent Fas-mediated caspase-3 expression and apoptosis of CB T cells. The pre-activated UCB and adult peripheral blood T cells behaved similarly with regard to death receptor expression, caspase-3 expression and apoptosis upon Fas-engagement. Although IL-15 promotes overall activated UCB T-cell survival, it did not particularly prevent Fas-mediated activation-induced cell death.

Tumor necrosis factor alpha (TNF-α) is important in promoting relative adrenal insufficiency (RAI) due to systemic inflammatory response syndrome (SIRS). We identified the TNF-α receptor involved in the inhibition of adrenal corticotrophin (ACTH)-stimulated hydrocortisone release by studying the expression of TNF-α receptors in adrenal cortex Y1 cells and the effect of downregulating TNF receptors on ACTH-stimulated hydrocortisone release.We used real-time PCR and immunocytochemistry to evaluate the expression of TNF receptors on Y1 cells. TNF-receptor 1 (TNF-R1) DNA fragments corresponding to the short hairpin RNA (shRNA)-sequences were synthesized and cloned into pcDNA(TM) 6.2-GW/EmGFP expression vector. Knockdown efficiency of TNF-R1 expression was evaluated in miRNA transfected and mock-miRNA transfected Y1 cells by quantitative real-time PCR (Q-PCR). Hydro-cortisone expression levels were determined in TNF-R1-knockdown and control Y1 cells treated with TNF-α and ACTH.Mouse adrenal cortex Y1 cells were positive for type I TNF-R1, but not type II TNF-receptor (TNF-R2). Blocking TNF-R1 expression resulted in loss of TNF-α-mediated inhibition of ACTH-stimulated hydrocortisone expression, suggesting a role for the TNF-R1 related signaling pathway in ACTH-stimulated hydrocortisone synthesis.The inhibitory effect of TNF-α on ACTH-stimulated hydrocortisone synthesis was mediated via TNF-R1 in adrenal cortex.

Because almost all pulmonary diseases are not caused by one gene, multiple gene transfection is required for current gene therapy. Adenovirus is an important gene therapy vector, but a short duration and the inability of repeated administration remain limitations. The aims of this study were to evaluate whether adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs gene expression and facilitates repeated vector administration to investigate the feasibility of a cotransfection strategy.F344 rats received intratracheal administration of 1 x 10(9) plaque-forming units of adenoviral vector encoding beta-galactosidase or both adenoviral vector encoding beta-galactosidase and adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin. In the expression study beta-galactosidase gene expression in the lung was examined by means of enzyme-linked immunosorbent assay on days 2, 7, 14, 28, and 56 (n = 4/day). In the repeated transfection study, soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase were readministered once (7 days after the first adenovirus administration) or twice (on days 7 and 14; n = 4/day). A 2-way factorial analysis of variance was used for statistical analysis.Soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolonged the duration of beta-galactosidase expression. However, antiadenovirus antibody production was significantly increased in the cotransfection group. In addition, there was no increase in beta-galactosidase expression after readministration of soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase.Adenoviral vector encoding soluble tumor necrosis factor alpha receptor immunoglobulin and beta-galactosidase cotransfection prolongs beta-galactosidase expression but does not increase beta-galactosidase expression after repeated administration. These results suggest that tumor necrosis factor alpha is one of the most important factors in regulating the duration of gene expression. The cotransfection approach is feasible, but the increase of antiadenovirus antibodies might make repeated cotransfection unfeasible.

To investigate the relationship between tumor necrosis factor receptor II (TNFR II) gene 196T/G polymorphism and preterm labor in Han population in Chengdu.Samples were collected from 96 subjects in corresponding period, 46 preterm labor pregnant women (we collected partial placental tissues of preterm labor, 21 cases of infectious group with chorioamnionitis, 25 cases of noninfectious group without chorioamnionitis), and 50 normal labor pregnant women. The DNA was extracted from each sample by using Chelex-100 method, then PCR-RFLP was performed to determine the TNFR II 196 gene polymorphism.1) TNFR II 196 genotype frequencies of 196M/M (TT), 196M/R (TG) + 196R/R (GG) were 71.7%, 21.7%+ 6.5% and 80.0%, 20.0%+0.0% in preterm labor and normal control group respectively. Allele frequencies of R (G), M (T) were 17.4%, 82.6% and 10.0%, 90.0%, respectively. There were no significant difference in frequencies of genotype and allele in TNFR II 196 gene polymorphism between two groups (P0.05, P0.05, respectively). 2) Close correlation was observed between the different genotypes and the chorioamnionitis (chi2 = 11.088, P0.05). The odds ratio (OR) for TG+GG genotype was 12.65, 95% CI 2.359, 67.848, with more than 12.65 times probability of chorioamnionitis than that of TT genotype group.Polymorphism in 196 site of TNFR II gene was not crucial in preterm labor genesis, TG (GG) genotype may contribute to susceptibility to chorioamnionitis in the process of preterm labor in Chinese Han population.

In order to define the receptor binding site and the structure-functional relationship of tumor necrosis factor (TNF), single amino acid substitutions were made by site-directed mutagenesis at selected residues of human tumor necrosis factor, using a phagemid mutagenesis/expression vector. The recombinant TNF mutants were compared to the wild type TNF in assays using crude bacterial lysates, for protein yield, solubility, subunit trimerization, receptor binding inhibition activity, and in vitro cytotoxic activity. All mutants which did not form cross-linkable trimer also showed little cytotoxic activity or receptor binding inhibition activity, indicating that trimer formation is obligatory for TNF-alpha activity. Most mutations of internal residues yielded no cross-linkable trimer, while most mutations of surface residues yielded cross-linkable trimer. Mutations at surface residues Leu29, Arg31, and Ala35 yielded cross-linkable trimers with good activities, except proline substitutions which may cause conformational changes in the polypeptide chain. This suggested that these residues are near the receptor binding site. Mutations at other strictly conserved internal residues such as Ser60, His78, and Tyr119 form cross-linkable trimer with little activity. These mutations may indirectly affect the receptor binding site by forming trimers with undetectable abnormalities. Mutants of surface residues Tyr87, Ser95, Ser133, and Ser147 affect receptor binding and cytotoxic activity but not trimer formation, suggesting that these residues are involved directly in receptor binding. The fact that residues Arg31, Ala35, Tyr87, Ser95, and Ser147, located on the opposite sides of a monomer, are clustered at the intersubunit grooves of TNF trimer supports the current notion that TNF receptor binding sites are trivalent and are located at the three intersubunit grooves. However, our finding that Ser133, which is outside the groove, can also be involved directly in receptor binding suggested that the receptor binding sites of TNF may not be confined to the intersubunit grooves, but extended to include additional surface residues.

The relevance of estrogen functions in lipid metabolism has been suggested in patients with estrogen-signaling deficiencies. Their importance was further implied by studies in estrogen-deficient mice (ArKO mice), which progressively developed hepatic steatosis. As circulating tumor necrosis factor (TNF)-alpha levels are known to positively correlate with disturbances in lipid metabolism, we investigated the impact of the loss of TNF-alpha signaling on carbohydrate and lipid metabolism in ArKO mice. Histological examinations of the livers of mice at 5 months of age revealed that ArKO male mice lacking the TNF-alpha receptor type 1 (TNFR1) gene (ArKO/TNFR1KO) or both the TNFR 1 and 2 genes (ArKO/TNFR12KO) developed more severe hepatic steatosis than ArKO or ArKO/TNFR2KO mice. Serum analyses demonstrated a clear increase in cholesterol and insulin levels in the ArKO/TNFR1KO mice compared with the ArKO mice. Glucose- and insulin-tolerance tests further revealed exacerbation of the systemic insulin resistant phenotype in the ArKO/TNFR1KO mice. Hepatic expression of lipogenic genes including fatty-acid synthase and stearoyl-Coenzyme A desaturase 1 were more markedly upregulated in the ArKO/TNFR1KO mice than the ArKO mice. These findings indicate that under estrogen-deficient physiological conditions, hepatic lipid metabolism would benefit from TNF-alpha mediated signaling via TNFR1.

The activation of the c-Jun N-terminal kinases and their substrate transcription factor c-Jun is central to the death of dopaminergic neurons of the substantia nigra pars compacta (SNC) but the underlying signal cascades are poorly understood. We have studied the impact of the p55 tumor necrosis factor-alpha receptor (TNF-R) 1 on the N-terminal phosphorylation of c-Jun and the survival of the dopaminergic SNC neurons after transection of the medial forebrain bundle. The axotomy raised the immunoreactivities of tumor necrosis factor-alpha, p75 TNF-R2 and ED1 (ectodysplasin A) in the substantia nigra equally in wildtype and knockout (ko) mice and of TNF-R1 in wildtype mice. Importantly, TNF-R1 ko significantly reduced the early phosphorylation of c-Jun between 18 h and 3 d post-axotomy but the functional deficiency of TNF-R1 did not affect the survival of the dopaminergic neurons up to day 30. These findings demonstrate that: (i) TNF-R1 is involved in the early cell body response after axon transection; (ii) TNF-R1 operates upstream of c-Jun N-terminal kinase/c-Jun, the central signal system of nerve fiber injury, and (iii) the failure of persistent reduction of activated c-Jun is linked to the failure of protection of dopaminergic SNC neurons by TNF-R1 ko.

We examined the influence of supplementation with selenomethionine on soluble tumor necrosis factor-alpha receptor type 1 (sTNF-R1) and C-reactive protein (CRP) concentrations in patients with psoriasis who were treated with narrowband ultraviolet B.Thirty-seven patients had narrowband ultraviolet B therapy five times a week and received 200 mug of selenium daily as selenomethionine (group 1, n = 19) or placebo (group 2, n = 18) for 4 wk. Assessment, performed at baseline, after 2 and 4 wk, and 4 wk after the end of treatment included measurement of the Psoriasis Area and Severity Index (PASI) and serum concentrations of selenium (micrograms per liter), sTNF-R1 (nanograms per milliliter), and CRP (milligrams per liter). Control sera were obtained from 20 healthy volunteers.Baseline PASI was 12.70 +/- 5.48 (13.02 +/- 6.25 in group 1 and 12.37 +/- 4.71 in group 2), selenium concentration was 50.55 +/- 9.54 (49.05 +/- 10.38 and 52.13 +/- 8.61, respectively), sTNF-R1 concentration was 1.91 +/- 0.38 (1.96 +/- 0.37 and 1.87 +/- 0.40, respectively), and CRP concentration was 25.34 +/- 8.27 (26.12 +/- 8.42 and 24.57 +/- 7.72). In controls, selenium concentration was 48.71 +/- 9.39 (P0.05 versus patients), sTNF-R1 concentration was 1.48 +/- 0.30 (P0.05), CRP concentration was6. The baseline sTNF-R1 level correlated to PASI value (r = 0.40, P0.05) and CRP concentration (r = 0.36, P0.05). The treatment resulted in an almost parallel decrease in PASI in both groups. At 4 wk after the end of treatment, selenium concentrations were 83.77 +/- 5.13 in group 1 and 52.12 +/- 7.54 in group 2 (P0.05), sTNF-R1 concentrations were 1.72 +/- 0.27 and 1.47 +/- 0.26 (P0.05), and CRP concentrations were 7.72 +/- 4.23 and 8.15 +/- 3.32, respectively (P0.05). Selenium concentration correlated inversely with CRP in group 1.The results confirm that sTNF-R1 and CRP concentrations are increated in active psoriasis and that supplementation with selenomethionine for 4 wk in safe doses is ineffacious as adjuvant therapy in patients with psoriasis.

Chlamydia psittaciwas found to modulate receptor expression for the cytokine receptors that are involved in the synergistic induction of indoleamine dioxygenase in epithelial cells. Increases in receptor expression were seen even with inactivatedChlamydia, suggesting that chlamydial antigens and not products of infection are important for up-regulating cytokine receptor expression.

The aim of the study was to examine the concentration of soluble tumor necrosis factor alpha type I (sTNF-R1) in patients with psoriasis vulgaris in relation to the severity of skin lesions and the effects of treatment.the study was conducted among 34 patients. The assessment of the severity of skin lesions (Psoriasis Area and Severity Index -PASI), and the serum concentration of sTNF-R1 (ELISA) were performed at baseline and after 2 weeks of treatment. Control sera were obtained from 30 healthy volunteers.pretreatment PASI was 12.98 +/- 5.77 and after two-week treatment 6.17 +/- 2.12 (p0.05), sTNF-R1 concentration 1.88 +/- 0.41 ng/mL and 1.65 +/- 0.46 ng/mL, respectively (p0.05). Serum concentration of sTNF-R1 was significantly lower in controls (1.48 +/- 0.30 ng/mL) as compared with its pre-treatment level in psoriasis patients. A significant correlation between pre-treatment PASI and concentration of sTNF-R1 was found (r = 0.40, p0.05).serum concentration of sTNF-R1 can be a sensitive marker of activity of psoriasis vulgaris and decreases following the effective treatment.

Background Obesity and insulin resistance are increasingly common problems in children. Tumor necrosis factor-alpha (TNF-alpha) has important effects on lipid and glucose metabolism. This effect may be mediated through soluble TNF-alpha receptor 2 (sTNFR2). Objective To investigate the relationship between insulin resistance and the TNF-alpha system in childhood obesity. Children and methods Twenty-one obese and six non-obese children were studied. Body mass index (BMI) z-scores, percent body fat (PBF) and waist to hip ratio (WHR) were determined. Fasting serum levels of total cholesterol, HDL-cholesterol, LDL-cholesterol, TNF-alpha and sTNFR2 were measured. A standard 2-hour oral glucose tolerance test (dose of glucose: 1.75 g/kg, max. 75 g) was done. Insulin resistance (IR) was estimated by fasting plasma insulin, plasma insulin at 120 min, homeostasis model assessment (HOMA) and insulin area under the curve (AUC) from OGTT. Insulin sensitivity was estimated by oral glucose insulin sensitivity (OGIS120). Results Among the obese participants, one child (5.2%) was found to have diabetes mellitus and four others (21.1%) impaired glucose tolerance (IGT). Obese children had significantly elevated sTNFR2 levels. Furthermore, the group of obese children with IGT and the patient with newly diagnosed diabetes mellitus together (n = 5) had significantly higher levels of serum sTNFR2 (2,865+/-320 pg/ml) than the rest of the obese (2,460+/-352 pg/ml; p = 0.016) or lean (1,969+/-362 pg/ml; p = 0.014) children. Serum sTNFR2 levels correlated positively with insulin AUC, HOMA IR, fasting plasma insulin, plasma insulin at 120 min, total cholesterol and LDL/ HDL ratio, and negatively with OGIS120. Multiple regression analysis revealed that age, WHR, sTNFR2 and LDL predicted 81% of the variability in glucose at 120 min. Conclusion sTNFR2 is a candidate marker of insulin resistance and glucose intolerance.

Tumor necrosis factor-alpha receptor knockout (TNF-alphaRKO) mice have homozygous deletions of the genes that code for both the 55- and 75-kD receptors. The mice are protected from the fibrogenic effects of bleomycin, silica, and inhaled asbestos. The asbestos-exposed animals exhibit reduced expression of other peptide growth factors such as transforming growth factor (TGF)-alpha, platelet-derived growth factors, and TGF-beta. In normal animals, these and other cytokines are elaborated at high levels during the development of fibroproliferative lung disease, but there is little information available that has allowed investigators to establish the role of the individual growth factors in disease pathogenesis. Here, we show that overexpression of TGF-beta(1) by means of a replication-deficient adenovirus vector induces fibrogenesis in the lungs of the fibrogenic-resistant TNF-alphaRKO mice. The fibrogenic lesions developed in both the KO and background controls within 7 d, and both types of animals exhibited similar incorporation of bromodeoxyuridine. Interestingly, airway epithelial cell proliferation appeared to be suppressed, perhaps due to the presence of the TGF-beta(1), a well-known inhibitor of epithelial mitogenesis. Before these experiments, there was no information available that would provide a basis for predicting whether or not TGF-beta(1) expression induces fibroproliferative lung disease in fibrogenic-resistant TNF-alphaRKO mice, an increasingly popular animal model.

Soluble (sTNF) and transmembrane (tmTNF) forms of TNFalpha (TNF) have distinct proinflammatory effects. We investigated whether tmTNF altered the synthesis of some proinflammatory proteins involved in atherothrombosis, in murine aortas and aortic endothelial cells (MAEC). Samples were obtained from wild-type (WT) mice and TNF-deficient mice that express a mutated non cleavable tmTNF transgene (tmTNFnc). The levels of secreted MCP-1, RANTES, IL-6, PAI-1, soluble ICAM-1, and soluble TNF receptor type 1 (TNFR1; CD120a) antigens, MMP-9 activity and of cell surface ICAM-1 were not significantly different between the two types of MAEC. The magnitude of endotoxin-stimulated production of RANTES, MCP-1 and IL-6 was similar in the two types of cells. Of note, the amount of synthesized TNF receptor type 2 (TNFR2; CD120b), measured by its secreted (in aorta and MAEC), intracellular and mRNA levels (in MAEC), was significantly 4-fold lower in tmTNFnc than in WT mice, both in basal and endotoxin-stimulated conditions. A neutralizing anti-TNF antibody or the recombinant murine TNF did not modify the magnitude of the difference in TNFR2 production between the two types of cells, suggesting a preponderant role of tmTNF in the down-regulation of TNFR2 synthesis. Macrophages of tmTNFnc mice also produced less TNFR2 than WT macrophages (-30%). Plasmas of tmTNFnc mice contained significantly less sTNFR2 than WT mice (-75%). In conclusion, an increase in tmTNF levels, rather than the lack of sTNF, significantly down-modulated TNFR2 synthesis in aortic endothelial cells, but had no major influence on the synthesis of some major pro-inflammatory and pro-atherothrombotic proteins.

interferon beta (IFN-beta) reduces relapse rate and disease progression in patients with the relapsing-remitting form of multiple sclerosis (RRMS). IFN-beta may act by upregulating the expression of anti-inflammatory components of the immune system.To determine whether weekly intramuscular (i.m.) injection of IFN-beta1a had a short- or long-term effect on the expression of naturally occurring soluble factors that play an immunosuppressive role within the cytokine network.serum levels of interleukin-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor alpha receptor type I and type II (sTNF-alphaRI and sTNF-alphaRII), and soluble E-selectin (sE-Sel) were followed over time in ten patients with RRMS who were treated with weekly i.m. injections of 30 mg (= 6 MU) of IFN-beta1a. Patient sera were sampled before, and 24, 48, 72, 96, and 168 hours after the first IFN-beta1a injection (short-term), and then at 1, 3, 6, 9 and 12 months after therapy initiation (long-term); highly sensitive, commercially available ELISA tests were used.serum levels of IL-1Ra, sTNF-alphaRI and sTNF-alphaRII, but not sE-Sel were significantly increased in both short- and long-term follow-up. Interestingly, IL-1Ra, sTNF-alphaRI and sTNF-alphaRII behaviors were completely different, suggesting that these naturally occurring immunoregulatory factors were differentially affected by IFN-beta1a.our study demonstrates that weekly i.m. injection of 30 mg of IFN-beta1a induces the expression of soluble mediators that may suppress the activities of pro-inflammatory cytokines such as IL-1 and TNF-alpha.

OBJECTIVE - Leptin and tumor necrosis factor (TNF)-α are associated with insulin resistance and cardiovascular disease. In vitro studies suggested that these effects may be mediated via overproduction of monocyte chemoattracting protein (MCP)-1/CCL2, which is a chemokine involved in the pathogenesis of atherosclerosis. RESEARCH DESIGN AND METHODS - In this study, fasting plasma leptin, soluble TNF-α receptor 2 (TNF-α-R2), and MCP-1/CCL2 concentrations were measured in 207 middle-aged women (age 61 ± 12 years, BMI 30.1 ± 6.6 kg/m2), including 53 patients with type 2 diabetes, 42 with impaired glucose tolerance, and 112 with normal glucose tolerance, to assess cross-sectionally their relationship with markers of atherosclerosis and, longitudinally over 7 years, whether their circulating levels were associated with cardiovascular disease (CVD) mortality. RESULTS - At baseline, leptin and TNF-α-R2 were not different among groups; meanwhile, MCP-1/CCL2 was increased in type 2 diabetes (P < 0.05). All showed significant associations with biochemical risk markers of atherosclerosis. In a univariate analysis, age, fasting insulin, leptin, and MCP-1/CCL2 were associated with CVD mortality at 7 years. When a multivariate analysis was performed, only age, leptin, and insulin retained an independent association with CVD mortality, with leptin showing a protective effect (hazard ratio 0.88; P < 0.02). CONCLUSIONS - In middle-aged women, MCP-1/CCL2, leptin, and TNF-α-R2 were all related to biochemical risk markers of atherosclerosis. MCP-1/CCL2 concentration was the only one to be increased in type 2 diabetes with respect to nondiabetic women and the only one to be associated with increased risk of CVD mortality after a 7-year follow-up period in the univariate analysis. In the multivariate analysis, neither MCP-1/CCL2 nor TNF-α-R2 was associated with CVD mortality, and inspection of the data showed that leptin, in both the univariate and multivariate analysis, was associated with a protective effect

The immune stimulatory unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce expression of cyclooxygenase-2 (cox-2). The present study demonstrates that CpG DNA can up-regulate cox-2 expression by post-transcriptional mechanisms in RAW264.7 cells. To determine the CpG DNA-mediated signaling pathway that post-transcriptionally regulates cox-2 expression, a cox-2 translational reporter (COX2-3'-UTR-luciferase) was generated by inserting sequences within the 3'-untranslated region (UTR) of cox-2 to the 3' end of the luciferase gene under control of the SV40 promoter. CpG DNA-induced COX2-3'-UTR-luciferase activity was completely inhibited by an endosomal acidification inhibitor chloroquine, a Toll-like receptor 9 antagonist inhibitory CpG DNA, or overexpression of a dominant negative (DN) form of MyD88. However, overexpression of DN-IRAK-1 or DN-TRAF6 resulted in substantial, but not complete, inhibition of the CpG DNA-induced COX2-3'-UTR-luciferase activity. Activation of all three MAPKs (ERK, p38, and JNK) was required for optimal COX2-3'-UTR-luciferase activity induced by CpG DNA. Overexpression of DN-TRAF6 suppressed CpG DNA-mediated activation of p38 and JNK, but not ERK, explaining the partial inhibitory effects of DN-TRAF6 on CpG DNA-induced COX2-3'-UTR-luciferase activity. Co-expression of DN-TRAF6 and N17Ras completely inhibited CpG DNA-induced COX2-3'-UTR-luciferase activity, indicating the involvement of Ras in CpG DNA-mediated ERK and COX2-3'-UTR regulation. Collectively, our results suggest that MyD88 and MAPKs play a key regulatory role in CpG DNA-mediated cox-2 expression at the post-transcriptional level and that TRAF6 is a diverging point in the Toll-like receptor 9-signaling pathway for CpG DNA-mediated MAPK activation.

OBJECTIVE—Tumor necrosis factor-α (TNF-α) is one of the proposed mediators of insulin resistance, upregulated in human obesity. Insulin resistance, however, might precede the development of obesity, especially in subjects with a family history of type 2 diabetes. Therefore, the aim of the present study was to assess plasma levels of TNF-α and soluble forms of its receptors (soluble TNF-α receptors 1 [sTNFR1] and 2 [sTNFR2]) and to evaluate the relationship of the TNF-α system with insulin resistance in lean, nondiabetic offspring of type 2 diabetic patients. RESEARCH DESIGN AND METHODS—We compared 20 lean offspring (BMI RESULTS—Both men and women in the offspring group were markedly more insulin-resistant and had higher plasma levels of sTNFR2 (all P < 0.05). TNF-α, sTNFR1, and other examined parameters did not differ between the studied groups. Both TNF-α receptors were related to waist-to-hip ratio (WHR), fat-free mass (FFM), plasma total cholesterol, HDL cholesterol, LDL cholesterol, and nonesterified fatty acids (NEFAs). sTNFR2, but not sTNFR1, was also associated with insulin sensitivity (r = −0.49, P = 0.001). This relationship remained significant after adjustment for WHR, FFM, plasma insulin, and NEFA. CONCLUSIONS—TNF-α system might be involved in modulating insulin action before the onset of obesity in subjects at high risk for type 2 diabetes.

To define the frequency of the exon 6 tumor necrosis factor-alpha (TNF-alpha) receptor II (TNFRII) gene polymorphism in severe and mild-moderate rheumatoid arthritis (RA) and its possible influence on anti-TNF-alpha treatment responsiveness.Two cohorts of patients with RA, the first (n = 97) defined as methotrexate responders (MTX-R) with mild-moderate synovitis, and the second (n = 78) defined as nonresponders to combination therapy and receiving anti-TNF-alpha treatment because of their severe and aggressive disease (TNF-T), were studied retrospectively and compared to age, sex, and ethnically matched controls (n = 84). In the prospective study, 66 patients with severe RA were followed over the first 6 months of anti-TNF-alpha therapy and their response was examined according to genotype.We observed a trend towards an increased frequency of the GG genotype in patients with severe RA (6.4%) in comparison with patients with mild-moderate disease (3.1%) and controls (1.2%). When looking at the response to anti-TNF-alpha therapy, we observed that after 12 weeks of treatment, 37.8% of the TT versus 10.7% of the TG/GG patients passed from high to medium-low disease activity (p = 0.03).In our cohorts of patients selected by response to the conventional therapy and by disease severity, our preliminary study results showed a trend towards a higher prevalence of the GG genotype for the exon 6 TNFRII polymorphism in the less responsive patients with more aggressive disease. We also found a lower degree of response to anti-TNF-alpha treatments in patients carrying the G allele.

Arsenic trioxide (As2O3) has been reported to induce apoptosis in human T-cell leukemia virus type-I (HTLV-I) infected T-cell lines and fresh adult T-cell leukemia (ATL) cells and to induce G1 phase accumulation in HTLV-I infected T-cell lines. The present study aimed to clarify the pathway of As2O3-induced apoptosis in HTLV-I infected T-cell lines, MT-1 and MT-2, and fresh ATL cells separated from peripheral blood of patients with acute or chronic type ATL. Cells were treated up to 72 h at clinically tolerable concentrations of As2O3 (1-2 micromol/l) shown to be safe in patients with acute promyelocytic leukemia (APL). Activation of caspases 3, 8, and 9, loss of mitochondrial transmembrane potential and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) were observed during As2O3 treatment. Furthermore, prior exposure to a broad-spectrum caspase inhibitor blocked As2O3-induced apoptosis but not G1 phase accumulation. While pre-treatment with a CD95 receptor-blocking antibody (Ab) or a TNF-alpha neutralizing Ab did not show such inhibitions in these cells. In conclusion, As2O3 induces apoptosis in HTLV-I infected T-cell lines and fresh ATL cells through CD95 or TNF-alpha receptor independent caspase activation.