Increased Reactive Oxygen Species and Tumor Necrosis Factor-Alpha Production by Monocytes are Associated with Elevated Levels of Uric Acid in Pre-Eclamptic Women

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Role of Paracoccidioides brasiliensis cell wall fraction containing beta-glucan in tumor necrosis factor-alpha production by human monocytes: correlation with fungicidal activity

The polysaccharide fraction of Paracoccidioides brasiliensis mycelial cell wall (F1 fraction), the active component of which is composed of beta-glucan, was investigated in regard to the activation of human monocytes for fungal killing. The cells were primed with interferon-gamma (IFN-gamma) or F1 (100 and 200 mug ml(-1)) or F1 (100 and 200 mug ml(-1)) plus IFN-gamma for 24 h and then evaluated for H2O2 release. In other experiments, the cells were pretreated with the same stimuli, challenged with a virulent strain of P. brasiliensis and evaluated for fungicidal activity and levels of tumor necrosis factor (TNF-alpha) in the supernatants. F1 increased the levels of H2O2 in a similar manner to IFN-gamma. However, a synergistic effect between these two activators was not detected. on the contrary, a significant fungicidal activity was only obtained after priming with IFN-gamma plus F1. This higher activity was associated with high levels of TNF-alpha in the supernatants of the cocultures. Overall, P. brasiliensis F1 fraction induced human monocytes to release relatively high levels of TNF-alpha, which, in combination with IFN-gamma, is responsible for the activation of human monocytes for effective killing of P. brasiliensis.

Smoking status and tumor necrosis factor-alpha mediated systemic inflammation in COPD patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Secretion of tumor necrosis factor-alpha by mouse peritoneal macrophages in the presence of dental sealers, sealapex and endomethasone

After filling root canals, the healing process depends on the chemical composition or physical-chemical properties of the material used, among other factors. All root canal sealers, whether solid or plastic, are foreign matter for the body if they remain in permanent contact with apical and periapical tissues. As a result, the first organic reaction that occurs is an attempt to phagocytize the material. During phagocytosis, macrophages release a large number of cell mediators into the area, among which are cytokines that are essential in intercellular communication and in many physiological and pathophysiological processes. One of these cytokines is tumor necrosis factor-alfa (TNF-α), which acts through links to specific receptors on the cell membrane initiating a cascade of events leading to induction, activation, or inhibition of numerous cytokine-regulated genes in the cell nucleus. The release of TNF-α in a cell culture of mouse peritoneal macrophages incubated with three concentrations (25, 50, and 100 mg/ml) of two endodontic sealers was measured. The solutions containing the calcium hydroxide-based root canal sealer (Sealapex) released fewer units of TNF-α than solutions containing the zinc oxide and eugenol-based sealer (Endomethasone).

Molecular basis for defining the pineal gland and pinealocytes as targets for tumor necrosis factor

The pineal gland, the gland that translates darkness into an endocrine signal by releasing melatonin at night, is now considered a key player in the mounting of an innate immune response. Tumor necrosis factor (TNF), the first pro-inflammatory cytokine to be released by an inflammatory response, suppresses the translation of the key enzyme of melatonin synthesis (arylalkylamine-N-acetyltransferase, Aanat). Here, we show that TNF receptors of the subtype 1 (TNF-R1) are expressed by astrocytes, microglia, and pinealocytes. We also show that the TNF signaling reduces the level of inhibitory nuclear factor kappa B protein subtype A (NFKBIA), leading to the nuclear translocation of two NFKB dimers, p50/p50, and p50/RelA. The lack of a transactivating domain in the p50/p50 dimer suggests that this dimer is responsible for the repression of Aanat transcription. Meanwhile, p50/RelA promotes the expression of inducible nitric oxide synthase (iNOS) and the production of nitric oxide, which inhibits adrenergically induced melatonin production. Together, these data provide a mechanistic basis for considering pinealocytes a target ofTNF and reinforce the idea that the suppression of pineal melatonin is one of the mechanisms involved in mounting an innate immune response. © 2011 Carvalho-Sousa, da Silveira Cruz-Machado, Tamura, Fernandes, Pinato, Muxel, Cecon and Markus.

Differential Gene Expression Profiles May Differentiate Responder and Nonresponder Patients with Rheumatoid Arthritis for Methotrexate (MTX) Monotherapy and MTX plus Tumor Necrosis Factor Inhibitor Combined Therapy

Objective. We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-alpha (anti-TNF) combined therapy. Methods. We evaluated 25 patients with RA taking MTX 15-20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received intliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. Results. Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF nonresponders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. Conclusion. Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1 CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study. (First Release July 1 2012; J Rheumatol 2012;39:1524-32; doi:10.3899/jrheum.120092)

Persistent Periodontal Disease Hampers Anti-Tumor Necrosis Factor Treatment Response in Rheumatoid Arthritis

Objective: This study aimed to evaluate prospectively the influence and the evolution of periodontal disease (PD) in rheumatoid arthritis (RA) patients submitted to anti-tumor necrosis factor (TNF) therapy. Methods: Eighteen patients with RA (according to the American College of Rheumatology criteria) were assessed for PD before (BL) and after 6 months (6M) of anti-TNF treatment: 15 infliximab, 2 adalimumab, and 1 etanercept. Periodontal assessment included plaque and gingival bleeding indices, probing pocket depth, cementoenamel junction, and clinical attachment level. Rheumatologic evaluation was performed blinded to the dentist's assessment: demographic data, clinical manifestations, and disease activity (Disease Activity Score using 28 joints [DAS28], erythrocyte sedimentation rate [ESR], and C-reactive protein [CRP]). Results: The median age and disease duration of patients with RA were 50 years (25-71 y) and 94% were female. Periodontal disease was diagnosed in 8 patients (44.4%). Comparing BL to 6M, periodontal parameters in the entire group remained stable (P > 0.05) throughout the study (plaque and gingival bleeding indices, probing pocket depth, cementoenamel junction, and clinical attachment level), whereas an improvement in most analyzed RA parameters was observed in the same period: DAS28 (5.5 vs. 3.9, P = 0.02), ESR (21 vs. 12.5 mm/first hour, P = 0.07), and CRP (7.8 vs. 2.8 mg/dL, P = 0.25). Further analysis revealed that this improvement was restricted to the group of patients without PD (DAS28 [5.5 vs. 3.6, P = 0.04], ESR [23.0 vs. 11.5 mm/first hour, P = 0.008], and CRP [7.4 vs. 2.1, P = 0.01]). In contrast, patients with PD had lack of response, with no significant differences in disease activity parameters between BL and 6M: DAS28 (5.2 vs. 4.4, P = 0.11), ESR (17.0 vs. 21.0, P = 0.56), and CRP (9.0 vs. 8.8, P = 0.55). Conclusions: This study supports the notion that PD may affect TNF blocker efficacy in patients with RA. The possibility that a sustained gingival inflammatory state may hamper treatment response in this disease has high clinical interest because this is a treatable condition.

Tumor necrosis factor sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. CONCLUSION: Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Immune response to influenza vaccination in children treated with methotrexate or/and tumor necrosis factor-alpha inhibitors

In children treated with immunosuppressive medication such as methotrexate and tumor necrosis factor-alpha (TNF-α) inhibitors, additional immunizations are recommended because of increased susceptibility to infections. However, it is unclear if adequate antibody response to vaccinations can be established in children receiving methotrexate and/or TNF-α inhibitors. In a prospective open label study, we assessed seroprotection and seroconversion following influenza vaccination during 2 seasons (6 strains) in 36 children with autoimmune disease treated either with methotrexate (n=18), TNF-α inhibitors (n=10) or both (n=8) and a control group of 16 immunocompetent children. Influenza antibody titers were determined by hemagglutinin inhibition assay, before and 4-8 weeks after vaccination. Post-vaccination seroprotection (defined as a titer ≥1:40) did not significantly differ between immunosuppressed and immunocompetent subjects. Seroconversion, defined as the change from a nonprotective (< 1:40) to a protective titer (≥1:40) with at least a 4-fold titer increase, was less likely to occur in immunosuppressed patients, although no significant difference from the control group was established. Safety evaluation of vaccination showed no serious adverse events. Children receiving methotrexate and/or TNF-α inhibitors can be safely and effectively immunized against influenza, with a seroprotection after vaccination comparable to immunocompetent children.

Tumor necrosis factor-alpha: alternative role as an inhibitor of osteoclast formation in vitro

TNFalpha is known to stimulate the development and activity of osteoclasts and of bone resorption. The cytokine was found to mediate bone loss in conjunction with inflammatory diseases such as rheumatoid arthritis or chronic aseptic inflammation induced by wear particles from implants and was suggested to be a prerequisite for the loss of bone mass under estrogen deficiency. In the present study, the regulation of osteoclastogenesis by TNFalpha was investigated in co-cultures of osteoblasts and bone marrow or spleen cells and in cultures of bone marrow and spleen cells grown with CSF-1 and RANKL. Low concentrations of TNFalpha (1 ng/ml) caused a >90% decrease in the number of osteoclasts in co-cultures, but did not affect the development of osteoclasts from bone marrow cells. In cultures with p55TNFR(-/-) osteoblasts and wt BMC, the inhibitory effect was abrogated and TNFalpha induced an increase in the number of osteoclasts in a dose-dependent manner. Osteoblasts were found to release the inhibitory factor(s) into the culture supernatant after simultaneous treatment with 1,25(OH)(2)D(3) and TNFalpha, this activity, but not its release, being resistant to treatment with anti-TNFalpha antibodies. Dexamethasone blocked the secretion of the TNFalpha-dependent inhibitor by osteoblasts, while stimulating the development of osteoclasts. The data suggest that the effects of TNFalpha on the differentiation of osteoclast lineage cells and on bone metabolism may be more complex than hitherto assumed and that these effects may play a role in vivo during therapies for inflammatory diseases.

Expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta in the brain during experimental group B streptococcal meningitis

We performed mRNA in situ hybridization for TNF-alpha and IL-1beta from infant rats with group B streptococcal meningitis. Induction of both cytokines was seen in the ependyma and the meninges at 4 h. Both cytokines were expressed in the brain parenchyma at 12 h. Induction of IL-1beta mRNA was seen in vessels within the brain cortex. Neutrophilic infiltrate at all time points examined was minimal and could not account for the observed cytokine expression.

Fatal hepatitis mediated by tumor necrosis factor TNFalpha requires caspase-8 and involves the BH3-only proteins Bid and Bim

Apoptotic death of hepatocytes, a contributor to many chronic and acute liver diseases, can be a consequence of overactivation of the immune system and is often mediated by TNFalpha. Injection with lipopolysaccharide (LPS) plus the transcriptional inhibitor D(+)-galactosamine (GalN) or mitogenic T cell activation causes fatal hepatocyte apoptosis in mice, which is mediated by TNFalpha, but the effector mechanisms remain unclear. Our analysis of gene-targeted mice showed that caspase-8 is essential for hepatocyte killing in both settings. Loss of Bid, the proapoptotic BH3-only protein activated by caspase-8 and essential for Fas ligand-induced hepatocyte killing, resulted only in a minor reduction of liver damage. However, combined loss of Bid and another BH3-only protein, Bim, activated by c-Jun N-terminal kinase (JNK), protected mice from LPS+GalN-induced hepatitis. These observations identify caspase-8 and the BH3-only proteins Bid and Bim as potential therapeutic targets for treatment of inflammatory liver diseases.

Comparison of drug retention rates and causes of drug discontinuation between anti-tumor necrosis factor agents in rheumatoid arthritis

OBJECTIVE: Tumor necrosis factor (TNF) inhibitors have revolutionized the treatment of severe rheumatoid arthritis (RA), yet drug discontinuation is common. The aim of this study was to compare treatment retention rates and specific causes of anti-TNF discontinuation in a population-based RA cohort. METHODS: All patients treated with etanercept, infliximab, or adalimumab within the Swiss Clinical Quality Management RA cohort between 1997 and 2006 were included in the study. Causes of treatment discontinuation were broadly categorized as adverse events (AEs) or nontoxic causes, and further subdivided into specific categories. Specific causes of treatment interruption were analyzed using a Cox proportional hazards model and adjusted for potential confounders. RESULTS: A total of 2,364 anti-TNF treatment courses met the inclusion criteria. Treatment discontinuation was reported 803 times: 309 with etanercept, 249 with infliximab, and 245 with adalimumab. Drug inefficacy represented the largest single cause of treatment discontinuation (55.8% of cases). The median time of receiving anti-TNF therapy was 37 months, but discontinuation rates differed between the 3 anti-TNF agents (P < 0.001), with shorter retention rates for infliximab (hazard ratio [HR] 1.24, 99% confidence interval [99% CI] 1.01-1.51). The specific causes of treatment discontinuation revealed an increased risk of AEs with infliximab (HR 1.4, 99% CI 1.003-1.96), mostly due to an increased risk of infusion or allergic reactions (HR 2.11, 99% CI 1.23-3.62). Other discontinuation causes were equally distributed between the anti-TNF agents. CONCLUSION: In this population, infliximab was associated with higher overall discontinuation rates compared with etanercept and adalimumab, which is mainly due to an increased risk of infusion or allergic reactions.

The processing of human pro-tumor necrosis factor

Human pro-TNF-$\alpha$ is a 26 kd type II transmembrane protein, and it is the precursor of 17 kd mature TNF. Pro-TNF release mature from its extracellular domain by proteolytic cleavage between resideu Ava ($-$1) and Val (+1). Both forms of TNF are biologically active and the native form of mature TNF is a bell-shaped trimer. The structure of pro-TNF was studied both in intact cell system and in an in vitro translation system by chemical crosslinking. We found that human pro-TNF protein exist as a trimer in intact cells (LPS-induced THP-1 cells and TNF cDNA transfected COS-3 cells) and this trimeric structure is assembled intracellularly, possibly in the ER. By analysis several deletion mutants, we observed a correlation between expression of pro-TNF cytotoxicity in a juxtacrine fashion and detection of the trimer, suggesting the trimeric structure is very important for its biologic activity. With a series of deletion mutants in the linking domain, we found that the small deletion did not block the cleavage and large deletion did regardless of the presence or absence of the native cleavage site, suggesting that the length of the residues between the plasma membrane and the base of the trimer determines the rate of the cleavage, possibly by blocking the accessibility of the cleavage enzyme to its action site. Our data also suggest that the native cleavage site is not sufficient for the release of mature TNF and alternative cleavage site(s) exists. ^

Improved bioassay for the detection of porcine tumor necrosis factor using a homologous cell line: PK(15)

Close similarities of various physiological parameters makes the pig one of the preferred animal models for the study of human diseases, especially those involving the cardiovascular system. Unfortunately, the use of pig models to study diseases such as viral hemorrhagic fevers and endotoxic shock syndrome have been hampered by the lack of the necessary immunological tools to measure important immunoregulatory cytokines such as tumor necrosis factor (TNF). Here we describe a TNF-bioassay which is based on the porcine kidney cell line PK(15). Compared to the widely used murine fibroblastoid cell line L929, the PK(15) cell line displays a 100-1000-fold higher sensitivity for porcine TNF-alpha, a higher sensitivity for human TNF-alpha, and a slightly lower sensitivity for murine TNF-alpha. Using a PK(15) bioassay we can detect recombinant TNF-alpha as well as cytotoxic activity in the supernatants of lipopolysaccharide (LPS)-activated porcine monocytes at high dilutions. This suggests that the sensitivity of the test should permit the detection of TNF in biological specimens such as pig serum.