Circulating tumour necrosis factor-α bioactivity in rheumatoid arthritis patients treated with infliximab: link to clinical response

Our objective was to clarify the heterogeneity in response to infliximab treatment in rheumatoid arthritis (RA); to this end, a bioassay was designed to explore the contribution of circulating tumour necrosis factor (TNF)-α bioactivity and its possible link to response. The bioassay is based on the induction of IL-6 and osteoprotegerin (OPG) production by synoviocytes in response to TNF-α. RA synoviocytes were cultured with TNF-α (5 ng/ml) and 42 RA plasma samples collected just before starting therapy. Levels of IL-6 and OPG were measured in supernatants. In 20 of the patients, plasma samples collected before and 4 hours after the first and the ninth infusions were tested in the same way. Plasma concentrations of TNF-α and p55 and p75 soluble receptors were measured using ELISA. TNF-α induced IL-6 and OPG production by synoviocytes, which was further increased with patient plasma dilutions and inhibited by infliximab. With plasma samples obtained before the first infusion, the IL-6-induced production was greater in patients with a good clinical response than in the poor responders (44.4 ± 23.3 ng/ml versus 27.4 ± 20.9 ng/ml; P = 0.05). This high circulating TNF-α bioactivity was strongly inhibited with the first infliximab infusion. The difference between IL-6 levels induced with plasma samples obtained before and 4 hours after the first infusion was greater in patients with a good clinical response (40.0 ± 23.7 ng/ml versus 3.4 ± 10.0 ng/ml; P = 0.001). Similar findings were obtained for OPG production (7.0 ± 6.2 ng/ml versus 0.0 ± 3.0 ng/ml; P < 0.05). Levels of circulating TNF-α bioactivity were predictive of clinical response to TNF-α inhibition, confirming a key role for TNF-α in these RA patients.     

Human tumour necrosis factor-α inhibits glucose transport in cultured ehrlich ascites tumour cells

Human recombinant tumour necrosis factor-α (rhTNF-α) arrested the growth of Ehrlich ascites tumour (EAT) cells in vitro. It suppressed cellular glucose uptake and decreased the membrane density of glucose transporters as measured by glucose-reversible cytochalasin B binding. The glucose transporters' affinity for substrate was also reduced. However, rhTNF-α treatment exerted no effect on the phosphoribosyl pyrophosphate level in EAT cells. The role of rhTNF-α on the inhibition of glucose transport of tumour cells is discussed.     

Deletion of tumor necrosis factor-α receptor type 1 exacerbates insulin resistance and hepatic steatosis in aromatase knockout mice

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)-α levels are known to positively correlate with disturbances in lipid metabolism, we investigated the impact of the loss of TNF-α 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-α receptor type 1 (TNFR1) gene (ArKO/TNFR1KO) or both the TNFR 1 and 2 genes (ArKO/TNFR1&2KO) 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-α mediated signaling via TNFR1.     

Inhibition of established collagen-induced arthritis with a tumour necrosis factor-α inhibitor expressed from a self-contained doxycycline regulated plasmid

Tumor necrosis factor (TNF)-α is produced by cells of the immune system and is a key mediator in immune and inflammatory reactions. Through interaction with widely expressed receptors (TNF receptor 1 and TNF receptor 2), TNF-α is able to orchestrate the expression of a range of downstream proinflammatory molecules. Over the past decade novel biologics that inhibit TNF-α have been developed as extremely effective treatments for rheumatoid arthritis. Structurally, these biologics are antibodies, or TNF receptors on an antibody backbone that bind TNF-α directly and are delivered to patients by repeated injection. Gene therapy offers an improved approach to delivering biologics as a single administration of their encoding genetic material. In the present study we demonstrate the therapeutic effect of a small molecular weight dimeric TNF receptor 2 (dTNFR) constitutively expressed from plasmid DNA, delivered intramuscularly with electroporation, after disease onset in a collagen-induced arthritis model. Regulated promoters that enable the production of a transgene to be controlled are more suited to the application of gene therapy in the clinic. Regulated expression of dTNFR from the plasmid pGTRTT was also therapeutic in the mouse collagen-induced arthritis model when the inducer doxycycline was also administered, whereas no therapeutic effect was observed in the absence of doxycycline. The therapeutic effect of dTNFR expressed from a constitutive or regulated plasmid was dependent on the degree of disease activity at the time of DNA injection. The observations of this study are considered with regard to the disease model, the magnitude of gene regulation, and the path to clinical application.     

Acute treatment with tumour necrosis factor-α induces changes in protein metabolism in rat skeletal muscle

Acute treatment of rats with recombinant tumour necrosis factor (TNF-) caused an enhanced proteolytic rate —measured as tyrosine released in the presence of cycloheximide — insoleus muscle (34%). The cytokine treatment also decreased the rate of protein synthesis in this muscle (22%) while it had no effect upon the same parameter inextensor digitorum longus (EDL) (26%) muscle. In addition, treatment of rats with TNF- increased amino acid uptake by transport system A in the incubated muscles both insoleus (45%) andEDL (99%) in the presence of insulin in the incubating medium. This effect was not associated with a direct action of TNF on muscle since the addition of different concentrations of the cytokine to the preparations did not alter the uptake of -(methyl)-aminoisobutyric acid by the incubated muscles. It can be concluded that acute TNF- treatment causes changes in protein metabolism in red-type muscles — suchsoleus — while little effects are seen in white-type muscles — such as EDL. The results presented may, to some extent, be related to the cachectic response associated with cancer and inflammation.     

Tumor necrosis factor-α receptor type 1, not type 2, mediates its acute responses in the kidney

Acute administration of tumor necrosis factor-α (TNF-α) resulted in decreases in renal blood flow (RBF) and glomerular filtration rate (GFR) but induced diuretic and natriuretic responses in mice. To define the receptor subtypes involved in these renal responses, experiments were conducted to assess the responses to human recombinant TNF-α (0.3 ng·min(-1)·g body wt(-1) iv infusion for 75 min) in gene knockout (KO) mice for TNF-α receptor type 1 (TNFαR1 KO, n = 5) or type 2 (TNFαR2 KO, n = 6), and the results were compared with those obtained in corresponding wild-type [WT (C57BL/6), n = 6] mice. Basal levels of RBF (PAH clearance) and GFR (inulin clearance) were similar in TNFαR1 KO, but were lower in TNFαR2 KO, than WT mice. TNF-α infusion in WT mice decreased RBF and GFR but caused a natriuretic response, as reported previously. In TNFαR1 KO mice, TNF-α infusion failed to cause such vasoconstrictor or natriuretic responses; rather, there was an increase in RBF and a decrease in renal vascular resistance. Similar responses were also observed with infusion of murine recombinant TNF-α in TNFαR1 KO mice (n = 5). However, TNF-α infusion in TNFαR2 KO mice caused changes in renal parameters qualitatively similar to those observed in WT mice. Immunohistochemical analysis in kidney slices from WT mice demonstrated that while both receptor types were generally located in the renal vascular and tubular cells, only TNFαR1 was located in vascular smooth muscle cells. There was an increase in TNFαR1 immunoreactivity in TNFαR2 KO mice, and vice versa, compared with WT mice. Collectively, these functional and immunohistological findings in the present study demonstrate that the activation of TNFαR1, not TNFαR2, is mainly involved in mediating the acute renal vasoconstrictor and natriuretic actions of TNF-α.     

Evaluating the efficacy of sequential biologic therapies for rheumatoid arthritis patients with an inadequate response to tumor necrosis factor-α inhibitors

Introduction  

The long-term treatment of rheumatoid arthritis (RA) most often involves a sequence of different therapies. The response to therapy, disease progression and detailed knowledge of the role of different therapies along treatment pathways are key aspects to help physicians identify the best treatment strategy. Thus, understanding the effectiveness of different therapeutic sequences is of particular importance in the evaluation of long-term RA treatment strategies. The objective of this study was to systematically review and quantitatively evaluate the relationship between the clinical response to biologic treatments and the number of previous treatments with tumor necrosis factor α (TNF-α) inhibitors.     

Soluble inflammatory markers as predictors of virological response in patients with chronic hepatitis C virus infection treated with interferon-α plus ribavirin

The host immune response plays an important role in viral clearance in patients who are chronically infected with hepatitis C virus (HCV) and are treated with interferon and ribavirin. Activation of the immune system involves the release of pro and anti-inflammatory molecules that can be measured in plasma samples. The present study aimed to evaluate the association between pretreatment plasma levels of chemokines and soluble tumor necrosis factor receptors (sTNF-R) and the virological response in treated patients with chronic hepatitis C infection. Forty-one chronically-infected HCV patients that were being treated with interferon-α (IFN-α) plus ribavirin were included in the study. Socio-demographic, clinical and laboratory data were collected and pretreatment plasma levels of chemokine CCL2, CCL3, CCL11, CCL24, chemokine CXCL9, CXCL10, sTNF-R1 and sTNF-R2 were measured. The virological response was assessed at treatment week 12, at the end of treatment and 24 weeks after treatment. Pretreatment CXCL10 levels were significantly higher in patients without an early virological response (EVR) or sustained virological response (SVR) compared to responders [512.9 pg/mL vs. 179.1 pg/mL (p = 0.011) and 289.9 pg/mL vs. 142.7 pg/mL (p = 0.045), respectively]. The accuracy of CXCL10 as a predictor of the absence of EVR and SVR was 0.79 [confidence interval (CI) 95%: 0.59-0.99] and 0.69 (CI 95%: 0.51-0.87), respectively. Pretreatment plasma levels of the other soluble inflammatory markers evaluated were not associated with a treatment response. Pretreatment CXCL10 levels were predictive of both EVR and SVR to IFN-α and ribavirin and may be useful in the evaluation of candidates for therapy.     

Tumor necrosis factor-α converting enzyme (TACE) increases RANKL expression in osteoblasts and serves as a potential biomarker of periodontitis

Periodontitis is a very prevalent disease. Therefore, biomarkers for the early and standard diagnoses of periodontitis are urgently needed. TACE is a membrane-bound metalloprotease. Although a recent study suggested that TACE levels in GCF are elevated during periodontal disease, the levels of TACE in GCF at different stages of chronic periodontitis have not been determined. Here, we analyzed the protein levels of TACE in GCF from periodontal disease subjects and confirmed that the protein levels of TACE were higher in the moderate periodontitis groups. TACE is known to be a NF-κB ligand that stimulates RANKL secretion in osteoblasts. To understand the effects of TACE on RANKL and OPG in osteoblasts, we treated MG63 cells with TACE. We observed an increase in RANKL protein expression but a decrease in OPG protein expression. Our data suggest that TACE can induce RANKL expression and promote osteoclastogenesis, thus worsening the outcome of periodontitis.     

Utility of plasma tumour necrosis factor-α and transforming growth factor-β1 as predictors of survival and treatment outcome in advanced non-small cell lung carcinoma

We hypothesized that plasma level of tumour necrosis factor (TNF)-α and transforming growth factor (TGF)-β1 may be a potential tool for diagnosis, prognosis and prediction of treatment outcome in non-small cell lung carcinoma (NSCLC). Plasma levels of TNF-α and TGF-β1 were quantified in 100 NSCLC patients and 100 controls. Association of TNF-α and TGF-β1 with response to therapy and survival was determined in 42 patients. An increased presence of TNF-α and TGF-β1 was observed in NSCLC compared with controls. TNF-α and TGF-β1 levels did not correlate with survival and response to chemotherapy. TNF-α and TGF-β1 do not appear to be reliable markers for predicting survival and response to therapy in advanced NSCLC.     

Metabolic effects of tumour necrosis factor-α on rat brown adipose tissue

Intravenous administration of a single dose (100 g/kg bw) of recombinant tumour necrosis factor- (TNF, cachectin) to rats increased the rate ofin vitro fatty acid synthesis in interscapular brown adipose tissue (IBAT) from both glucose and alanine, without changes in the oxidation of these substrates to¹⁴CO₂. Lactate production and glycerol release were also unaffected by treatment with the cytokine. Additionally, the presence of TNF in the incubation media did not affect fatty acid synthesis, suggesting an indirect effect of the cytokine. The activities of different enzymes of glucose and alanine metabolism such as hexokinase, phosphofructokinase, pyruvate kinase, glucose-6-phosphate dehydrogenase and alanine transaminase, did not suffer changes as a consequence of TNF administration. The same applied to the enzymatic activities involved in fatty acid synthesis such as fatty acid synthase, acetyl-CoA carboxylase and ATP-citrate lyase. Conversely, citrate levels in IBAT were increased in animals treated with TNF, suggesting that it could be the cause for the increased fatty acid synthesis in this tissue.     

Effects of tumour necrosis factor-α (cachectin) on glucose metabolism in the rat

Intravenous administration of a single dose (20 g) of recombinant tumour necrosis factor- (TNF, cachectin) to rats decreased the rate of intestinal glucose absorption. In vivo, the oxidation of [U-¹⁴C]glucose to ¹⁴CO₂ was significantly increased by the cytokine. In addition, [¹⁴C]lipid accumulation from [U-¹⁴C]glucose was increased both in liver and brown adipose tissue of the TNF-injected animals. The decrease observed in intestinal glucose absorption was not associated with changes in intestinal metabolism. There was no difference in glucose metabolism by isolated enterocytes from either control or TNF-injected rats whether in the absence or presence of different concentrations of the cytokine in the incubation medium. In contrast, tumour necrosis factor altered the rate of gastric emptying as measured by the gastrointestinal distribution of [³H]inulin following an intragastric glucose load. These results suggest that the cytokine profoundly alters glucose metabolism by increasing its whole-body oxidation rate and delaying intestinal absorption through a reduced gastric emptying.     

Tumor necrosis factor-α as a therapeutic target for diabetic nephropathy

Activation of innate immunity with the subsequent development of a chronic low-grade inflammatory response is now recognized as a critical factor in the pathogenesis of diabetes mellitus and diabetic complications, including diabetic nephropathy. In the setting of diabetic nephropathy, there is now evidence of the relevant contribution of pro-inflammatory cytokines, with special participation of tumor necrosis factor-α (TNF-α). This new pathogenic perspective leads to new therapeutic implications derived from modulation of inflammation and inflammatory cytokines. Experimental studies have shown the beneficial renal actions derived from TNF-α inhibition with the use of soluble TNF-α receptor fusion proteins, chimeric monoclonal antibodies and pentoxifylline (PTF). Clinical application of this strategy is nowadays limited to PTF administration, which has demonstrated significant beneficial effects in patients with diabetic nephropathy. Overall, these studies indicate that inhibition of TNF-α might be an efficacious treatment for renal disease secondary to diabetes mellitus.     

Commitment to apoptosis induced by tumour necrosis factor-α is dependent on caspase activity

Tumour Necrosis Factor binding at the cell surface induces a complex series of signaling events culminating in the caspase cascade, which is central to apoptosis. However, recent work from several laboratories has questioned caspase involvement in commitment to cell death. We have therefore investigated the involvement of caspases in the crucial commitment stage of tumour necrosis factor-induced apoptosis in human T-leukaemic CEM-C7 cells and breast carcinoma MCF-7 cells, using both peptide-based and viral caspase inhibitors. Our observations converge on the conclusion that commitment to death in these systems is dependent on caspase activity, e.g. baculovirus p35 produces over 50-fold protection of colony-forming ability, the most stringent criterion of cell survival. These observations strongly support the view that the caspase family is of great biological and medical significance, since caspase dysfunction resulting in failure to commit to cell death after treatment with tumour necrosis factor or other stimuli may contribute to cancer development.     

Fullerenes and their derivatives as inhibitors of tumor necrosis factor-α with highly promoted affinities

Tumor necrosis factor-α (TNF-α) is a cell signalling protein involved in systemic inflammation in infectious and other malignant diseases. Physiologically, it plays an important role in regulating host defence, but its overexpression can lead to serious illnesses including cancer, autoimmune disease and inflammatory disease. Gadolinium-based metallofullerenols, e.g., Gd@C₈₂(OH) _x (x?≈?22), are well known for their abundant biological activities with low toxicity experimentally and theoretically; however, their activity in direct TNF-α inhibition has not been explored. In this work, we investigated the inhibiting effects of four types of fullerene-based ligands: fullerenes, fullerenols, metallofullerenes, and metallofullerenols. We reported previously that fullerenes, metallofullerenes and their hydroxylated derivatives (fullerenols) can reside in the same pocket of the TNF-α dimer as that of SPD304—a known inhibitor of TNF-α [He et al. (2005) Science 310:1022, 18]. Ligand docking and binding free energy calculations suggest that, with a similar nonpolar interaction dominated binding pattern, the fullerene-based ligands, C₆₀, C₆₀(OH)₁₂, Gd@C₆₀, C₈₂, C₈₂(OH)₁₂, Gd@C₈₂, Gd@C₈₂(OH)₁₃ and Gd@C₈₂(OH)₂₁, have larger affinity than currently known inhibitors, and could be used to design novel inhibitors of TNF-α in the future.

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Graphical Abstract Fullerene-material/TNF-α

     

Influence of depression symptoms on serum tumor necrosis factor-α of patients with chronic low back pain

Introduction  

Patients with chronic low back pain (cLBP) have high rates of comorbid psychiatric disorders, mainly depression. Recent evidence suggests that depressive symptoms and pain, as interacting factors, have an effect on the circulating levels of inflammatory markers relevant to coronary artery disease. Our previous work showed a higher serum level of an inflammatory marker tumour necrosis factor-alpha (TNFα) in patients with cLBP, which did not correlate with intensity of low back pain alone. In the present study we investigated the cross-sectional associations of depressive symptoms, low back pain and their interaction with circulating levels of TNFα.     

Selective down-regulation of the Cqα/G11α G-protein family in tumour necrosis factor-α induced cell death

Investigations into the regulation of heterotrimeric GTP-binding protein a-subunits in models of tumour necrosis factor- (TNF)-induced cell death, revealed the selective down-regulation of the G_q/G₁₁ family of G-proteins. The human HeLa and murine L929 cells treated with recombinant human TNF for up to 24 h displayed down-regulated G_q/G₁₁ family protein levels, but not G_s, G_i and G_o protein levels as determined by Western analyses. This effect of TNF was observed in a concentration - and time-dependent manner, consistent with the profiles of TNF-induced cell death observed. Moreover, the functioning of G_q/G₁₁ family proteins were found to be impaired in TNF-treated cells, as measured by agonist-induced [Ca²⁺],_i release. In contrast, G_s activity was unaltered by TNF-treatment, determined by measurement of agonist-induced intracellular cyclic AMP generation. These findings in TNF-induced cytotoxic models, indicate a novel 'cross-talk' mechanism by which TNF alters Ca²⁺-signalling mechanisms, which may contribute towards the apoptotic and necrotic cell death.