Stable human regulatory T cells switch to glycolysis following TNF receptor 2 costimulation.

Following activation, conventional T (Tconv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (Treg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymus-derived Treg (tTreg) cells can become glycolytic in response to tumour necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tTreg cells, but not in Tconv cells. Glycolysis in CD3-TNFR2-activated tTreg cells is driven by PI3-kinase-mTOR signalling and supports tTreg cell identity and suppressive function. In contrast to glycolytic Tconv cells, glycolytic tTreg cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2hiCD4+CD25hiCD127lo effector T cells, which were FOXP3+IKZF2+, revealed an increase in glucose consumption and intracellular lactate levels, thus identifying them as glycolytic tTreg cells. Our study links TNFR2 costimulation in human tTreg cells to metabolic remodelling, providing an additional avenue for drug targeting.

Antidepressant treatment resistance is associated with increased inflammatory markers in patients with major depressive disorder.

BACKGROUND: One third of patients with major depressive disorder (MDD) fail to respond to currently available antidepressant medications. Inflammation may contribute to treatment non-response through effects on neurotransmitter systems relevant to antidepressant efficacy. In post-hoc analyses, increased concentrations of inflammatory markers prior to treatment predict poor antidepressant response. However, limited data exists on whether depressed patients with multiple failed treatment trials in their current episode of depression exhibit increased inflammation. METHODS: Plasma concentrations of inflammatory markers were measured in unmedicated, medically stable patients with MDD (n = 98) and varying numbers of adequate antidepressant treatment trials in the current depressive episode as measured by the Massachusetts General Hospital Antidepressant Treatment Response Questionnaire. Covariates including age, sex, race, education, body mass index (BMI) and severity of depression were included in statistical models where indicated. RESULTS: A significant relationship was found between number of failed treatment trials and tumor necrosis factor (TNF), soluble TNF receptor 2 (sTNF-R2) and interleukin (IL)-6 (all p < 0.05 in multivariate analyses). Post hoc pairwise comparisons with correction for multiple testing revealed that patients with 3 or more failed trials in the current episode had significantly higher plasma TNF, sTNF-R2 and IL-6 compared to individuals with 0 or 1 trial (all p < 0.05). High sensitivity c-reactive protein was also associated with a greater number of treatment failures, but only in models with BMI excluded. CONCLUSIONS: Measuring inflammatory markers and targeting inflammation or its downstream mediators may be relevant for depressed patients with multiple failed antidepressant treatment trials in their current depressive episode.

Generation of a sensitive TNFR2-specific murine assays system.

Tumor necrosis factor (TNF)/TNF receptors (TNFR1/TNFR2) are considered to be potential drug targets to treat refractory diseases, including autoimmune diseases and malignant tumors. However, their specific functions, especially in the case of TNFR2, are poorly understood. In this study, we constructed a mouse TNFR2 (mTNFR2)-mediated biological assay system that shows no effects of mouse TNFR1 (mTNFR1) in order to screen mTNFR2-selective stimulating agents. Mouse TNFR1(-/-)R2(-/-) preadipocytes were transfected with the gene encoding the mTNFR2/mouse Fas (mFas) chimeric receptor in which the extracellular and transmembrane domains of mTNFR2 were fused to the intracellular domain of mFas. Our results demonstrated that this cell line exhibits highly sensitive mTNFR2-mediated cytotoxic effects. We propose that this mTNFR2-mediated biological assay system would be a useful tool to screen for mTNFR2-selective stimulating agents.

Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

BACKGROUND: Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. METHODS: Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. RESULTS: CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. CONCLUSION: Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

Suppression of soluble T cell-associated proteins by an anti-interferon-α monoclonal antibody in adult patients with dermatomyositis or polymyositis.

OBJECTIVE: The aim of this study was to identify serum markers that are modulated by an investigational anti-IFN-α mAb, sifalimumab, in adult DM or PM patients. METHODS: In a phase 1b clinical trial, sera were collected from a total of 48 DM or PM adult patients receiving either placebo for 3 months or sifalimumab for 6 months. Samples were tested for 128 selected proteins using a multiplex luminex immunoassay. Muscle biopsies from selected patients were stained for T cell infiltration using an anti-CD3 antibody. RESULTS: A robust overexpression of multiple serum proteins in DM or PM patients was observed, particularly in patients with an elevated baseline type I IFN gene signature in the blood or muscle. Neutralization of the type I IFN gene signature by sifalimumab resulted in coordinated suppression of T cell-related proteins such as soluble IL-2RA, TNF receptor 2 (TNFR2) and IL-18. Muscle biopsies from two patients with the highest serum protein suppression were selected and found to have a pronounced reduction of muscle T cell infiltration. Down-regulation of IL-2RA correlated with favourable manual muscle test 8 (MMT-8) alterations in sifalimumab-dosed patients. CONCLUSION: A reduced level of multiple T cell-associated proteins after sifalimumab but not placebo administration suggests a suppressive effect of blocking type I IFN signalling on T cell activation and chemoattraction that may lead to a reduction of T cell infiltration in the muscle of myositis patients. Further, soluble IL-2RA changes from baseline may serve as a responsive and/or predictive marker for type I IFN-targeted therapy in adult DM or PM patients.

Intrathecal ultra-low dose naloxone enhances the antihyperalgesic effects of morphine and attenuates tumor necrosis factor-α and tumor necrosis factor-α receptor 1 expression in the dorsal horn of rats with partial sciatic nerve transection.

BACKGROUND: Glutamate homeostasis and microglia activation play an important role in the development and maintenance of neuropathic pain. We designed this investigation to examine whether ultra-low dose naloxone administered alone or in combination with morphine could alter the concentration of the excitatory amino acids (EAAs) glutamate and aspartate, as well as the expression of tumor necrosis factor-α (TNF-α) and its receptors (TNFR1 and TNFR2) in the spinal cord dorsal horn of rats with partial sciatic nerve transection (PST). METHODS: Male Wistar rats underwent intrathecal catheter implantation for drug delivery and were divided in 7 groups: sham-operated + saline (sham), PST + saline (S), PST + 15 ng naloxone (n), PST + 15 µg naloxone (N), PST + 10 µg morphine (M), PST + 15 ng naloxone + 10 µg morphine (Mn), PST + 15 µg naloxone + 10 µg morphine (MN). Thermal withdrawal latency and mechanical withdrawal threshold, TNF-α and TNFR expression in the spinal cord and dorsal root ganglia, and EAAs glutamate and aspartate concentration in cerebrospinal fluid dialysates were measured. RESULTS: Ten days after PST, rats developed hyperalgesia (P < 0.0001) and allodynia (P < 0.0001), and increased TNF-α (P < 0.0001) and TNFR1 expression (P = 0.0009) were measured in the ipsilateral spinal cord dorsal horn. The antihyperalgesic and antiallodynic effects of morphine (10 µg) were abolished by high-dose naloxone (15 µg; P = 0.0031) but enhanced by ultra-low dose naloxone (15 ng; P = 0.0015), and this was associated with a reduction of TNF-α (P < 0.0001) and TNFR1 (P = 0.0009) expression in the spinal cord dorsal horn and EAAs concentration (glutamate: P = 0.0001; aspartate: P = 0.004) in cerebrospinal fluid dialysate. Analysis of variance (ANOVA) or Student t test with Bonferroni correction were used for statistical analysis. CONCLUSIONS: Ultra-low dose naloxone enhances the antihyperalgesia and antiallodynia effects of morphine in PST rats, possibly by reducing TNF-α and TNFR1 expression, and EAAs concentrations in the spinal dorsal horn. Ultra-low dose naloxone may be a useful adjuvant for increasing the analgesic effect of morphine in neuropathic pain conditions.

Estradiol modulates tumor necrosis factor-induced endothelial inflammation: role of tumor necrosis factor receptor 2.

The sex hormone estradiol (E(2)) appears to mediate both anti-atherogenic and pro-inflammatory effects in premenopausal women, suggesting a complex immunomodulatory role. Tumor necrosis factor (TNF) is a key pro-inflammatory cytokine involved in the pathogenesis of atherosclerosis and other inflammatory diseases. Alterations at the TNF receptors (TNFRs) and their downstream signaling/transcriptional pathways can affect inflammatory responses. Given this background, we hypothesized that chronic E(2) exposure would alter endothelial inflammatory response involving modulation at the levels of TNFRs and signaling pathways. HUVECs were used as the model system. Pre-treatment with E(2) did not significantly alter TNF-induced upregulation of pro-inflammatory molecules ICAM-1 (3-6 times) and VCAM-1 (5-7 times). However, pharmacological inhibition of transcriptional pathways suggested a partial shift from NF-ĸB (from 97 to 64%) towards the JNK/AP-1 pathway in ICAM-1 upregulation on E(2) treatment. In contrast, VCAM-1 expression remained NF-ĸB dependent in both control (∼96%) and E(2) treated (∼85%) cells. The pro-inflammatory TNF effects were mediated by TNFR1. Interestingly, E(2) pre-treatment increased TNFR2 levels in these cells. Concomitant TNFR2 activation (but not TNFR1 activation alone) led to the shift towards JNK/AP-1-mediated ICAM-1 upregulation in E(2)-treated cells, suggesting the effects of chronic E(2) to be dependent on TNFR2 signaling.

Creation of an improved mutant TNF with TNFR1-selectivity and antagonistic activity by phage display technology.

Tumor necrosis factor-alpha (TNF), which binds two types of TNF receptors (TNFR1 and TNFR2), regulates the onset and exacerbation of autoimmune diseases such as rheumatoid arthritis and Crohn's disease. In particular, TNFR1-mediated signals are predominantly related to the induction of inflammatory responses. We have previously generated a TNFR1-selective antagonistic TNF-mutant (mutTNF) and shown that mutTNF efficiently inhibits TNFR1-mediated bioactivity in vitro and attenuates inflammatory conditions in vivo. In this study, we aimed to improve the TNFR1-selectivity of mutTNF This was achieved by constructing a phage library displaying mutTNF-based variants, in which the amino acid residues at the predicted receptor binding sites were substituted to other amino acids. From this mutant TNF library, 20 candidate TNFR1-selective antagonists were isolated. Like mutTNF, all 20 candidates were found to have an inhibitory effect on TNFR1-mediated bioactivity. However, one of the mutants, N7, displayed significantly more than 40-fold greater TNFR1-selectivty than mutTNF. Therefore, N7 could be a promising anti-autoimmune agent that does not interfere with TNFR2-mediated signaling pathways.

Interferon beta-1a induces tumor necrosis factor receptor 1 but decreases tumor necrosis factor receptor 2 leukocyte mRNA levels in relapsing-remitting multiple sclerosis.

OBJECTIVE: Members of the tumor necrosis factor (TNF) receptor superfamily play a major role in the pathogenesis of multiple sclerosis. To elucidate the role of TNF receptors, in 53 relapsing-remitting multiple sclerosis patients, we measured the TNF receptor 1 and receptor 2 (TNF-R1 and TNF-R2) mRNA levels in peripheral blood leukocytes in natural history (n = 27) and during administration of interferon (IFN) beta-1a (n = 26). METHODS: Every 3 months for the duration of 1 year peripheral blood leukocytes were investigated by quantitative reverse transcription polymerase chain reaction. Every 6 months, MRI scans of the brain were analyzed semiquantitatively. RESULTS: At baseline, clinical expanded disability status scale score and TNF-R1 mRNA level were correlated. In the therapy group, the difference between T2 lesion load at baseline and after 12 months correlated negatively with the difference between TNF-R1 mRNA level at baseline and after 12 months. Subcutaneously applied IFN beta increased the amount of TNF-R1 mRNA, but partly decreased the amount of TNF-R2 mRNA in clinically and subclinically defined responders to therapy after 1 year compared to baseline. CONCLUSION: This result might support the notion that due to different signal transduction properties of both receptors in the natural course of multiple sclerosis, TNF-alpha signaling in leukocytes via TNF-R1 might be beneficial, but detrimental via proinflammatory TNF-R2: part of the therapeutic efficacy of current first-line standard therapy with IFN might be due to the modulation of signal transduction pathways.

Relationship between circulating tumor necrosis factor system and bone mass before and after estrogen plus progestogen therapy.

OBJECTIVE: The aim of his study was to investigate the relationship between the circulating tumor necrosis factor (TNF) system and bone mineral density (BMD) before and after estrogen plus progestogen therapy (EPT). METHODS: Serum levels of TNF-alpha, TNF-beta, soluble TNF receptor (sTNFR) 1, sTNFR2, and bone turnover markers and BMDs at the lumbar spine and proximal femur were measured in 192 postmenopausal Korean women. Among all women, 70 were treated with sequential EPT for 1 year. RESULTS: BMDs at all skeletal sites and bone turnover markers were not correlated with serum TNF and sTNFR. After adjustment for age, years since menopause, and body mass index, serum TNF-beta levels were significantly lower in osteoporotic women than in normal women, whereas serum levels of TNF-alpha and sTNFR did not differ among normal, osteopenic, and osteoporotic postmenopausal women. After 6 months of EPT, serum TNF-beta levels increased significantly (P < 0.05), whereas serum TNF-alpha, sTNFR1, and sTNFR2 levels were unchanged. The 1-year changes in BMD at the lumbar spine and proximal femur after EPT were not correlated with the basal levels of serum TNF-alpha and sTNFR and their changes 6 months after EPT. CONCLUSIONS: In the circulating TNF system, only serum TNF-beta levels were lower in osteoporotic postmenopausal women compared with normal postmenopausal women and increased after EPT, but changes in circulating TNF and sTNFR after EPT had no association with changes in bone markers and BMD. The circulating TNF system may not be clinically useful for predicting BMD and bone response after EPT.

Use of atorvastatin as an anti-inflammatory treatment in Crohn's disease.

BACKGROUND AND PURPOSE: Experimental and clinical investigations have revealed that statins can downregulate both acute and chronic inflammatory processes. Whether statins express anti-inflammatory activities in the treatment of Crohn's disease is unknown. EXPERIMENTAL APPROACH: Ten patients were given 80 mg atorvastatin once daily for 13 weeks and then followed up for 8 weeks after the treatment. The anti-inflammatory effects of statin were assessed by measuring levels of plasma C-reactive protein (CRP), soluble (s) CD14, tumour necrosis factor (TNF)-alpha, sTNFRI and II, CCL2 and 8 and the mucosal inflammation by faecal calprotectin. Circulating monocytes were subgrouped and their chemokine receptor expression of CCR2 and CX(3)CR1 were analysed. KEY RESULTS: In 8 of 10 patients, atorvastatin treatment reduced CRP (P=0.008) and sTNFRII (P=0.064). A slight decrease in plasma levels of sCD14, TNF-alpha and sTNFRI was observed in 7/10 patients and faecal calprotectin was reduced in 8/10 patients. We also observed that the treatment diminished expression of CCR2 and CX(3)CR1 on monocyte populations (P=0.014). At the follow-up visit, 8 weeks after the atorvastatin treatment was terminated, CRP levels had returned to those seen before the treatment. CONCLUSIONS AND IMPLICATIONS: Our findings imply that atorvastatin therapy reduces inflammation in patients with Crohn's disease and, therefore, encourage further investigations of statin-mediated protective effects in inflammatory bowel diseases.

The influence of phytic acid on TNF-alpha and its receptors genes' expression in colon cancer Caco-2 cells.

Inositol hexaphosphate (IP6, phytic acid) is a naturally occurring carbohydrate abundantly present in high-fiber diets and it is also contained in almost all mammalian cells. It plays an important role in signal transduction, cell proliferation and differentiation. Some natural substances have been shown to elicit an impact on the expression of TNF-alpha and its receptors in cancer cells. TNF-alpha represents cytokine very often deregulated at the level of both gene expression and signal transmission through TNF-alpha receptors (TNFRI and TNFRII). The aim of the present study was to analyze the IP6 influence on the transcription of genes coding for TNF-alpha and its receptors in human colon cancer cells line Caco-2 Real-time QRT-PCR based on TaqMan methodology was applied to analyze quantitatively the transcript levels of these genes. Three concentrations (1, 2.5 and 5 mM) of IP6 were used for Caco-2 cells stimulation for 1, 6, 12 and 24 h. The results showed that IP6 modulated the expression of the listed genes at transcription level in a dose and time dependent manner. The enhanced TNFRI and decreased TNF-alpha and TNFRII transcription in Caco-2 cells stimulated for 12 h with IP6 seems to be the presumptive evidence for anti-inflammatory and antitumor activity of IP6.

Lovastatin induces neuroprotection through tumor necrosis factor receptor 2 signaling pathways.

Statins are widely used as medication to lower cholesterol levels in human patients. In addition, it was recently reported that they also reduce the incidence of stroke and progression of Alzheimer's disease when prophylactically administered. To date there is only limited information available on how statins exert this beneficial effect. In this study we investigated the neuroprotective effect of lovastatin in primary cortical neurons. We found that lovastatin protects cortical neurons in a concentration-dependent manner against glutamate-mediated excitotoxicity. Interestingly, lovastatin with or without glutamate and/or tumor necrosis factor-alpha (TNF-alpha) increased TNF receptor 2 (TNF-R2) expression in cortical neurons. It was previously shown that activation of TNF-R2 signaling, which includes phosphorylation of protein kinase B (PKB)/Akt and activation of nuclear factor-kappa B (NF-kappaB), protects neurons against ischemic or excitotoxic insults. To investigate if lovastatin-induced neuroprotection was mediated by TNF-R2 signaling, primary cortical neurons were isolated from TNF-R1(-/-) or TNF-R2(-/-) mice. We could show that lovastatin is neuroprotective in TNF-R1(-/-) neurons, while protection is completely absent in TNF-R2(-/-) neurons. Furthermore, lovastatin-mediated neuroprotection led to an increase in PKB/Akt and NF-kappaB phosphorylation, whereas inhibition of PKB/Akt activation entirely abolished lovastatin-induced neuroprotection. Thus, lovastatin-induced neuroprotection against glutamate-excitotoxicity via activation of TNF-R2-signaling pathways.

Simple and highly sensitive assay system for TNFR2-mediated soluble- and transmembrane-TNF activity.

Drugs that target tumor necrosis factor-alpha (TNF) are particularly important in the treatment of severe inflammatory progression in rheumatoid arthritis, Crohn's disease and psoriasis. Despite the central role of the TNF/TNF receptor (TNFR) in various disease states, there is a paucity of information concerning TNFR2 signaling. In this study, we have developed a simple and highly sensitive cell-death based assay system for analyzing TNFR2-mediated bioactivity that can be used to screen for TNFR2-selective drugs. Using a lentiviral vector, a chimeric receptor was engineered from the extracellular and transmembrane domain of human TNFR2 and the intracellular domain of mouse Fas and the recombinant protein was then expressed in TNFR1(-/-)R2(-/-) mouse preadipocytes. Our results demonstrate that this chimeric receptor is capable of inducing apoptosis by transmembrane- as well as soluble-TNF stimuli. Moreover, we found that our bioassay based on cell death phenotype had an approximately 80-fold higher sensitivity over existing bioassays. We believe our assay system will be an invaluable research tool for studying TNFR2 and for screening TNFR2-targeted drugs.

Lipopolysaccharide-induced upregulation of tumor necrosis factor-alpha (TNF-alpha) and TNF receptors in rat sciatic nerve.

The proinflammatory and lipopolysaccharide (LPS)-inducible cytokine tumor necrosis factor alpha (TNF-alpha) has been shown to enhance primary sensory nociceptive signaling. However, the precise cellular sites of TNF-alpha and TNF receptors synthesis are still a matter of controversy. Therefore, we focused our study on TNF-alpha, TNFR1, and TNFR2 protein synthesis and expression patterns in sciatic nerve of controls and rats under systemic challenge with LPS. The enzyme-linked immunosorbent (ELISA) assay showed that the protein level of TNF-alpha reached peak at 6 h. Double immunofluorescence revealed that LPS-induced expression of TNF-alpha exclusively located in a subpopulation of Schwann cells, endothelial cells, and macrophages, which increased at late time point in the rat sciatic nerve. Positive staining of TNF receptors were also found in Schwann cells and a few endothelial cells. These observations have demonstrated the production of this proinflammatory cytokine by peripheral nerve glia especially Schwann cells. Synthesized TNF-alpha might directly act on peripheral nerve glia via TNF receptors, but the inherent mechanisms remain unknown. Further studies are needed to confirm the pathogenic role of tumor necrosis factor in the early stage of inflammation.

TNF-alpha inhibits macrophage clearance of apoptotic cells via cytosolic phospholipase A2 and oxidant-dependent mechanisms.

Removal of apoptotic cells from inflammatory sites is an important step in the resolution of inflammation. Both murine and human macrophages stimulated with TNF-alpha or directly administered arachidonic acid showed an impaired ability to ingest apoptotic cells (efferocytosis). The inhibition was shown to be due to generation of reactive oxygen species, was blocked with a superoxide dismutase mimetic, MnTBAP, and was mimicked by direct addition of H2O2. To determine the mechanism of TNF-alpha-stimulated oxidant production, bone marrow-derived macrophages from gp91(phox)-deficient mice were examined but shown to still produce oxidants and exhibit defective apoptotic cell uptake. In contrast, a specific cytosolic phospholipase A2 inhibitor blocked the oxidant production and reversed the inhibited uptake. The suppressive effect of endogenous or exogenous oxidants on efferocytosis was mediated through activation of the GTPase, Rho. It was reversed in macrophages pretreated with C3 transferase to inactivate Rho or with an inhibitor of Rho kinase. During maturation of human monocyte-derived macrophages, only mature cells exhibited TNF-alpha-induced suppression of apoptotic cell clearance. The resistance of immature macrophages to such inhibition was shown to result not from defective generation of oxidants, but rather, from lack of response of these cells to the oxidants. Overall, the data suggest that macrophages in a TNF-alpha- and oxidant-rich inflammatory environment are less able to remove apoptotic cells and, thereby, may contribute to the local intensity of the inflammatory response.

TNF-alpha in cancer treatment: molecular insights, antitumor effects, and clinical utility.

Tumor necrosis factor alpha (TNF-alpha), isolated 30 years ago, is a multifunctional cytokine playing a key role in apoptosis and cell survival as well as in inflammation and immunity. Although named for its antitumor properties, TNF has been implicated in a wide spectrum of other diseases. The current use of TNF in cancer is in the regional treatment of locally advanced soft tissue sarcomas and metastatic melanomas and other irresectable tumors of any histology to avoid amputation of the limb. It has been demonstrated in the isolated limb perfusion setting that TNF-alpha acts synergistically with cytostatic drugs. The interaction of TNF-alpha with TNF receptor 1 and receptor 2 (TNFR-1, TNFR-2) activates several signal transduction pathways, leading to the diverse functions of TNF-alpha. The signaling molecules of TNFR-1 have been elucidated quite well, but regulation of the signaling remains unclear. Besides these molecular insights, laboratory experiments in the past decade have shed light upon TNF-alpha action during tumor treatment. Besides extravasation of erythrocytes and lymphocytes, leading to hemorrhagic necrosis, TNF-alpha targets the tumor-associated vasculature (TAV) by inducing hyperpermeability and destruction of the vascular lining. This results in an immediate effect of selective accumulation of cytostatic drugs inside the tumor and a late effect of destruction of the tumor vasculature. In this review, covering TNF-alpha from the molecule to the clinic, we provide an overview of the use of TNF-alpha in cancer starting with molecular insights into TNFR-1 signaling and cellular mechanisms of the antitumor activities of TNF-alpha and ending with clinical response. In addition, possible factors modulating TNF-alpha actions are discussed.

Effects of AM3 (Inmunoferon) on increased serum concentrations of interleukin-6 and tumour necrosis factor receptors I and II in cyclists.

The aims of this study were to examine the changes in plasma concentrations of inflammatory cytokines induced by training and competition in professional cyclists. We report the serum concentrations of interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-a), tumour necrosis factor receptors I and II (TNFR-I and -II) in a prospective, randomized, double-blind trial involving the administration of AM3 (Inmunoferon), an oral booster immunomodulator, or placebo to 16 professional cyclists (n = 8 in each group) for 65 consecutive days. Serum was collected just before treatment began (baseline), at the end of pre-competition training, before the mountain stage of the competition (60 days), 4 h after finishing this stage (62 days), and 18 h after the fifth and last day of competition (65 days). To determine the normal levels of cytokines and soluble TNF receptors, individual samples from 14 moderately trained healthy controls were studied. After 60 days of training, the serum concentrations of IL-6 did not differ significantly from those at the beginning of the study for either group of cyclists (placebo and AM3). A significant rise was seen in IL-6 concentrations in both the AM3 and placebo groups at 62 days, 4 h after finishing the mountain stage. The increase was significantly greater in the placebo group than in the AM3 group. At 65 days of treatment, 18 h after the fifth and last day of competition, IL-6 concentrations were similar to those recorded at the end of the training, but were significantly higher in the placebo group than in the AM3 group. At the end of training, serum TNFR-I concentrations in both groups of cyclists were significantly lower than at baseline. The concentrations of serum TNFR-I and -II both 4 h after finishing the mountain stage and 18 h after the fifth and last day of competition were significantly higher than those recorded after training in both groups. Professional cycling competition is associated with increases in serum IL-6 and TNFR-I and -II concentrations. Inmunoferon treatment reduced significantly the concentrations of IL-6 but not those of TNFR-I and -II.

Tamoxifen protects against acute tumor necrosis factor alpha-induced cardiac injury via improving mitochondrial functions.

Tamoxifen is the most commonly used antiestrogen for the treatment of breast cancer. Several clinical trials demonstrate that tamoxifen reduces the risk of heart disease and osteoporosis. However, the mechanism by which tamoxifen causes cardioprotection is unclear. Because increased levels of tumor necrosis factor alpha (TNFalpha) in tissue and/or plasma have been observed in virtually all forms of cardiac injury, we investigated whether tamoxifen prevents cardiac injury in a murine model of acute TNFalpha challenge. Five- to six-week-old female mice were injected (ip) with tamoxifen at 0.25 mg/kg daily for 3 or 7 days before receiving an injection of TNFalpha. Ultrastructural examination of cardiac tissues revealed remarkable protection against TNFalpha-induced mitochondrial damage in tamoxifen pretreated mice. Tamoxifen treatment significantly improved the mitochondrial respiratory function and enhanced superoxide-scavenging activity of mitochondria. These findings reveal a novel mitochondria-mediated mechanism by which tamoxifen exerts its cardiac protection effect against acute TNFalpha-induced heart injury.

Cationic cell-penetrating peptides interfere with TNF signalling by induction of TNF receptor internalization.

Cationic cell-penetrating peptides (CPPs) have been used widely as delivery vectors for the import of molecules that otherwise do not cross the plasma membrane of eukaryotic cells. In this work, we demonstrate that the three cationic CPPs, Antennapedia homeodomain-derived peptide (Antp), nona-arginine and Tat-derived peptide, inhibit tumour necrosis factor (TNF)-mediated signal transduction. This inhibition is based on the downregulation of TNF receptors at the cell surface by induction of internalization. In contrast to TNF-dependent receptor internalization, no receptor activation occurs. The receptor downregulation is not restricted to the CPPs. Remarkably, the HIV-1 Tat protein itself also induces the internalization of TNF receptors. The dynamin dependence of the internalization, as well as the fact that epidermal growth factor receptors are also internalized, suggest a general induction of clathrin-dependent endocytosis as the mechanism of action. The significance of these findings for the use of cationic CPPs in the import of bioactive peptides is demonstrated here using a conjugate consisting of Antp and a Smac protein-derived cargo peptide. The cargo alone, when introduced into cells by electroporation, enhanced TNF-induced apoptosis by inhibiting the anti-apoptotic action of IAPs (inhibitor of apoptosis proteins). For the Antp-Smac conjugate at concentrations below 40 muM the inhibitory effect of the Antp peptide compensated for the pro-apoptotic activity of the cargo, and led to the protection of cells against TNF-mediated apoptosis. These data provide important new information for the use of cationic CPPs for the cellular delivery of bioactive molecules.