P55 TNF Receptor Research Articles

Hepatic Cytochrome P450 Expression during Citrobacter Rodentium Infection in Tumor Necrosis Factor Receptor p55‐/‐ Mice

Citrobacter rodentium is the rodent equivalent of enteropathogenic Escherichia coli infection in humans. The proinflammatory cytokine tumor necrosis factor‐α (TNFα) is induced in mice during C. rodentium infection and may be involved in regulating cytochrome P450 (P450) expression during inflammation. In this study, female mice lacking the gene for the p55 receptor for TNFα, as well as their C57BL/6 controls, were orally infected with C. rodentium and sacrificed 7 days later. CYP2B9 and 3A41 mRNA levels were down‐regulated, and CYP2D22, 3A11 and 3A13 mRNA levels remained near control levels in both the C57BL/6 and the TNF p55 ‐/‐ mice. Hepatic CYP4A10 and 4A14 mRNAs were down‐regulated more (less than 20% of control) in the TNF p55‐/‐ mice than in the C57BL/6 mice, (50% of control). Similarly, CYP2D9 mRNA levels were more highly induced in TNF p55 ‐/‐ (18‐fold) than in C57Bl/6 (2‐fold) mice, although CYP2D protein levels were near control expression levels for both strains. CYP2E1 and 3A25 displayed similar trends in which their mRNA levels were down‐regulated only in the C57BL/6 but not in the TNF p55‐/‐ mice. CYP2E1 protein expression levels followed the same trend as its mRNA. The data suggest that TNFα may play a role in down‐regulating specific hepatic P450s during C. rodentium infection. Supported by NIH grant DK072372.

Leukemic Phase of Follicular Lymphoma after Treatment with Etanercept in a Patient with Psoriasis

Etanercept, a fully human soluble recombinant p75 tumor necrosis factor (TNF) receptor that blocks the binding of TNF to cell surface receptors, is approved for the treatment of psoriatic arthritis and other rheumatic conditions in the US and Europe. Ever since the introduction of anti-TNF treatment in patients with inflammatory autoimmune diseases, there have been concerns about a possible tumor-promoting effect of such a measure. We report a rare case of leukemic phase of follicular lymphoma in a 62-year-old man with moderate-to-severe plaque psoriasis receiving long-term treatment with etanercept for 3 years. Although the association is intriguing, no causal relationship is suggested.

Microglia Protect Neurons against Ischemia by Synthesis of Tumor Necrosis Factor

Microglia and infiltrating leukocytes are considered major producers of tumor necrosis factor (TNF), which is a crucial player in cerebral ischemia and brain inflammation. We have identified a neuroprotective role for microglial-derived TNF in cerebral ischemia in mice. We show that cortical infarction and behavioral deficit are significantly exacerbated in TNF-knock-out (KO) mice compared with wild-type mice. By using in situ hybridization, immunohistochemistry, and green fluorescent protein bone marrow (BM)-chimeric mice, TNF was shown to be produced by microglia and infiltrating leukocytes. Additional analysis demonstrating that BM-chimeric TNF-KO mice grafted with wild-type BM cells developed larger infarcts than BM-chimeric wild-type mice grafted with TNF-KO BM cells provided evidence that the neuroprotective effect of TNF was attributable to microglial- not leukocyte-derived TNF. In addition, observation of increased infarction in TNF-p55 receptor (TNF-p55R)-KO mice compared with TNF-p75R and wild-type mice suggested that microglial-derived TNF exerts neuroprotective effects through TNF-p55R. We finally report that TNF deficiency is associated with reduced microglial population size and Toll-like receptor 2 expression in unmanipulated brain, which might also influence the neuronal response to injury. Our results identify microglia and microglial-derived TNF as playing a key role in determining the survival of endangered neurons in cerebral ischemia.

Tumor necrosis factor receptor–associated periodic syndrome: Toward a molecular understanding of the systemic autoinflammatory diseases

Almost a decade has elapsed since the concept of autoinflammation was advanced to describe conditions characterized by ostensibly unprovoked inflammation, without the hightiter autoantibodies or antigen-specific T-cells found in known autoimmune diseases. This concept was introduced following the identification of mutations in the p55 tumor necrosis factor receptor (TNFR1) in patients with a dominantly inherited syndrome of systemic inflammation (TRAPS) (1). Systemic autoinflammatory diseases are caused by aberrant activation of the innate immune system. Some result in recurrent episodes of fever, localized serositis and skin rashes. Two hereditary recurrent fevers, familial Mediterranean fever (FMF) and the TNF receptor-associated periodic syndrome (TRAPS) were prototypes for this diagnostic category. In the past 10 years this concept has been extended to include a number of Mendelian disorders, including other hereditary recurrent fevers, as well as more common diseases with complex modes of inheritance including Behcet's disease and Crohn's disease (2). When the molecular basis of TRAPS was discovered it was considered an exceedingly rare disease with only a few families described in the literature. Over the past decade, more than 50 disease-associated mutations have been identified in hundreds of patients from a variety of ethnic backgrounds (3). Genotype-phenotype studies showed that mutations at cysteine residues are associated with a more severe phenotype and higher incidence of amyloidosis (4). Although, most TRAPS-associated mutations are fully penetrant, two TNFR1 variants, P46L and R92Q, have been also identified in asymptomatic family members, and at a low frequency in healthy populations. Tumor necrosis factor (TNF) is a pleiotropic cytokine that mediates a wide range of cellular activities primarily through its interaction with TNF receptor 1 (TNFR1; TNFRSF1A; CD120a; p55/p60). Triggering the TNF signaling cascade results in an array of responses, including apoptosis, inflammation and modulation of the immune response. TNFR1 is constitutively expressed on the cell surface in most tissues. It is a large polypeptide with an extracellular domain, consisting of 4 cysteine-rich domains (CRD), a transmembrane domain, and an intracellular death domain (DD). Each CRD includes 3 pairs of cysteine residues that form intramolecular disulfide bonds important in maintaining the 3dimensional conformation of the extracellular part of the receptor. Thus, a substitution at a cysteine residue has the potential to disrupt the relevant disulfide bond and result in significant structural perturbation. The first CRD, sometimes called the pre-ligand association domain (PLAD), allows for TNF-independent interactions of TNFR1 molecules,

The Effects of Cilostazol on Exercise-induced Ischaemia–reperfusion Injury in Patients with Peripheral Arterial Disease

Cilostazol improves walking distance in peripheral arterial disease (PAD) patients. The study objectives were to assess the effects of cilostazol on walking distance, followed by the additional assessment of cilostazol on exercise-induced ischaemia-reperfusion injury in such patients. PAD patients were prospectively recruited to a double-blinded, placebo-controlled trial. Patients were randomised to receive either cilostazol 100mg or placebo twice a day. The primary end-point was an improvement in walking distance. Secondary end-points included the assessment of oxygen-derived free-radical generation, antioxidant consumption and other markers of the inflammatory cascade. Initial and absolute claudication distances (ICDs and ACDs, respectively) were measured on a treadmill. Inflammatory response was assessed before and 30 min post-exercise by measuring lipid hydroperoxide, ascorbate, alpha-tocopherol, beta-carotene, P-selectin, intracellular and vascular cell-adhesion molecules (I-CAM and V-CAM), thromboxane B(2) (TXB(2)), interleukin-6, interleukin-10, high-sensitive C-reactive protein (hsCRP), albumin-creatinine ratio (ACR) and urinary levels of p75TNF receptor. All tests were performed at baseline and 6 and 24 weeks. One hundred and six PAD patients (of whom 73 were males) were recruited and successfully randomised from December 2004 to January 2006. Patients who received cilostazol demonstrated a more significant improvement in the mean percentage change from baseline in ACD (77.2% vs. 26.6% at 6 weeks, p=0.026 and 161.7% vs. 79.0% at 24 weeks, p=0.048) as compared to the placebo. Cilostazol reduced lipid hydroperoxide levels compared to a placebo-related increase before and after exercise (6 weeks: pre-exercise: -11.8% vs. +5.8%, p=0.003 and post-exercise: -12.3% vs. +13.9%, p=0.007 and 24 weeks: pre-exercise -15.5% vs. +12.0%, p=0.025 and post-exercise: -9.2% vs. +1.9%, p=0.028). beta-Carotene levels were significantly increased in the cilostazol group, compared to placebo, before exercise at 6 and 24 weeks (6 weeks: 34.5% vs. -7.4%, p=0.028; 24 weeks: 34.3% vs. 17.7%, p=0.048). Cilostazol also significantly reduced P-selectin, I-CAM and V-CAM levels at 24 weeks as compared to baseline (p<0.05). There was no difference between treatment groups for ascorbate, alpha-tocopherol, interleukin-6 and -10, hsCRP and p75TNF receptor levels. Cilostazol significantly improves ACD, in addition to attenuating exercise-induced ischaemia-reperfusion injury, in PAD patients.

Soluble Lymphotoxin Plays a Critical Role in Acute Graft-Versus-Host Disease

TNF is a key cytokine in the effector phase of both graft-versus-host disease (GVHD) and the graft-versus leukemia (GVL) effect after bone marrow transplantation (BMT). TNF neutralizing antibodies are now established as effective therapeutic adjuncts for the treatment of severe acute GVHD. TNF signals through the p55 and p75 TNF receptors (TNFR), which are also receptors for the soluble lymphotoxin homotrimer (LTα3). The membrane-bound lymphotoxin heterotrimeric molecule (LTα1β2) signals through the LTβ receptor. The function of these molecules in GVHD remains unknown. Pharmacological agents are available which block either TNF alone, or both TNF and LTα3 and elucidating the roles of these molecules in GVHD is essential for the design of rational therapeutic strategies.We have employed multiple well established preclinical models of GVHD to investigate the roles of these molecules. We first used a functional bioassay to confirm that a human TNFR:Fc construct was capable of preventing physiological activity of both recombinant murine TNF and LTα3. Irradiated B6D2F1 recipients were then transplanted with allogeneic B6.TNF−/− bone marrow and T cells, then treated with control Ig or the TNFR:Fc. Suprisingly, the TNFR:Fc provided significant protection from GVHD mortality in the absence of donor derived TNF (median survival 25.5 vs 35 days, P<0.0002). Having observed a potentially TNF-independent but TNFR dependent pathway in the generation of GVHD we next examined mRNA expression by real-time PCR. This demonstrated that the majority of LTα mRNA was within donor CD4 T cells and that expression was unbalanced (> 3 logs more LTα than LTβ). We subsequently developed the first reported ELISA to assess murine LTα3 protein and were able to clearly demonstrate LTα3 production by wild-type (WT) CD4 T cells seven days after BMT. To further examine this molecule, we performed experiments using WT, LTα−/− or LTβ−/− donor grafts in two BMT systems (B6 → B6D2F1 and B6 → BALB/c). Recipients of LTα−/− grafts were protected from GVHD (P<0.01) in both systems while LTβ−/− grafts induced GVHD that was equivalent to, or more severe than in recipients of WT grafts. This data confirms soluble LTα3 but not membrane bound LTα1β2 as an additional pathogenic molecule in GVHD. Using a luciferase-expressing, host-type leukemia (P815) in the B6 → B6D2F1 system, relapse occurred at an identical rate in recipients of WT and LTα−/− grafts confirming that GVL is maintained in the absence of donor derived soluble LTα3. Thus the combined neutralization of both LTα3 and TNF represents an important and logical therapeutic intervention in transplant medicine.

Cytokine activation is predictive of mortality in Zambian patients with AIDS-related diarrhoea

BackgroundMortality in Zambian AIDS patients is high, especially in patients with diarrhoea, and there is still unacceptably high mortality in Zambian patients just starting anti-retroviral therapy. We set out to determine if high concentrations of serum cytokines correlate with mortality.MethodsSerum samples from 30 healthy controls (HIV seropositive and seronegative) and 50 patients with diarrhoea (20 of whom died within 6 weeks) were analysed. Concentrations of tumour necrosis factor receptor p55 (TNFR p55), macrophage migration inhibitory factor (MIF), interleukin (IL)-6, IL-12, interferon (IFN)-γ and C-reactive protein (CRP) were measured by ELISA, and correlated with mortality after 6 weeks follow-up.ResultsApart from IL-12, concentrations of all cytokines, TNFR p55 and CRP increased with worsening severity of disease, showing highly statistically significant trends. In a multivariable analysis high TNFR p55, IFN-γ, CRP and low CD4 count (CD4 count <100) were predictive of mortality. Although nutritional status (assessed by body mass index, BMI) was predictive in univariate analysis, it was not an independent predictor in multivariate analysis.ConclusionHigh serum concentrations of TNFR p55, IFN-γ, CRP and low CD4 count correlated with disease severity and short-term mortality in HIV-infected Zambian adults with diarrhoea. These factors were better predictors of survival than BMI. Understanding the cause of TNFR p55, IFN-γ and CRP elevation may be useful in development of interventions to reduce mortality in AIDS patients with chronic diarrhoea in Africa.

Soluble TNFR II/IgG1 Fc fusion protein treatment in the LPS-mediated septic shock of rats

Tumor necrosis factor-α (TNF-α) is thought to play a major role in systemic inflammation associated with sepsis. A potent TNF antagonist, a recombinant fusion protein that consists of the soluble TNF receptor (p75) linked to the Fc portion of human IgG1 (sTNFR II/IgG1 Fc fusion protein, sTNFR:Fc), has been shown to provide rapid and sustained improvement in local inflammation diseases by binding TNF-α and preventing its proinflammatory activities. To explore the potential therapeutic efficacy for septic shock of sTNFR:Fc, we investigate the effect of this molecule on the survival rate, blood pressure, serum TNF-α bioactivity as well as the expression of TNF-α at mRNA level in the liver in a LPS-induced rat septic shock model. Blood pressure of the rats was monitored by multi-channel creature signal analysis system. Serum TNF-α level and bioactivity was assessed using an enzyme-linked immunoassay and a L929 cytotoxicity assay, respectively. The expression of TNF-α mRNA in liver was examined by semi-quantitative RT-PCR. sTNFR:Fc administered to rats 24 h before LPS challenge ablated the rise in serum TNF-α bioactivity that occurs in response to LPS and protected against hypotension and death. These results indicate that TNF-α is a mediator of fatal septic shock, and suggest that sTNFR:Fc offer a potential therapy of systemic infection.

The TNF-&amp;#945; System: Functional Aspects in Depression, Narcolepsy and Psychopharmacology

Changes of the tumor necrosis factor-alpha (TNF-α) system have been shown to be involved in the development of psychiatric disorders and are additionally associated with changes in body weight as well as endocrine and metabolic changes in psychiatric patients. TNF-α might, for example, contribute to the pathogenesis of depression by an activation of the hypothalamo-pituitary-adrenocortical (HPA) axis, an activation of neuronal serotonin transporters and the stimulation of the indoleamine 2,3-dioxygenase which leads to tryptophan depletion. On the other hand, during an acute depressive episode, an elevated HPA axis activity may suppress TNF-α system activity, while after remission, when HPA axis activity has normalized the suppression of the TNF-α system has been shown not to be apparent any more.In narcoleptic patients, soluble TNF receptor (sTNF-R) p75 plasma levels have been shown to be elevated, suggesting a functional role of the TNF-α system in the development of this disorder.Additionally, psychotropic drugs influence the TNF-α system as well as the secretion and the effect of hormones which counteract or interact with the TNF-α system such as the intestinal hormone ghrelin. However, only preliminary studies with restricted sample sizes exist on these issues, and many open questions remain.

TRIF and IRF-3 Binding to the TNF Promoter Results in Macrophage TNF Dysregulation and Steatosis Induced by Chronic Ethanol

Chronic ethanol (EtOH) abuse results in the development of steatosis, alcoholic hepatitis, and cirrhosis. Augmented TNF-alpha production by macrophages and Kupffer cells and signaling via the p55 TNF receptor have been shown to be critical for these effects of chronic EtOH; however, the molecular mechanisms leading to augmented TNF-alpha production remain unclear. Using cell culture models and in vivo studies we demonstrate that chronic EtOH results in increased TNF-alpha transcription, which is independent of NF-kappaB. Using reporter assays and chromatin immunoprecipitation we found that this increased transcription is due to increased IRF-3 binding to and transactivation of the TNF promoter. As IRF-3 is downstream from the TLR4 adaptor TIR-domain-containing adapter-inducing IFN-beta (Trif), we demonstrate that macrophages from Trif-/- mice are resistant to this dysregulation of TNF-alpha transcription by EtOH in vitro as well as EtOH-induced steatosis and TNF dysregulation in vivo. These data demonstrate that the Trif/IRF-3 pathway is a target to ameliorate liver dysfunction associated with chronic EtOH.

P55 Tumour necrosis factor receptor in bone marrow-derived cells promotes atherosclerosis development in low-density lipoprotein receptor knock-out mice

Tumour necrosis factor (TNF) is a pivotal pro-inflammatory cytokine with a clear pathogenic role in many chronic inflammatory diseases, and p55 TNF receptor (TNFR) mediates the majority of TNF responses. The aim of the current study was to investigate the role of p55 TNFR expression in bone marrow-derived cells, in atherosclerotic lesion development. Irradiated low-density lipoprotein receptor knock-out mice were reconstituted with either p55 TNFR knock-out or control haematopoietic stem cells to generate chimeras deficient or wild-type for p55 TNFR specifically in bone marrow-derived cells, including macrophages. Upon high fat feeding, p55 TNFR knock-out transplanted mice developed smaller atherosclerotic lesions. These lesions were characterized by the presence of smaller foam cells and a reduced macrophage foam cell area. They did not differ in other compositional characteristics as determined by quantification of inflammatory T-cell and neutrophil influx, apoptotic and necrotic cell death, and collagen content. In vitro studies confirmed a significant defect in modified lipoprotein endocytosis by p55 TNFR knock-out macrophages due to reduced scavenger receptor class A expression. Interestingly, plasma cytokine/chemokine profile analysis indicated that monocyte chemoattractant protein-1 (MCP-1) levels, a major chemokine involved in atherogenesis, were consistently and significantly lower in p55 TNFR knock-out transplanted mice compared with controls, before and after high fat feeding. p55 TNFR expression in bone marrow-derived cells contributes to the development of atherosclerosis by enhancing lesional foam cell formation and by promoting the expression of pro-atherosclerotic chemokines such as MCP-1.

Localization of tumour necrosis factor-alpha (TNF-alpha) and its receptors in normal and psoriatic skin: epidermal cells express the 55-kD but not the 75-kD TNF receptor.

The distribution of TNF-alpha, p55 TNF receptor (TNF-R) and p75 TNF-R in normal skin and uninvolved and lesional skin from psoriasis patients has been investigated, using specific mono- and polyclonal antibodies. In normal skin, and uninvolved and lesional skin from psoriasis patients, p55 TNF-R is associated with epidermal keratinocytes and a network of upper dermal dendritic cells. This suggests that the actions of TNF-alpha on epidermal cells in vivo are mediated by binding to the p55 TNF-R. In lesional psoriasis skin, there was staining of the parakeratotic stratum corneum and increased expression of p55 TNF-R in association with upper dermal blood vessels. Staining for p75 TNF-R in normal skin was restricted to eccrine sweat ducts and dermal dendritic cells, and was absent from the epidermis. In lesional psoriasis skin, there was staining for p75 TNF-R in association with upper dermal blood vessels and perivascular infiltrating cells. TNF-alpha in normal skin was predominantly localized to the basal cell layers of the epidermis, and was seen in association with eccrine ducts and sebaceous glands. In lesional psoriasis skin, and to a lesser extent in uninvolved psoriasis skin, TNF-alpha was distributed throughout the epidermis, and was also specifically localized to upper dermal blood vessels. Up-regulation of TNF-alpha, p55 TNF-R and p75 TNF-R on dermal blood vessels in psoriasis may play an important role in the pathogenesis of this condition by promoting cutaneous recruitment of inflammatory cells.

Elevated tumour necrosis factor-alpha (TNF-alpha) biological activity in psoriatic skin lesions.

Lesions of the common inflammatory skin disease psoriasis are characterized by epidermal hyperproliferation, leucocyte adhesion molecule expression and leucocyte infiltration. The local release of proinflammatory cytokines, such as TNF-alpha, may play an important role in the induction of these events. We have, therefore, analysed aqueous extracts of lesional and uninvolved (clinically normal) stratum corneum for the presence of TNF-alpha immunoreactivity and biological activity. TNF-alpha immunoreactivity and bioactivity were consistently higher in lesional compared with uninvolved samples. By using an anti-TNF-alpha neutralizing antibody it was demonstrated that the biological activity measured was due to the presence of TNF-alpha alone. Concentrations of soluble TNF receptors (p55 and p75) were also higher in lesional stratum corneum extracts, with the p55 form predominating. The plasma of psoriatic patients was also found to contain elevated concentrations of soluble p55 compared with normal controls. These results confirm the presence of immunoreactive TNF-alpha and, for the first time, conclusively demonstrate TNF-alpha biological activity and quantifiable concentrations of soluble TNF receptors (p55 and p75) in lesional psoriatic samples. TNF-alpha recovery from stratum corneum probably reflects synthesis in deeper, viable layers, where it is likely to exert its biological effects. Local and systemic release of soluble TNF receptors, in particular p55, may serve to regulate the effects of TNF-alpha in psoriasis.

Update on the use of etanercept across a spectrum of rheumatoid disorders

Etanercept is a soluble TNF receptor p75 fusion protein which is approved for subcutaneous use (50 mg weekly) in the treatment of patients with active rheumatoid arthritis (RA), juvenile RA, ankylosing spondylitis, and psoriatic arthritis. Etanercept binds to both TNFalpha and lymphotoxin and has quite a short mean half-life (70 hours). Numerous randomized clinical trials have demonstrated its efficacy to improve signs and symptoms in early and established RA and other inflammatory arthritis. Furthermore, etanercept has shown its ability to prevent radiographic progression and to improve health-related quality of life in patients with RA and psoriatic arthritis. A combination of etanercept plus methotrexate was more efficacious than etanercept monotherapy in RA patients but there is currently no evidence that such rheumatic combination is better than monotherapy in other disorders. Etanercept was generally well tolerated both in controlled trials with withdrawal rates being similar to the comparator groups and in large observational studies. Infections and injection-site reactions were the most frequently reported events. Serious infections were slightly increased but the occurrence of tuberculosis seemed less frequent than with anti-TNF monoclonal antibodies (infliximab and adalimumab). The benefit-risk ratio of etanercept appeared to be very positive, and this drug has now emerged as a major therapy in patients with active inflammatory arthritis. Furthermore, it is more frequently considered as an emerging and valuable option in patients with early disease.

Depression, comorbidities and the TNF-α system

Depression has frequently been reported to be associated with other physical diseases and changes in the cytokine system. We aimed to investigate associations between a medical history of depression, its comorbidities and cytokine plasma levels in the Bavarian Nutrition Survey II (BVS II) study sample and in patients suffering from an acute depressive episode. The BVS II is a representative study of the Bavarian population aged 13–80 years. The disease history of its 1050 participants was assessed through face-to-face interviews. A sub-sample of 568 subjects and 62 additional acutely depressed inpatients of the Max Planck Institute of Psychiatry participated in anthropometric measurements and blood sampling. Tumor necrosis factor-α (TNF-α) and soluble TNF receptor (sTNF-R) p55 and sTNF-R p75 plasma levels were measured using enzyme-linked immunosorbent assays. A history of depression was associated with a higher incidence of high blood pressure, peptic ulcer, dyslipoproteinemia, osteoporosis, allergic skin rash, atopic eczema and thyroid disease. Within the BVS II sample, participants with a history of depression differed from subjects who had never had depression with regard to sTNF-R p55 and sTNF-R p75 levels even when controlling for age, BMI and smoking status. Acutely depressed inpatients showed even higher levels of sTNF-R p55 and sTNF-R p75 than subjects in the normal population. TNF-α levels were also significantly elevated in acutely depressed patients. These results confirm earlier studies regarding the comorbidities of depression and support the hypothesis that activation of the TNF-α system may contribute to the development of a depressive disorder.

The Role of TRAF2 Binding to the Type I Interferon Receptor in Alternative NFκB Activation and Antiviral Response

Type I interferons (IFNs) play critical roles in the host defense by modulating gene expression through the IFN-dependent activation of STAT and NFkappaB transcription factors. Previous studies established that IFN activates NFkappaB through a classical NFkappaB pathway that results in IkappaBalpha degradation and formation of p50-containing NFkappaB complexes, as well as an alternative pathway that involves NFkappaB-inducing kinase and TRAF2, which results in the formation of p52-containing NFkappaB complexes. In this study, we examined the interaction of TRAF proteins with the type I IFN receptor. We found that TRAF2 was directly coupled to the signal-transducing IFNAR1 subunit of the IFN receptor. By immunoprecipitation, overexpression of epitope-tagged IFNAR1 constructs, and glutathione S-transferase pulldown experiments, we demonstrate that TRAF2 rapidly binds to the IFNAR1 subunit of the IFN receptor upon IFN binding. The membrane proximal half of the IFNAR1 subunit was found to directly bind TRAF2. Moreover, analysis of mouse embryo fibroblasts derived from TRAF2 knock-out mice demonstrated that TRAF2 plays a critical role in the activation of the alternative NFkappaB pathway by IFN, but not the classical NFkappaB pathway, as well as in the antiviral action of IFN. Our results place TRAF2 directly in the signaling pathway transduced through the IFNAR1 subunit of the IFN receptor. These findings provide an important insight into the molecular mechanisms by which IFN generates signals to induce its biological effects.

Gene therapy directed at the neuroimmune component of chronic pain with particular attention to the role of TNF alpha.

Identification that neuroimmune activation in the spinal cord is an important factor in the development of chronic pain has opened the possibility that gene transfer of anti-inflammatory peptides may be used to reduce pain neurotransmission. We review the published evidence regarding gene transfer to meninges to express the anti-inflammatory peptide interleukin 10, and gene transfer to dorsal root ganglia using replication incompetent HSV vectors to express interleukin 4, interleukin 10, or the soluble (p55) tumor necrosis factor receptor (sTNFR). The results of these experiments suggest a novel role for "reverse signaling" through the full-length membrane form of TNFalpha in spinal glia in the modulation of chronic pain.

Effect of anti-TNF-alpha on laser-induced choroidal neovascularization

To investigate the role of TNF-alpha in the development of laser-induced choroidal neovascularization (CNV) in the mouse. Laser photocoagulation was used to induce CNV in wild-type C57BL/6J mice by making four separate choroidal bums in each eye. Animals were treated 3 days before or after laser injury with recombinant TNF receptor P75 (etanercept, 5 microg/h, group 1, n = 12), chimeric monoclonal antibody (infliximab, 5 microg/h, group 2, n = 12) for 7 days by intraperitoneally implanted osmotic pumps. PBS was used as control (group 3, n = 12). The left eyes were removed for histopathologic examination and the right eyes were removed for flatmounts immunohistochemistry immediately after fluorescein angiography. In mice treated with medications 3 days before laser injury, left eyes were collected at 1 or 2 weeks after laser injury. In mice treated with medications 3 days after laser injury, left eyes were collected at 10 days after laser injury. CNV responses were compared by flatmount analysis of CNV-related fluorescence area and by determination of fluorescein angiographic leakage. The level of protein expression of TNF-alpha was semiquantitatively evaluated by Western blot analysis of the choroidal and RPE layer from mice with or without laser treatment. Western blotting demonstrated that TNF-alpha was highly expressed in choroidal and RPE cells of wild type mice 1 week after laser treatment as compared to the control mice without laser treatment. Etanercept and infliximab administrated 3 days before laser-damage significantly reduced CNV size and pathological fluorescein leakage in comparison to the control group one and two weeks after laser injury. Only etanercept administered 3 days after laser injury still significantly reduced the development of CNV lesions. Histopathological examination confirmed that CNV lesions in treated mice had smaller diameter and thinner center as compared to the control animals. Anti-TNF-alpha treatment reduces the size and leakage of laser-induced CNV. These results suggest the involvement of TNF-alpha in the development of laser-induced CNV and its potential use as a therapeutic agent in the age related macular degeneration.

A pilot study of combination anti-cytokine and anti-lymphocyte biological therapy in rheumatoid arthritis

Immunological tolerance in humans using anti-T-cell monoclonal antibodies (mAbs) may be hampered by a pro-inflammatory microenvironment. All clinical trials of such therapies in rheumatoid arthritis (RA), however, have selected patients with active disease at baseline. Concurrent neutralization of inflammation with a TNFalpha antagonist should maximize the potential of anti-T-cell mAbs to induce tolerance in RA. To evaluate the safety of combining a TNFalpha antagonist and CD4 mAb in RA. An iterative pilot study focused on the safety of such combination therapy. Eight poor prognosis, seropositive RA patients were treated with combined CD4 and TNFalpha blockade. Prolonged CD4 blockade was achieved with a humanized mAb, and TNFalpha blockade with a p55 TNF receptor fusion protein. There was a low incidence of classical first-dose reactions to the CD4 mAb, possibly reflecting concomitant TNFalpha blockade. An unusual anaphylactoid reaction was seen, however, and one patient developed a probable allergic reaction after several infusions. Skin rashes were common, as previously reported with CD4 mAb monotherapy. No serious infections were documented during follow-up, despite CD4+ lymphopenia in some patients. Most patients appeared to demonstrate improved RA disease control after the study. After 17-49 months after therapy, one patient was in remission, one remained off disease modifying anti-rheumatic drugs and five had stable disease, three on previously ineffective doses of methotrexate. We report, for the first time in man, immunotherapy with a combination of an anti-cytokine and an anti-T-cell reagent. We witnessed an unusual first-dose reaction but there were no significant infectious complications.

TNFR gene-modified mesenchymal stem cells attenuate inflammation and cardiac dysfunction following MI

Objectives. To investigate the protective effect of tumor necrosis factor receptor (TNFR) gene modified mesenchymal stem cells (MSCs) transplantation against inflammation and cardiac dysfunction following acute myocardial infarction (AMI). Design. MSCs were extracted from the tibias and femurs of rats and transfected with recombinant adeno-associated viral (rAAV) expressing EGFP (enhanced green fluorescent protein) or p75 (human 75 kilodalton) TNFR at multiplicity of infection of 105 particles/cell. Rats with AMI induced by occlusion of the left coronary artery were randomized to MSCs-TNFR transplantation group, MSCs-EGFP transplantation group and MI control group. Results. The effects of MSCs-TNFR transplantation on cardiac inflammation and left ventricular dysfunction were observed after 2 weeks of MI. We found that: 1) MSCs-TNFR transplantation attenuated protein production and gene expression of inflammatory cytokines TNF-α, IL-1β and IL-6; 2) MSCs-TNFR transplantation inhibited cardiomyocytes apoptosis and 3) MSCs-TNFR transplantation improved left ventricular function. Conclusions. The experimental data show that transplantation with rAAV-TNFR transfected MSCs improves left ventricular function following MI through anti-apoptotic and anti-inflammatory mechanisms.