P75 TNF-R Research Articles

Immunex Corp. v. Sandoz Inc. United States Court of Appeals of the Federal Circuit, 2020 964 F.3d 1049

Immunex Corp. v. Sandoz Inc. United States Court of Appeals of the Federal Circuit, 2020 964 F.3d 1049

TNF receptor p55 signaling modulates IL-12/23p40 production in Yersinia enterocolitica-induced reactive arthritis.

TNF receptor p55 signaling modulates IL-12/23p40 production in Yersinia enterocolitica-induced reactive arthritis.

Hypothalamo–pituitary–adrenal axis, glucose metabolism and TNF-α in narcolepsy

Narcolepsy with cataplexy is caused by a deficiency in the production of hypocretin, which regulates sleep and wakefulness and also influences appetite, neuroendocrine functions and metabolism. In this case-control study 11 patients with narcolepsy and cataplexy and 11 healthy adults underwent an oral glucose tolerance test and dexamethason-inhibition cortisol releasing hormone stimulation test. The average age of patients and controls was 35.1 ± 13.2 and 41.0 ± 2.9 years, respectively, the body mass index (BMI) 28.1 ± 6.6 and 25.5 ± 4.7. We did not find evidence of a significantly increased prevalence of disturbed glucose tolerance in narcolepsy patients. After hypothalamo-pituitary–adrenal axis suppression, the number of non-suppressors did not differ between the groups, indicating normal negative feedback sensitivity. The level of cortisol after dexamethason suppression was significantly lower in narcoleptic patients suggesting a slight basal down-regulation and/or a slightly increased negative feedback sensitivity of the major endocrine stress system in narcolepsy. Following CRH stimulation, there were no significant differences in levels of ACTH or cortisol, and in adrenocortical responsivity to ACTH. Finally, narcoleptic patients displayed significantly higher plasma levels of TNF- α , solubleTNFR p55, soluble TNFR p75 and IL-6 after adjustment for BMI. The present study confirms that narcolepsy by itself is not associated with disturbances of glucose metabolism, but goes along with a subtle dysregulation of inflammatory cytokine production. We also found that dynamic HPA system response is not altered, whereas negative feedback to dexamethasone might be slightly enhanced. This study was supported by the grant of the Czech Ministry of Health NT 13238-4/2012, PRVOUK-P26/LF1/4 and by the European Union Framework 6 (MCRTN-CT-2004-512362).

PW02-003 - Efficacy of anakinra in etanercept-resistant TRAPS

Tumor necrosis factor receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease inherited in an autosomal dominant fashion. TRAPS develops secondary to mutations in TNFRSF1A. Associated symptoms include periodic attacks of peritonitis, constipation, arthritis in large joints, arthralgia, migratory rash with underlying myalgia, periorbital edema, conjunctivitis, splenomegaly, and increased risk for AA amyloidosis. Typically, attacks last from days to weeks. The common treatment modalities are corticosteroids, the p75 TNFR:Fc fusion protein, etanercept, and IL-1 antagonists. Recent studies suggest that multiple cytokines are involved in the pathogenesis of TRAPS. To date, there are limited data comparing the efficacy of etanercept and IL-1 inhibitors in TRAPS.

Design, expression and characterization of a novel coexpression system of two antiarthritic molecules

The complexity of rheumatoid arthritis (RA) pathogenesis makes combined blockade of multiple targets an attractive therapeutic strategy. The combination therapy with anti-TNF plus anti-T-cell has been mostly reported to provide greater efficacy than anti-TNF alone. TNFR (p75)-Fc fusion protein, which has been proven effective in clinics, is chosen as the TNF antagonist in this study. CTLA4-FasL fusion molecule, which has been well characterized in our previous studies for its suppressive effect in rat arthritis model, is chosen as the T-cell antagonist. In this study, furin cleavage site and 2A self-processing sequence were introduced to link upstream TNFR-Fc and downstream CTLA4-FasL and mediate separate coexpression of the two fusion proteins in a single recombinant adeno-associated virus (rAAV) vector. Using this expression system, we generated two fusion proteins with same size as their individual counterparts in vitro and in vivo, and the proteins desirably retained their parent biological activities. In vivo results demonstrated that furin-2A technology is able to regulate separate coexpression of these proteins under arthritic inflammatory conditions. This study describes a single rAAV vector for production of two antiarthritic molecules antagonizing both TNF and T cells, which may serve as an attractive expression system for RA gene therapy.

Lack of TNFR p55 Results in Heightened Expression of IFN-γ and IL-17 during the Development of Reactive Arthritis

Reactive arthritis (ReA) is a type of arthritis originating from certain gastrointestinal or genitourinary infections. In previous studies, we reported the development of progressive Yersinia enterocolitica-induced ReA in mice lacking TNFR p55; however, the mechanisms underlying this effect are still uncertain. In this study, we investigated the impact of TNFR p55 deficiency in modulating Ag-specific Th1 and Th17 responses during this arthritogenic process. We found more severe ReA in TNFRp55(-/-) mice compared with their wild-type (WT) counterparts. This effect was accompanied by increased levels of Yersinia LPS in the joints of knockout mice. Analysis of the local cytokine profile revealed greater amounts of IFN-γ and IL-17 in arthritic joints of TNFRp55(-/-) mice compared with WT mice at day 21 postinfection. Moreover, altered IL-17 and IFN-γ production was observed in mesenteric and inguinal lymph nodes of Yersinia-infected TNFRp55(-/-) mice, as well as in spleen cells obtained from infected mice and restimulated ex vivo with bacterial Ags. Increased levels of cytokine secretion were associated with a greater frequency of CD4(+)IL-17(+), CD4(+)IFN-γ(+), and IL-17(+)IFN-γ(+) cells in TNFRp55(-/-) mice compared with WT mice. Remarkably, Ab-mediated blockade of IL-17 and/or IFN-γ resulted in reduced joint histological scores in TNFRp55(-/-) mice. A mechanistic analysis revealed the involvement of p40, a common subunit of heterodimeric IL-12 and IL-23, in the generation of augmented IFN-γ and IL-17 production under TNFR p55 deficiency. Taken together, these data indicate that, in the absence of TNFR p55 signaling, Th1 and Th17 effector cells may act in concert to sustain the inflammatory response in bacterial-induced arthritogenic processes.

The Values of Serum and Ascetic Fluid Tumor Necrosis Factor-Alpha (TNF- alpha), TNF-Receptor 1 and C-Reactive Protein (CRP) in Patients with Cirrhotic Ascites

Spontaneous bacterial peritonitis (SBP) is defined as an infection of initially sterile ascitic fluid (AF) without a detectable, surgically treatable source of infection. It is a frequent and severe complication of cirrhotic ascites. Because of the high morbidity and mortality of SBP, the rapid and accurate diagnosis is required. Aim: The present study aimed to measure the levels of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor (TNF-r p55) and C-reactive protein (CRP) in both ascetic fluid and serum of patients with sterile and infected cirrhotic ascites to show their diagnostic values as compared to ascitic fluid culture and polymorphnuclear leukocyte (PMN) count. Patients and methods: TNF-alpha, TNF-r p55 and CRP were measured in both ascetic fluid and serum of 20 patients with spontaneous bacterial peritonitis (SBP), in addition to 22 patients with sterile cirrhotic ascites. Results: The results of clinical examination showed a significant difference as regard abdominal pain, fever jaundice, upper GIT bleedings, encephalopathy and Sclerotherapy among both groups. The serum levels of CRP and TNF-alpha were significantly higher in patients with SBP as comparing to patients have sterile ascites, but TNF-r p55 serum level showed no significant difference. On evaluation of ascetic fluid parameters, total leucocytic count (TLC), plymorphnuclear (PMN) count , CRP, TNF-alpha, TNF-r p55 are significantly higher in SBP patient group than group of sterile ascites. Sensitivity and specificity of ascitic fluid PMN (cut-off value >250 cells/ mm3) in discriminating infected ascites from sterile ascites were 70% and 86.4%, respectively. Sensitivity and specificity of ascitic fluid CRP (cut-off value >1.0 mg/dL) in discriminating infected ascites from sterile ascites were 85% and 72.7%, respectively. Sensitivity and specificity of ascitic fluid TNF-alpha (cut off value >12 pg/ml) in discriminating infected ascites from sterile ascites were 80% and 63.6%, respectively. Sensitivity and specificity of TNF-r p55 (cut-off value >6.2 pg/ml) in discriminating infected ascites from sterile ascites were 75% and 68.2%, respectively. Conclusion: We concluded that, the elevated serum and ascetic fluid levels of CRP, TNF-alpha and TNF-r may suggest their role in the pathogenesis of ascetic fluid infection and their higher sensitivity and specificity make them to be good discriminators in ascetic fluid infection (especially a cheap and easy ascitic fluid CRP levels). Thus may help in rapid diagnosis and early start empirical antibiotic therapy without waiting the culture results.

The Values of Serum and Ascetic Fluid Tumor Necrosis Factor-Alpha (TNF- alpha), TNF-Receptor 1 and C-Reactive Protein (CRP) in Patients with Cirrhotic Ascites

Spontaneous bacterial peritonitis (SBP) is defined as an infection of initially sterile ascitic fluid (AF) without a detectable, surgically treatable source of infection. It is a frequent and severe complication of cirrhotic ascites. Because of the high morbidity and mortality of SBP, the rapid and accurate diagnosis is required. Aim: The present study aimed to measure the levels of tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor (TNF-r p55) and C-reactive protein (CRP) in both ascetic fluid and serum of patients with sterile and infected cirrhotic ascites to show their diagnostic values as compared to ascitic fluid culture and polymorphnuclear leukocyte (PMN) count. Patients and methods: TNF-alpha, TNF-r p55 and CRP were measured in both ascetic fluid and serum of 20 patients with spontaneous bacterial peritonitis (SBP), in addition to 22 patients with sterile cirrhotic ascites. Results: The results of clinical examination showed a significant difference as regard abdominal pain, fever jaundice, upper GIT bleedings, encephalopathy and Sclerotherapy among both groups. The serum levels of CRP and TNF-alpha were significantly higher in patients with SBP as comparing to patients have sterile ascites, but TNF-r p55 serum level showed no significant difference. On evaluation of ascetic fluid parameters, total leucocytic count (TLC), plymorphnuclear (PMN) count , CRP, TNF-alpha, TNF-r p55 are significantly higher in SBP patient group than group of sterile ascites. Sensitivity and specificity of ascitic fluid PMN (cut-off value >250 cells/ mm3) in discriminating infected ascites from sterile ascites were 70% and 86.4%, respectively. Sensitivity and specificity of ascitic fluid CRP (cut-off value >1.0 mg/dL) in discriminating infected ascites from sterile ascites were 85% and 72.7%, respectively. Sensitivity and specificity of ascitic fluid TNF-alpha (cut off value >12 pg/ml) in discriminating infected ascites from sterile ascites were 80% and 63.6%, respectively. Sensitivity and specificity of TNF-r p55 (cut-off value >6.2 pg/ml) in discriminating infected ascites from sterile ascites were 75% and 68.2%, respectively. Conclusion: We concluded that, the elevated serum and ascetic fluid levels of CRP, TNF-alpha and TNF-r may suggest their role in the pathogenesis of ascetic fluid infection and their higher sensitivity and specificity make them to be good discriminators in ascetic fluid infection (especially a cheap and easy ascitic fluid CRP levels). Thus may help in rapid diagnosis and early start empirical antibiotic therapy without waiting the culture results.

Signaling through the TNF-R2 (p75) but not TNF-R1 (p55) is absolutely required for in vivo CTL maturation in parent-into-F1 mice. (34.4)

Abstract The transfer of B6 splenocytes into normal BDF1 mice results in donor cytotoxic T lymphocytes (CTL) that eliminate host B cells by two weeks and is a useful model of in vivo CTL generation. We previously demonstrated that in vivo anti-TNF mAb treatment prevents CTL and B cell elimination, shifting disease phenotype to a lupus-like disease. To determine if TNF acts directly on naïve donor T cells or whether TNF acts indirectly to promote CTL, we transferred p55 and/or p75 TNF-R defective or wild type (WT) B6 donor cells into WT F1 hosts. At two weeks after transfer host splenocyte populations were assessed by flow cytometry. P55->F1 mice exhibited host B cell elimination comparable to WT->F1 mice (~10% of control F1 values) whereas p75 and p55/p75->F1 mice exhibited a significant increase in host B cells (>150% control) consistent with CTL failure and lupus-like disease. Host DC (CD11c+) and macrophage (CD11b+) values paralleled those of B cells for p55 or p75->F1 mice. Compared to WT->F1 mice, peak day 7 serum IFN-g was significantly reduced only for p75->F1 mice. Defective elimination of host populations in p75->F1 mice was not due to failure of donor CD8 T cells to engraft or to express an effector phenotype (CD44hi, CD62Llo). These results support the conclusion that TNF signaling through the p75 receptor is critical for in vivo CD8 CTL maturation in normal unirradiated mice whereas p55 signaling has a minimal if any role.

TNF receptor p55 and IL-872 and IL-877 isoforms: blood and urine levels in breast cancer patients

In the preliminary study reported here, 37 patients with breast cancer and 10 healthy volunteers were analyzed for soluble TNF-R p55 and two variants of IL-8 consisting of 72 and 77 amino acid residues (IL-872 and IL-877, respectively) in their blood and urine with novel ELISA test systems. The clinical/prognostic values of determining these inflammatory cytokines at different stages of the cancer process appeared to depend on the treatment course being evaluated. In contrast to expectations, it was noted that there was a stabile tendency for decreased TNF-R p55 and IL-872 levels in the plasma and urine of breast cancer patients as compared with levels observed with healthy controls. Moreover, patients that underwent polychemotherapy treatments were notable for significant decreases in IL-872 and TNF-R p55 levels in their blood plasma; these findings contrasted with significant increases in these parameters in these patients’ urine. Interestingly, the IL-877 isoform that now appeared both in the urine and plasma of patients was not detectable before initiation of the polychemotherapy. In spite of all these findings, individual fluctuations among these parameters still do not allow us to establish, at this time, any strong correlations between these values with any particular breast cancer stage or a type of treatment. Nonetheless, while the results here are preliminary, they demonstrate that testing for TNF-R, along with IL-8 isoforms, in the blood plasma and urine could potentially present a valid means for monitoring of the overall immune and disease progress/remission status in breast cancer patients. Ongoing studies with larger patient sample sizes, as well as collecting and analyzing samples at multiple timepoints—to minimize the potential influence of any inherent variability in cytokine levels in humans—will hopefully allow us to specify what these preliminary results reported here suggest, i.e., the potential utility of this experimental approach for determining disease progression or efficacy of treatment in cancer patients.

Absence of p55 TNF Receptor Reduces Atherosclerosis, but Has No Major Effect on Angiotensin II Induced Aneurysms in LDL Receptor Deficient Mice

BackgroundThe aim of the current study was to investigate the role of p55 TNF Receptor (p55 TNFR), the main signaling receptor for the pro-inflammatory cytokine tumor necrosis factor (TNF), in the development of two vascular disorders: atherosclerosis and angiotensin (Ang) II-induced abdominal aortic aneurysms (AAA).Methodology/Principal Findingsp55 TNFR deficient mice were crossed to an LDL receptor deficient background and were induced for the development of either atherosclerosis or AngII-induced AAA, and compared to littermate controls, wild-type for p55 TNFR expression. p55 TNFR deficient mice developed 43% smaller atherosclerotic lesions in the aortic sinuses compared to controls. Moreover, expression of CD68, a macrophage specific marker, exhibited a 50% reduction in the aortic arches. Decreased atherosclerosis correlated with a strong down-regulation in the expression of adhesion molecules, such as VCAM-1 and ICAM-1, by p55 TNFR deficient endothelium. In addition, expression levels of the pro-inflammatory cytokines and chemokines TNF, IL-6, MCP-1 and RANTES were significantly reduced in aortas of p55 TNFR deficient mice. In contrast, in the AngII-induced model of AAA, p55 TNFR deficiency correlated with a slight trend towards increased aneurismal lethality, but the incidence of aortic rupture due to a dissecting aneurysm, and the expansion of the suprarenal aorta were not significantly different compared to controls.Conclusion/SignificanceWe found that p55 TNFR expression promotes atherosclerosis, among other mechanisms, by enhancing expression of endothelial adhesion molecules, while it seems to have no major role in the development of AngII-induced AAA.

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.

Blockade of tumor necrosis factor in collagen-induced arthritis reveals a novel immunoregulatory pathway for Th1 and Th17 cells

IL-17 is implicated in the pathogenesis of rheumatoid arthritis (RA) and has previously been shown to be induced by tumor necrosis factor (TNF) in vitro. The aim of this study was to assess the impact of TNF inhibition on IL-17 production in collagen-induced arthritis, a model of RA. TNF blockade using TNFR-Fc fusion protein or anti-TNF monoclonal antibody reduced arthritis severity but, unexpectedly, expanded populations of Th1 and Th17 cells, which were shown by adoptive transfer to be pathogenic. Th1 and Th17 cell populations were also expanded in collagen-immunized TNFR p55−/− but not p75−/− mice. The expression of IL-12/IL-23 p40 was up-regulated in lymph nodes (LN) from p55−/− mice, and the expansion of Th1/Th17 cells was abrogated by blockade of p40. Treatment of macrophages with rTNF also inhibited p40 production in vitro. These findings indicate that at least one of the ways in which TNF regulates Th1/Th17 responses in arthritis is by down-regulating the expression of p40. Finally, although TNF blockade increased numbers of Th1 and Th17 cells in LN, it inhibited their accumulation in the joint, thereby providing an explanation for the paradox that anti-TNF therapy ameliorates arthritis despite increasing numbers of pathogenic T cells.

Treatment with infliximab may contribute to the development of peripheral neuropathy among the patients with rheumatoid arthritis

Sir, We have read with the interest the paper of Tektonidu et al. [1] reporting the two cases of peripheral neuropathy in patients with rheumatoid arthritis treated with anti-tumor necrosis factor (anti-TNF) alpha monoclonal antibody infliximab. The paper addresses the problem of potential side effects of infliximab upon peripheral nervous system. In spite of the fact that peripheral nervous system damage is not often reported in patients with rheumatoid arthritis on anti-TNF alpha treatment, the observation is of great clinical values when taking into consideration thousands of patients treated with a such an agent [2]. The role of TNF alpha in pathogenesis of nervous system damage is not fully elucidated. TNF alpha is thought to play a significant role in the pathogenesis of inflammatory demyelinating disease of the central nervous system, which was demonstrated in humans and in animal models [3–5]. If it is true, anti-TNF alpha agents would exert protective activity against demyelination of central and peripheral nervous systems. Many facts however suggest otherwise. The study with lenarcept, a soluble dimeric p55 TNFR–IgG fusion protein in patients with multiple sclerosis (MS), was terminated prematurely because of unexpected exacerbation of the disease. The study was designed to verify a good therapeutic response observed in animal model of MS [6]. These facts are instructive into two ways: Firstly, it is difficult to translate the results from animal models directly to man, and secondly, toxic effect of anti-TNF alpha blockers may be due to imbalance of TNF alpha and TNF alpha receptor levels in the patients. Rapid changes in concentrations of TNF alpha may thus potentially evoke or worsen underlying latent demyelinating process. As the problem of peripheral neuropathy in patients receiving anti-TNF alpha agents was not explained well in prospective studies, we undertook the 1 year observational study. During this period of time, 28 patients (26 women and 2 men) with active, refractory to standard diseasemodifying anti-rheumatic drug (DMARD) therapy were investigated. All patients were treated with infliximab within 1 year (cumulative dose, 36 mg/kg of body mass). Before administration of the first dose of infliximab and after 12 months of therapy, electromyography was carried out in all patients. The examination included measurement of motoneuronal conduction velocity and amplitude in the following nerves: median, peroneal, tibial and ulnar and sensory neuronal velocity, and amplitude in the median, sural, and ulnar nerves. Before the first administration of infliximab, all patients were characterized by normal nervous function. There were no disturbances in conduction velocity and amplitude in examined nerves. After 12 months of the treatment with TNF alpha antagonist, we observed statistically significant changes in following electromyographic parameters: an increased motoneuronal amplitude in median nerve (4.93 vs 8.06 mV) and a decrease in conduction velocity in the nerves of lower limbs; tibial and sural 50.88 vs 47.08 m/s, and 48.73 vs 44.93 m/s respectively [7]. These results were still in normal limits, and the patients continued to be asymptomatic. Data obtained from our study suggests, however, that anti-TNF alpha treatment exerts impact on peripheral nervous system function and, in some predisposing circumstances, may result in overt neuropathy. In the light of the Clin Rheumatol (2007) 26:1595–1596 DOI 10.1007/s10067-007-0657-3

Regulation of IL-1 and TNF Receptor Expression and Function by Endogenous Macrophage Migration Inhibitory Factor

Macrophage migration inhibitory factor (MIF) has a key role in regulation of innate and adaptive immunity and is implicated in sepsis, tumorigenesis, and autoimmune disease. MIF deficiency or immunoneutralization leads to protection against fatal endotoxic, exotoxic, and infective shock, and anti-inflammatory effects in other experimental models of inflammatory disease. We report a novel regulatory role of MIF in type 1 IL-1R and p55 TNFR expression and function. Compared with wild-type cells, MIF-deficient cells were hyporesponsive to IL-1- and TNF-induced MAPK activity, AP-1 activity, and cellular proliferation, while NF-kappaB function was preserved. Hyporesponsiveness of MIF-deficient cells was associated with down-regulation of cytokine receptor expression, which was restored by reconstitution of either an upstream kinase of MAPK, MAPK/ERK kinase, or MIF. These data suggest that endogenous MIF is required for cytokine activation of MAPK/AP-1 and cytokine receptor expression. This autocrine regulatory pathway defines an important amplifying role of endogenous MIF in cytokine-mediated immune and inflammatory diseases and provides further molecular evidence for the critical role of MIF in cellular activation.

Plasma Levels of Tumor Necrosis Factor α and Soluble Tumor Necrosis Factor Receptors in Patients With Narcolepsy

Narcolepsy is a disabling sleep disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucinations, and sleep paralysis. Recent studies suggest that the immune system might play a pathogenic role pointing to a possible involvement of inflammatory cytokines. We investigated a sample of 30 patients with narcolepsy in comparison with 120 sex- and age-matched and 101 sex-, body mass index (BMI)-, and age-matched randomly selected normal controls. In these groups, plasma concentrations of tumor necrosis factor alpha (TNF-alpha) and its soluble receptors p55 and p75 (soluble TNF receptor [sTNF-R] p55 and sTNF-R p75) were measured using commercial enzyme-linked immunosorbent assays. The narcoleptic patients showed a significantly higher BMI compared with controls of the same age. Soluble TNF-R p75 levels were consistently elevated in the narcoleptic patients compared with their sex- and age-matched (P = .001) as well as sex-, BMI-, and age-matched counterparts (P = .003). Female narcoleptic patients exhibited higher sTNF-R p55 levels compared with their sex- and age-matched controls (P = .01), but this difference disappeared when comparing patients with sex-, BMI-, and age-matched normal controls. Tumor necrosis factor alpha levels did not differ significantly between groups. Narcoleptic patients show increased plasma levels of sTNF-R p75, suggesting a functional alteration of the TNF-alpha cytokine system, further corroborating a possible pathogenic role of the immune system in this sleep disorder.

Stoichiometry of LTβR Binding to LIGHT

LTbetaR is a member of the TNF receptor family of proteins. It binds to two different cell surface ligands, LIGHT, a homotypic trimer, and LTalpha1beta2, a heterotypic trimer. We have measured the affinities of the dimeric IgG fusion protein, LTbetaRIgG, and monomeric LTbetaR protein binding to both LIGHT and LTalpha1beta2 using surface plasmon resonance and found values of <0.1 and 38 nM for LIGHT and <0.1 and 48 nM for LTalpha1beta2, respectively. We also determined the stoichiometries of binding for both forms of the receptor LTbetaRIgG and LTbetaR binding to LIGHT. The data obtained from several biophysical methods are consistent with receptor polypeptide to trimeric ligand ratios of 2:1. The determined masses of the complexes using SEC-LS corresponded to a single LTbetaRIgG bound to a LIGHT trimer, or two LTbetaR bound per LIGHT. Sedimentation velocity of varied ratios of LTbetaR to a fixed concentration of LIGHT were analyzed by SEDANAL and were successfully fit with a model with two tight binding sites on LIGHT and one poor affinity site. Isothermal calorimetric titration of LIGHT with either LTbetaR or LTbetaRIgG also demonstrated stoichiometries of 1:2 and 1:1, respectively. The binding of LTbetaR to LIGHT was endothermic and, hence, entropy-driven. TNFR p55 (extracellular domain) complexed with the trimeric ligand, TNFbeta, exhibits a 3:1 receptor/ligand stoichiometry. This complex has been used as the prototypical model setting the receptor-ligand complexation paradigm for the entire TNF family. The LTbetaR/LIGHT binding stoichiometry of 2:1 demonstrated here does not fit the paradigm. This has numerous implications for cell biology including signaling requiring only dimerization of LTbetaR rather than trimerization as expected from the structural paradigm.

Infliximab acts directly on human osteoclast precursors and enhances osteoclast formation induced by receptor activator of nuclear factor κB ligand in vitro

Infliximab is known to protect against the development of joint destruction. In the present study, we sought to determine whether Infliximab acts directly on human osteoclast precursors and influences monocyte-osteoclast differentiation induced by receptor activator of nuclear factor kappaB ligand (RANKL) in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from rheumatoid arthritis (RA) patients and normal controls were cultured in the presence of RANKL and macrophage colony stimulating factor. Infliximab, antihuman tumor necrosis factor alpha (TNFalpha), antihuman TNF soluble receptor p55 (TNFR p55), and antihuman TNF soluble receptor p75 (TNFR p75) antibodies were added. Osteoclast formation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP) staining cells and the extent of lacunar resorption. Addition of Infliximab resulted in a marked increase in the number of TRAP-positive multinucleated cells (TRAP(+) MNCs) and in the extent of lacunar resorption compared with the control cultures. Antihuman TNFalpha antibody showed the same effect; however, the addition of neither TNFR p55 nor TNFR p75 antibody affected the extent of TRAP(+) MNCs and lacunar resorption. Our results suggest that infliximab acts directly on early osteoclast precursors, and stimulates osteoclast formation and lacunar resorption induced by RANKL in vitro.

Infliximab acts directly on human osteoclast precursors and enhances osteoclast formation induced by receptor activator of nuclear factor κB ligand in vitro

Infliximab is known to protect against the development of joint destruction. In the present study, we sought to determine whether Infliximab acts directly on human osteoclast precursors and influences monocyte-osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL) in vitro. Peripheral blood mononuclear cells (PBMCs) isolated from rheumatoid arthritis (RA) patients and normal controls were cultured in the presence of RANKL and macrophage colony stimulating factor. Infliximab, antihuman tumor necrosis factor α (TNFα), antihuman TNF soluble receptor p55 (TNFR p55), and antihuman TNF soluble receptor p75 (TNFR p75) antibodies were added. Osteoclast formation was determined by assessing the number of tartrate-resistant acid phosphatase (TRAP) staining cells and the extent of lacunar resorption. Addition of Infliximab resulted in a marked increase in the number of TRAP-positive multinucleated cells (TRAP+ MNCs) and in the extent of lacunar resorption compared with the control cultures. Antihuman TNFα antibody showed the same effect; however, the addition of neither TNFR p55 nor TNFR p75 antibody affected the extent of TRAP+ MNCs and lacunar resorption. Our results suggest that infliximab acts directly on early osteoclast precursors, and stimulates osteoclast formation and lacunar resorption induced by RANKL in vitro.

Roles of Tumor Necrosis Factor Alpha (TNF-α) and the p55 TNF Receptor in CD1d Induction and Coxsackievirus B3-Induced Myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.