Targeting an inflammation-amplifying cell population can attenuate osteoarthritis-associated pain.

BACKGROUND: Understanding of pain in osteoarthritis, its genesis, and perception is still in its early stages. Identification of precise ligand-receptor pairs that transduce pain and the cells and tissues in which they reside will elucidate new therapeutic approaches for pain management. Our recent studies had identified an inflammation-amplifying (Inf-A) cell population that is expanded in human OA cartilage and is distinctive in the expression of both IL1R1 and TNF-R2 receptors and active Jnk signaling cascade. METHODS: In this study, we have tested the function of the cartilage-resident IL1R1+TNF-R2+ Inf-A cells in OA. We have identified that the IL1R1+TNF-R2+ Inf-A cells expand in aged mice as well as after anterior cruciate ligament tear upon tibia loading and OA initiation in mice. We targeted and modulated the Jnk signaling cascade in InfA through competitive inhibition of Jnk signaling in mice and human OA explants and tested the effects on joint structure and gait in mice. RESULTS: Modulation of Jnk signaling led to attenuation of inflammatory cytokines CCL2 and CCL7 without showing any structural improvements in the joint architecture. Interestingly, Jnk inhibition and lowered CCL2 and 7 are sufficient to significantly improve the gait parameters in treated PTOA mice demonstrating reduced OA-associated pain. Consistent with the mice data, treatment with JNK inhibitor did not improve human OA cartilage explants. CONCLUSION: These studies demonstrate that Inf-A, an articular-cartilage resident cell population, contributes to pain in OA via secretion of CCL2 and 7 and can be targeted via inhibition of Jnk signaling.

Vitexin along with verapamil downregulates efflux pump P-glycoprotein in macrophages and potentiate M1 to M2 switching via TLR4-NF-κB-TNFR2 pathway in lipopolysaccharide treated mice.

The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.

Distepharinamide, a novel dimeric proaporphine alkaloid from Diploclisia glaucescens, inhibits the differentiation and proliferative expansion of CD4+Foxp3+ regulatory T cells.

BACKGROUND: CD4+Foxp3+ regulatory T cells (Tregs) represent the primary cellular mechanism of tumor immune evasion. Elimination of Treg activity by the pharmacological agent may enhance anti-tumor immune responses. However, Treg-eliminating agents, especially those with small molecules, are rarely reported. PURPOSE: To identify small molecule inhibitors of Treg cells from natural products. METHODS: Compounds from Diploclisia glaucescens were isolated by column chromatography, and structures were identified by spectroscopic evidence and quantum calculations. The tet-On system for Foxp3-GFP expression in Jurkat T cells was generated to screen Treg inhibitors based on Foxp3 expression. The effect of the compound on TNF-induced proliferative expansion of naturally occurring Tregs (nTregs) and TGF-ß-induced generation of Tregs (iTregs) from naive CD4+ Tcells was further examined. RESULTS: A novel dimeric proaporphine alkaloid, designated as distepharinamide (DSA) with a symmetric structure isolated from the stems of D. glaucescens, restrained the doxycycline (Doxy)-induced Foxp3-tGFP expression, decreased the half-life of Foxp3 mRNA as well as reduced the mRNA levels of chemokine receptors (CCR4, CCR8 and CCR10) in Jurkat T cells with inducible Foxp3-tGFP expression. In lymphocytes or purified Tregs from wild-type C57BL/6 mice or from C57BL/6-Tg(Foxp3-DTR/EGFP)23.2Spar/Mmjax mice, DSA markedly inhibited TNF-induced proliferative expansion of Tregs present in the unfractionated CD4+ T cells, accompanied by the down-regulation of TNFR2, CD25 and CTLA4 expression on Tregs. Furthermore, DSA potently inhibited TGF-ß-induced differentiation of Foxp3-expressing iTregs. Importantly, the expression of Foxp3 mRNA by both nTregs and iTregs was decreased by DSA treatment. Nevertheless, DSA at the same concentrations did not inhibit the proliferation of conventional CD4+ and CD8+ T cells stimulated by anti-CD3/CD28 antibodies. CONCLUSION: DSA, a novel dimeric proaporphine alkaloid, potently inhibited the expansion of nTregs and generation of iTregs. Therefore, DSA or its analogs may merit further investigation as novel immunotherapeutic agents.

Nigellidine (Nigella sativa, black-cumin seed) docking to SARS CoV-2 nsp3 and host inflammatory proteins may inhibit viral replication/transcription and FAS-TNF death signal via TNFR 1/2 blocking.

Tissue damage occurs in COVID-19 patients due to nsp3-induced Fas-FasL interaction/TNF-related apoptosis. Presently, possible therapeutic-drug, nigellidine against was screened by bioinformatics studies COVID-19. Atomic-Contact-Energy (ACE) and binding-blocking effects were explored of nigellidine (Nigella sativa L.) in the active/catalytic sites of viral-protein nsp3 and host inflammatory/apoptotic signaling-molecules Fas/TNF receptors TNFR1/TNFR2. A control binding/inhibition of Oseltamivir to influenza-virus neuraminidase was compared here. In AutoDock, Oseltamivir binding-energy (BE) and inhibition-constant (KI) was -4.12 kcal/mol and 959.02. The ACE values (PatchDock) were -167.02/-127.61/-124.91/-122.17/-54.81/-47.07. The nigellidine BE/KI with nsp3 was -7.61 and 2.66, respectively (ACE values were -221.40/-215.62/-113.28). Nigellidine blocked FAS dimer by binding with a BE value of -7.41 kcal/mol. Its strong affinities to TNFR1 (-6.81) and TNFR2 (-5.1) are demonstrated. Our present data suggest that nigellidine may significantly block the TNF-induced inflammatory/Fas-induced apoptotic death-signaling in comparison with a positive-control drug Oseltamivir. Further studies are necessary before proposing nigellidine as medical drug.

Characterization of the Active Components of the Multimerized sTNFRIIAdiponectin Fusion Protein Showing Both TNFα-Antagonizing and Glucose Uptake-Promoting Activities.

BACKGROUND: The sTNFRII-adiponectin fusion protein previously showed strong TNFα antagonistic activity. However, the fusion protein exists as mixture of different multimers. The aim of the present study was to characterize its active components. METHODS: In this study, the fusion protein was isolated and purified by Ni-NTA affinity and gel exclusion chromatography, and further identified by Coomassie staining and western blotting. The TNFα antagonistic and glucose uptake-promoting activities were determined in vitro. The glucose detection kit and 2- NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose) were used to measure their effects on glucose metabolism (including glucose consumption and glucose uptake in HepG2 and H9C2 cells). The effect of the fusion protein on glucose uptake was also examined in free fatty acid (FFA)- induced insulin resistance cell model. RESULTS: The sTNFRII-adiponectin fusion protein was found to exist in three forms: 250 kDa (hexamer), 130 kDa (trimer), and 60 kDa (monomer), with the final purity of 90.2%, 60.1%, and 81.6%, respectively. The fusion protein could effectively antagonize the killing effect of TNFα in L929 cells, and the multimer was found to be superior to the monomer. In addition, the fusion protein could increase glucose consumption without impacting the number of cells (HepG2, H9C2 cells) in a dosedependent manner. Mechanistically, glucose uptake was found to be enhanced by the translocation of GLUT4. However, it could not improve glucose uptake in the cell model of insulin resistance. CONCLUSION: In summary, the active components of the fusion protein are hexamers and trimers. The hexamer and trimer of sTNFRII-adiponectin fusion protein had both TNFα-antagonizing and glucose uptake-promoting activities, although neither of them could improve glucose uptake in the cell model of insulin resistance.

Effects of bovine tumor necrosis factor alpha decoy receptors on cell death and inflammatory cytokine kinetics: potential for bovine inflammation therapy.

BACKGROUND: Refractory diseases, including bacterial infections, are causing huge economic losses in dairy farming. Despite efforts to prevent and treat those diseases in cattle, including the use of antimicrobials, it is not well controlled in the field. Several inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), play important roles in disease progression; thus, blocking these cytokines can attenuate the acute and sever inflammation and may be a novel strategy for treatment. However, biological drugs targeting inflammatory cytokines have not been used in cattle. Therefore, in this study, bovine sTNFR1 and sTNFR2 IgG1 Fc-fusion proteins (TNFR1-Ig and TNFR2-Ig) were produced, and their anti-inflammatory functions were analyzed in vitro, to develop decoy receptors for bovine TNF-α. RESULTS: Both TNFR1-Ig and TNFR2-Ig were shown to bind with TNF-α, and TNFR2-Ig showed higher affinity toward TNF-α than TNFR1-Ig. We next stimulated murine fibroblast-derived cells (L929 cells) with TNF-α to induce cell death and analyzed cell viability in the presence of TNFR-Ig proteins. Both TNFR1-Ig and TNFR2-Ig suppressed TNF-α-induced cell death, significantly improving cell viability. In addition, cell death induced by TNF-α was suppressed, even at low TNFR2-Ig concentrations, suggesting TNFR2-Ig has higher activity to suppress TNF-α functions than TNFR1-Ig. Finally, to examine TNFR2-Ig's anti-inflammatory, we cultured peripheral blood mononuclear cells from cattle with TNF-α in the presence of TNFR2-Ig and analyzed the gene expression and protein production of the inflammatory cytokines IL-1ß and TNF-α. TNFR2-Ig significantly reduced the gene expression and protein production of these cytokines. Our results suggest that TNFR2-Ig inhibits inflammatory cytokine kinetics by blocking TNF-α to transmembrane TNFR, thereby attenuating excessive inflammation induced by TNF-α. CONCLUSIONS: Collectively, the findings of this study demonstrated the potential of TNFR2-Ig as a novel therapeutic for inflammatory diseases, such as bovine clinical mastitis. Further investigation is required for future clinical application.

[Recombinant human tumor necrosis factor receptor type â ¡-IgG Fc fusion protein for treatment of occupational medicamentosa-like dermatitis induced by trichloroethylene].

Objective: To investigate the efficacy and safety of the recombinant human tumor necrosis factor receptor Ⅱ-IgG Fc fusion protein (rhTNFR: Fc, etanercept) for the treatment of occupational medicamentosa-like dermatitis induced by trichloroethylene (OMLDT) . Methods: In September 2011 to February 2016, 12 patients with OMLDT were treated with etanercept 25 mg, subcutaneous injection, twice per week, doubling of first dose. The course of treatment was 6 weeks. The drug eruption area and severity index (DASI) score, the proportion of patients achieving a 50%, 75% and 90% reduction in DASI (DASI50, DASI75, DASI90) and the serum level of TNF-α were used to assess the efficacy at different times. Adverse reactions were also recorded and evaluated. The results were statistically analyzed by nonparametric Friedman test and repetitive measurement ANOVA using the software SPSS19.0. Results: After 4 weeks treatment, the DASI score decreased form 56.33±7.02 to 0.50±0.91 (P<0.01) . The DASI50, DASI75 and DASI90 were all increased to 12 (100%) . The serum level of TNF-α decreased form (43.74±41.62) pg/ml to (3.03±0.47) pg/ml (P<0.01) . Statistically significant difference was observed from the above indexes. There were no adverse reactions in clinical application. Conclusion: Recombinant human tumor necrosis factor receptor Ⅱ-IgG Fc fusion protein may be a safe and effective drug in the treatment of OMLDT.

Acute liver injury attenuation of a novel recombinant sTNFR through blocking hepatic apoptosis.

CONTEXT: Tumor necrosis factor (TNF) α plays a key role in acute liver injury (ALI) induced by injection of d-galactosamine (D-Gal)/lipopolysaccharide (LPS). A novel recombinant trimeric sTNFRII, sTNFRII-gAD, has been tested to be effective in ameliorating ALI, when administered prior to ALI establishment. This study aims to validate the protective effect of sTNFRII-gAD when given after ALI setup and further explore its effect on hepatic apoptosis. MATERIALS AND METHODS: The treatments were carried out concomitantly with ALI establishment with clinically approved sTNFRII-Fc (the dimeric sTNFRII) as a positive control. Lethality, liver weight, and serum alanine transaminase were measured, and histological analysis was performed to evaluate liver injury induced by D-Gal/LPS. Additionally, Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) and Western blot analyses of caspase-3 were used to examine hepatocellular apoptosis. RESULTS: sTNFRII-gAD given after D-Gal/LPS injection turned out to attenuate animal mortality significantly (p < 0.01), and had better hepatic protection. In terms of apoptosis, both sTNFRII-gAD and sTNFRII-Fc displayed noticeable improvement of apoptosis evidenced by dramatic decline of active caspase-3 compared to the control group. CONCLUSIONS: The results demonstrated that sTNFRII-gAD therapeutically diminished the lethality induced by D-Gal/LPS, possibly through blocking hepatic apoptosis initiated by TNFα. Of note, sTNFRII-gAD was superior to sTNFRII-Fc in some respects, indicating a promising alternative for the therapeutic strategy against the diseases associated with excessive TNFα.

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.

Kinematic and dynamic gait compensations in a rat model of lumbar radiculopathy and the effects of tumor necrosis factor-alpha antagonism.

INTRODUCTION: Tumor necrosis factor-α (TNFα) has received significant attention as a mediator of lumbar radiculopathy, with interest in TNF antagonism to treat radiculopathy. Prior studies have demonstrated that TNF antagonists can attenuate heightened nociception resulting from lumbar radiculopathy in the preclinical model. Less is known about the potential impact of TNF antagonism on gait compensations, despite being of clinical relevance. In this study, we expand on previous descriptions of gait compensations resulting from lumbar radiculopathy in the rat and describe the ability of local TNF antagonism to prevent the development of gait compensations, altered weight bearing, and heightened nociception. METHODS: Eighteen male Sprague-Dawley rats were investigated for mechanical sensitivity, weight-bearing, and gait pre- and post-operatively. For surgery, tail nucleus pulposus (NP) tissue was collected and the right L5 dorsal root ganglion (DRG) was exposed (Day 0). In sham animals, NP tissue was discarded (n = 6); for experimental animals, autologous NP was placed on the DRG with or without 20 µg of soluble TNF receptor type II (sTNFRII, n = 6 per group). Spatiotemporal gait characteristics (open arena) and mechanical sensitivity (von Frey filaments) were assessed on post-operative Day 5; gait dynamics (force plate arena) and weight-bearing (incapacitance meter) were assessed on post-operative Day 6. RESULTS: High-speed gait characterization revealed animals with NP alone had a 5% decrease in stance time on their affected limbs on Day 5 (P ≤0.032). Ground reaction force analysis on Day 6 aligned with temporal changes observed on Day 5, with vertical impulse reduced in the affected limb of animals with NP alone (area under the vertical force-time curve, P <0.02). Concordant with gait, animals with NP alone also had some evidence of affected limb mechanical allodynia on Day 5 (P = 0.08) and reduced weight-bearing on the affected limb on Day 6 (P <0.05). Delivery of sTNFRII at the time of NP placement ameliorated signs of mechanical hypersensitivity, imbalanced weight distribution, and gait compensations (P <0.1). CONCLUSIONS: Our data indicate gait characterization has value for describing early limb dysfunctions in pre-clinical models of lumbar radiculopathy. Furthermore, TNF antagonism prevented the development of gait compensations subsequent to lumbar radiculopathy in our model.

Attenuation of inflammatory events in human intervertebral disc cells with a tumor necrosis factor antagonist.

STUDY DESIGN: The inflammatory responses of primary human intervertebral disc (IVD) cells to tumor necrosis factor α (TNF-α) and an antagonist were evaluated in vitro. OBJECTIVE: To investigate an ability for soluble TNF receptor type II (sTNFRII) to antagonize TNF-α-induced inflammatory events in primary human IVD cells in vitro. SUMMARY OF BACKGROUND DATA: TNF-α is a known mediator of inflammation and pain associated with radiculopathy and IVD degeneration. sTNFRs and their analogues are of interest for the clinical treatment of these IVD pathologies, although information on the effects of sTNFR on human IVD cells remains unknown. METHODS: IVD cells were isolated from surgical tissues procured from 15 patients and cultured with or without 1.4 nmol/L TNF-α (25 ng/mL). Treatment groups were coincubated with varying doses of sTNFRII (12.5-100 nmol/L). Nitric oxide (NO), prostaglandin E2 (PGE2), and interleukin-6 (IL6) levels in media were quantified to characterize the inflammatory phenotype of the IVD cells. RESULTS: Across all patients, TNF-α induced large, statistically significant increases in NO, PGE2, and IL6 secretion from IVD cells compared with controls (60-, 112-, and 4-fold increases, respectively; P < 0.0001). Coincubation of TNF-α with nanomolar doses of sTNFRII significantly attenuated the secretion of NO and PGE2 in a dose-dependent manner, whereas IL6 levels were unchanged. Mean IC50 values for NO and PGE2 were found to be 35.1 and 20.5 nmol/L, respectively. CONCLUSION: Nanomolar concentrations of sTNFRII were able to significantly attenuate the effects of TNF-α on primary human IVD cells in vitro. These results suggest this sTNFR to be a potent TNF antagonist with potential to attenuate inflammation in IVD pathology.

Restoration of regenerative osteoblastogenesis in aged mice: modulation of TNF.

Skeletal changes accompanying aging are associated with both increased risk of fractures and impaired fracture healing, which, in turn, is due to compromised bone regeneration potential. These changes are associated with increased serum levels of selected proinflammatory cytokines, e.g., tumor necrosis factor alpha (TNF-alpha). We have used a unique model of bone regeneration to demonstrate (1) that aged-related deficits in direct bone formation can be restored to young mice by treatment with TNF blockers and (2) that the cyclin-dependent kinase inhibitor p21 is a candidate for mediation of the osteoinhibitory effects of TNF. It has been hypothesized recently that TNF antagonists may represent novel anabolic agents, and we believe that the data presented here represent a successful test of this hypothesis.

[Protective effects of rhu TNFR: Fc against the lipopolysaccharide induced intestinal damage of rats and its underlying mechanism].

To investigate the protective effects of recombinant human tumor necrosis factor receptor II: IgG Fc fusion protein (rhu TNFR: Fc) against the lipopolysaccharide (LPS) induced intestinal damage of rats and its underlying mechanism. SD rats were randomly divided into four groups: control group, rhuTNFR: Fc group, LPS group and rhu TNFR: Fc + LPS group. Mean arterial pressure (MAP) was continuously monitored and the mortality rates were assessed. The levels of TNF-alpha and its bioactivity in the serum were assessed by ELISA and flow cytometry respectively. Pathologic changes of intestinal tissue were observed by HE staining. The rats of control and rhu TNFR: Fc group all survived with stable MAP, and the low level and bioactivity of TNF-alpha in the serum were maintained. While 83% of the rats in LPS group died by 6 h with the levels and bioactivity of TNF-alpha increasing significantly. In rhu TNFR: Fc + LPS group, the mortality rate of rats dropped to 33%. The TNF-alpha level increased compared with control group but its bioactivity decreased significantly compared with LPS group. The MPO activity and content of MDA decreased significantly. The status of pathological manifestation in the intestine was also ameliorated. These data suggest that rhu TNFR: Fc could protect rats from the acute intestine injury induced by LPS through ablating the rise in serum TNF-alpha level and bioactivity as well as anti-oxidation.

[Induction of apoptosis by tumor necrosis factor receptors 2 transgene in human laryngeal squamous cacinoma in nude mice animal model].

OBJECTIVE: To investigate the effect of tumor necrosis factor receptors II (TNFR II) in vivo transgene with topical injection of TNF alpha in inducing apoptosis and cell killing of laryngeal squamous carcinoma in nude mice animal model. METHOD: Laryngeal carcinoma implantation animal model was established on nude mice. In vivo gene transfection of TNFR II was carried out using liposome as a carrier. TNF alpha was topically injected into tumor. Goss measurement of tumor, flow cytometry, immunohistochemistry, tunel and transmission electron microscopy were conducted to observe the expression of TNFR II protein and the apoptosis of tumor cells, and the effects of tumor killing and growth inhibition was objectively evaluated. RESULT: Nude mice models bearing laryngeal carcinoma was established in 94.5% animals. After in vivo gene transfection, the expression of TNFR II protein reach the highest level at 48 hours, and remain in a substantially high level within 72 hours. Immunohistochemistry showed the expression of TNFR II is mainly on the cell membrane of the transfected tumor cells. Topical injection of 2000 U TNF alpha was most efficient in inducing tumor cell apoptosis, cell inhibition and cell killing. The tumor volume, weight, and tumor/body ratio in TNFR II transfected group were (1161.333 +/- 166.555) mm3, (1.100 +/- 0.832) g and 0.044 +/- 0.332, respectively, with a corresponding high level of tumor cell apoptosis rate (38.226 +/- 13.671) %, all of which were significantly higher than that in non-transfected group. Tunel and ultrastructural observations demonstrated apoptosis-related changes in the transfected tumor cells. CONCLUSION: Up-regulation of TNFR II expression by in vivo gene transfection on tumor cells can remarkably enhance the tumor cell killing effect of topical injection of TNF-alpha. In vivo transgene of TNFR II in combination with topical injection of TNF alpha may become a effective gene therapy method in treating laryngeal cancer.

Selective stimulation of either tumor necrosis factor receptor differentially induces pain behavior in vivo and ectopic activity in sensory neurons in vitro.

Recent studies suggest that tumor necrosis factor-alpha (TNF) sensitizes primary afferent neurons, and thus facilitates neuropathic pain. Here, we separately examined the roles of tumor necrosis factor receptor (TNFR) 1 and 2 by parallel in vivo and in vitro paradigms using proteins that selectively activate TNFR1 or TNFR2 (R1 and R2). In vivo, intrathecally injected R1, but not R2 slightly reduced mechanical and thermal withdrawal thresholds in rats, whereas co-injection resulted in robust, at least additive pain-associated behavior. In vitro, the electrophysiological responses of dorsal root ganglia (DRG) from rats with spinal nerve ligation were measured utilizing single-fiber recordings of teased dorsal root filaments. In naïve DRG, only R1 (10-1000 pg/ml) induced firing in Ass- and Adelta-fibers, whereas R2 had no effect. In injured DRG, both R1 and R2 at significantly lower concentrations (1 pg/ml) increased discharge rates of Adelta-fibers. Most interesting, in adjacent uninjured DRG, R2 and not R1, increased ectopic activity in both Ass- and Adelta-fibers. We conclude that TNFR1 may be predominantly involved in the excitation of sensory neurons and induction of pain behavior in the absence of nerve injury, TNFR2 may contribute in the presence of TNFR1 activation. Importantly, the effects of individually applied R1 and R2 on injured and adjacent uninjured fibers imply that the role of TNFR2 in the excitation of sensory neurons increases after injury.

The role of soluble TNF receptors for TNF-alpha in uveitis.

PURPOSE: To investigate the presence of soluble tumor necrosis factor receptors (sTNF-Rs) and TNF-alpha in the ocular fluids of patients with uveitis and the capacity of sTNF-Rs to affect TNF-alpha production by intraocular T cells. METHODS: Ocular fluid samples were collected from patients with active and inactive uveitis, as well as from control subjects without uveitis. The sTNF-Rs and TNF-alpha levels were measured by enzyme-linked immunosorbent assay (ELISA). T-cell clones (TCCs) were established from intraocular infiltrating cells, and the TCCs were cocultured with recombinant sTNF-Rs or TNF-alpha. The supernatants were measured by ELISA. The neutralization of sTNF-R production by TCCs was evaluated with an anti-human TNF-R antibody. RESULTS: Significantly higher amounts of sTNF-R1 and -R2 were present in the ocular fluids of patients with active uveitis than in the ocular fluids of those with inactive uveitis and in control subjects. Significantly higher amounts of TNF-alpha were present in the ocular fluids of patients with active uveitis than in those with inactive uveitis. Recombinant sTNF-Rs enhanced TNF-alpha production by TCCs in a dose-dependent manner. Similarly, recombinant TNF-alpha enhanced sTNF-Rs production by the TCCs, and production was neutralized with anti-human TNF-R antibody. CONCLUSIONS: sTNF-Rs are present in the ocular fluids of patients with uveitis. Intraocular levels of sTNF-Rs are significantly increased in patients with uveitis, particularly in those with active uveitis. The data suggest that intraocular sTNF-Rs may play a regulatory role in ocular inflammation such as occurs in uveitis.

Human agonistic antibody to tumor necrosis factor-related apoptosis-inducing ligand receptor 2 induces cytotoxicity and apoptosis in prostate cancer and bladder cancer cells.

OBJECTIVES: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. In this study, we investigated the susceptibility of human prostate cancer and bladder cancer cells to HGS-ETR2, a human monoclonal agonistic antibody specific for TRAIL-R2. METHODS: The cell surface expression of TRAIL-R1 and TRAIL-R2 on prostate cancer and bladder cancer cells was determined using flow cytometry. Cytotoxicity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and caspase activities were measured by a quantitative colorimetric assay. RESULTS: HGS-ETR2 effectively induced apoptotic cell death in DU145, PC3, and LNCaP human prostate cancer cells and J82 and T24 human bladder cancer cells. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell surface expression of the two TRAIL receptors, in that TRAIL-R2, but not TRAIL-R1, was frequently expressed in the prostate cancer and bladder cancer cells. HGS-ETR2 significantly activated the caspase cascade, including caspase-3, -6, -8, and -9, which were the downstream molecules of the death receptors in prostate cancer cells. Caspase-3, -6, and -9 were also significantly activated with HGS-ETR2-induced apoptosis in the bladder cancer cells. CONCLUSIONS: These findings suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent against prostate cancer and bladder cancer.