Human Tumor Necrosis Factor Receptor Research Articles

Cognitive Impact of Neurotropic Pathogens: Investigating Molecular Mimicry through Computational Methods

Neurotropic pathogens, notably, herpesviruses, have been associated with significant neuropsychiatric effects. As a group, these pathogens can exploit molecular mimicry mechanisms to manipulate the host central nervous system to their advantage. Here, we present a systematic computational approach that may ultimately be used to unravel protein–protein interactions and molecular mimicry processes that have not yet been solved experimentally. Toward this end, we validate this approach by replicating a set of pre-existing experimental findings that document the structural and functional similarities shared by the human cytomegalovirus-encoded UL144 glycoprotein and human tumor necrosis factor receptor superfamily member 14 (TNFRSF14). We began with a thorough exploration of the Homo sapiens protein database using the Basic Local Alignment Search Tool (BLASTx) to identify proteins sharing sequence homology with UL144. Subsequently, we used AlphaFold2 to predict the independent three-dimensional structures of UL144 and TNFRSF14. This was followed by a comprehensive structural comparison facilitated by Distance-Matrix Alignment and Foldseek. Finally, we used AlphaFold-multimer and PPIscreenML to elucidate potential protein complexes and confirm the predicted binding activities of both UL144 and TNFRSF14. We then used our in silico approach to replicate the experimental finding that revealed TNFRSF14 binding to both B- and T-lymphocyte attenuator (BTLA) and glycoprotein domain and UL144 binding to BTLA alone. This computational framework offers promise in identifying structural similarities and interactions between pathogen-encoded proteins and their host counterparts. This information will provide valuable insights into the cognitive mechanisms underlying the neuropsychiatric effects of viral infections.Graphical

Efficacy and safety of tripterygium wilfordii Hook F plus TNF inhibitor for active rheumatoid arthritis: A multicentre, randomized, double-blind, triple-dummy controlled trial

An investigator-initiated, multicentre, randomized, double-blind, triple-dummy, controlled trial was conducted at 14 tertiary rheumatology centers in China to evaluate the efficacy and safety of Tripterygium wilfordii Hook F (TwHF) with recombinant human TNF receptor IgGFc fusion protein (rhTNFR-Fc) in active Rheumatoid Arthritis (RA). Primary endpoint was the proportion of patients achieved a 50% improvement of American College of Rheumatology criteria (ACR50) in TwHF+rhTNFR-Fc vs. methotrexate (MTX) group at week 12. ACR50 was achieved in 57.1% (72/126), 41.3% (52/126), 23.0% (29/126), and 26.2% (33/126) patients receiving TwHF+rhTNFR-Fc, MTX + rhTNFR-Fc, TwHF and MTX monotherapy, respectively, at week 12 (TwHF+rhTNFR-Fc vs. other three groups, all p < 0.05). No statistical difference in serious adverse events or adverse events leading to discontinuation of study across all groups was documented. TwHF+rhTNFR-Fc was superior to MTX for active RA, and was more effective than MTX + rhTNFR-Fc on ACR50, with a similar safety profile.Trial registration:ClinicalTrials.govNCT03589833.

Deletion of the non-adjacent genes UL148 and UL148D impairs human cytomegalovirus-mediated TNF receptor 2 surface upregulation.

Human cytomegalovirus (HCMV) is a prototypical β-herpesvirus which frequently causes morbidity and mortality in individuals with immature, suppressed, or senescent immunity. HCMV is sensed by various pattern recognition receptors, leading to the secretion of pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα). TNFα binds to two distinct trimeric receptors: TNF receptor (TNFR) 1 and TNFR2, which differ in regard to their expression profiles, affinities for soluble and membrane-bound TNFα, and down-stream signaling pathways. While both TNF receptors engage NFκB signaling, only the nearly ubiquitously expressed TNFR1 exhibits a death domain that mediates TRADD/FADD-dependent caspase activation. Under steady-state conditions, TNFR2 expression is mainly restricted to immune cells where it predominantly submits pro-survival, proliferation-stimulating, and immune-regulatory signals. Based on the observation that HCMV-infected cells show enhanced binding of TNFα, we explored the interplay between HCMV and TNFR2. As expected, uninfected fibroblasts did not show detectable levels of TNFR2 on the surface. Intriguingly, however, HCMV infection increased TNFR2 surface levels of fibroblasts. Using HCMV variants and BACmid-derived clones either harboring or lacking the ULb' region, an association between TNFR2 upregulation and the presence of the ULb' genome region became evident. Applying a comprehensive set of ULb' gene block and single gene deletion mutants, we observed that HCMV mutants in which the non-adjacent genes UL148 or UL148D had been deleted show an impaired ability to upregulate TNFR2, coinciding with an inverse regulation of TACE/ADAM17.

Carotenoids as potential inhibitors of TNFα in COVID-19 treatment.

Tumor necrosis factor-alpha (TNF-α) is a multifunctional pro-inflammatory cytokine, responsible for autoimmune and inflammatory disorders. In COVID-19 patients, increased TNF-α concentration may provoke inflammatory cascade and induce the initiation of cytokine storm that may result in fatal pneumonia and acute respiratory distress syndrome (ADRS). Hence, TNFα is assumed to be a promising drug target against cytokine storm in COVID-19 patients. In the present study, we focused on finding novel small molecules that can directly block TNF-α-hTNFR1 (human TNF receptor 1) interaction. In this regards, TNF-α-inhibiting capacity of natural carotenoids was investigated in terms of blocking TNF-α-hTNFR1 interaction in COVID-19 patients with the help of a combination of in silico approaches, based on virtual screening, molecular docking, and molecular dynamics (MD) simulation. A total of 125 carotenoids were selected out of 1204 natural molecules, based on their pharmacokinetics properties and they all met Lipinski's rule of five. Among them, Sorgomol, Strigol and Orobanchol had the most favorable ΔG with the best ADME (absorption, distribution, metabolism, excretion) properties, and were selected for MD simulation studies, which explored the complex stability and the impact of ligands on protein conformation. Our results showed that Sorgomol formed the most hydrogen bonds, resulting in the highest binding energy with lowest RMSD and RMSF, which made it the most appropriate candidate as TNF-α inhibitor. In conclusion, the present study could serve to expand possibilities to develop new therapeutic small molecules against TNF-α.

Diagnosis and treatment of 10 cases of idiopathic chondrolysis of the hip.

To analyze the clinical characteristics, X-ray and magnetic resonance imaging manifestations, and treatment efficacy of idiopathic chondrolysis of the hip. Ten patients with idiopathic chondrolysis of the hip treated at our hospital from September 2013 to April 2022 were collected, and their clinical features, X-ray and magnetic resonance imaging manifestations, and treatment outcomes were analyzed. Their main clinical features included single hip pain, claudication gait, and pelvic tilt, without specific clinical symptoms and signs. Laboratory tests such as blood analysis, erythrocyte sedimentation rate, C-reactive protein, rheumatoid factor, autoimmune test, coagulation function, interferon release test for tuberculosis infection, and tuberculosis antibody test were normal. In the late stage, the imaging shows degeneration or ankylosis of the joint. The disease progresses rapidly, and joint degeneration can occur in adolescence without effective treatment. Herein, seven patients were treated with recombinant human tumor necrosis factor receptor-antibody type II fusion protein. Among them, four (according to Camarnat magnetic resonance imaging classification, three were Stage I and one was Stage II) showed improved symptoms and function, while two (Stage III) had progressed. One patient (Stage I) who received recombinant human tumor necrosis factor receptor had no significant relief of symptoms, for whom anterior capsular release surgery significantly improved the hip joint motion. Of the four patients who underwent hip release surgery, three showed progression. Idiopathic chondrolysis of the hip has no specific clinical and laboratory tests and a high misdiagnosis rate. Thus, early magnetic resonance imaging is a critical reference for diagnosis. We recommend that patients with magnetic resonance imaging Stage II or earlier be actively treated with recombinant human tumor necrosis factor receptor and start treatment as early as possible. In the third stage, the treatment effect is mediocre, and the narrowed hip space is difficult to change.

Production of recombinant human tumor necrosis factor receptor- IgG1 Fc domain fusion protein expressed by genetically CHO- DG44 cells

BACKGROUND: TNF-α (Tumor necrosis factor-alpha) plays a vital role in the human immune system. However, abnormal activity and overexpression of TNF-α are some of the causes of allergic and autoimmune diseases. Inhibiting the activity of this molecule is one of the novel pathologies for these diseases. The development of this recombinant protein is expected to reduce the financial burden of treating inflammatory rheumatic diseases. OBJECTIVE: The study’s objective was to generate and evaluate the biological activity of TNFR-Fc, construct of fusions an extracellular part TNF-α receptor (p75) and an Fc fragment of human immunoglobulin G1, expressed from the CHO-DG44 cell system. METHODS: The recombinant TNFR- Fc plasmid was constructed and identified by PCR, restriction enzyme digestion, and sequencing. A stable cell line for expression of TNFR-Fc was selected by limiting dilution cloning. Structural characterization, the binding affinity of TNFR-Fc to TNF-α, the neutralizing the cytotoxic activity- induced TNF-α, and the TNF-α-induced apoptosis suppression of TNFR- Fc were analyzed by SDS/PAGE Western blotting, ELISA, WST assay, Immunofluorescence, and flow cytometry. RESULTS: Preliminary analysis of the structural characteristics showed that TNFR-Fc is a low- glycosylated protein and perhaps in dimeric form. Furthermore, the recombinant TNFR-Fc can interact with its ligand TNF-α with a dissociation constant Kd 0.25±0.03μM equivalent to that of the original drug, Etanercept. We also demonstrated that TNFR-Fc expressed from CHO-DG44 was able to neutralize TNF-α- induced cytotoxic activity and inhibited p53-related apoptosis in vitro, similar to Etanercept. CONCLUSIONS: These data collectively suggested that TNFR-Fc potently blocks TNF-α, which could be a novel therapeutic strategy for cytokine-driven diseases.

Verloren negatively regulates the expression of IMD pathway dependent antimicrobial peptides in Drosophila

Drosophila immune deficiency (IMD) pathway is similar to the human tumor necrosis factor receptor (TNFR) signaling pathway and is preferentially activated by Gram-negative bacterial infection. Recent studies highlighted the importance of IMD pathway regulation as it is tightly controlled by numbers of negative regulators at multiple levels. Here, we report a new negative regulator of the IMD pathway, Verloren (Velo). Silencing of Velo led to constitutive expression of the IMD pathway dependent antimicrobial peptides (AMPs), and Escherichia coli stimulation further enhanced the AMP expression. Epistatic analysis indicated that Velo knock-down mediated AMP upregulation is dependent on the canonical members of the IMD pathway. The immune fluorescent study using overexpression constructs revealed that Velo resides both in the nucleus and cytoplasm, but the majority (~ 75%) is localized in the nucleus. We also observed from in vivo analysis that Velo knock-down flies exhibit significant upregulation of the AMP expression and reduced bacterial load. Survival experiments showed that Velo knock-down flies have a short lifespan and are susceptible to the infection of pathogenic Gram-negative bacteria, P. aeruginosa. Taken together, these data suggest that Velo is an additional new negative regulator of the IMD pathway, possibly acting in both the nucleus and cytoplasm.

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白联合糖皮质激素治疗急性泛发性发疹性脓疱病一例

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白联合糖皮质激素治疗急性泛发性发疹性脓疱病一例

Recombinant expression, purification and characterization of human soluble tumor necrosis factor receptor 2

TNFR2 is aberrantly expressed on various cancer cells and highly immunosuppressive regulatory T cells (Tregs) accumulated in tumor microenvironment. As an oncoprotein and a stimulator of the immune checkpoint Tregs that promote cancer cell survival and tumor growth, TNFR2 is considered to be a prospective target for cancer immunotherapy with the blockers developed to simultaneously inhibit abundant TNFR2+ tumor-associated Tregs and directly kill TNFR2-expressing tumors. The soluble ectodomain of TNFR2 has also been successfully applied in clinical treatment for TNF-related autoimmune diseases. Research practices on these therapeutic strategies need recombinant protein of human soluble TNFR2 (hsTNFR2); however, mass production of such biologics using eukaryotic cells is generally high-cost in culture materials and growth conditions. This study aimed to establish an efficient methodology to prepare bioactive hsTNFR2 through a prokaryotic expression system. Recombinant vector pMCSG7-hsTNFR2 was constructed and the His-tagged fusion protein expressed in E. coli was enriched in inclusion bodies. Recombinant hsTNFR2 was denatured, refolded, and then purified by affinity chromatography, tag removal, ion-exchange chromatography and gel filtration chromatography. A protein yield of 8.4 mg per liter of bacterial culture liquid with a purity of over 97% was obtained. Purified hsTNFR2 exhibited strong affinity to human TNF-α with a KD of 10.5 nM, and inhibited TNF-α-induced cytotoxicity in L929 cells with an EC50 of 0.57 μg/ml. The biological activity assessed in vitro indicated that this soluble protein can be promisingly used in drug discovery for immunotherapy of TNFR2+ cancers and treatment of autoimmune diseases featured by TNF-α overload.

Effect of a Recombinant Dimeric Tumor Necrosis Factor Receptor on Inflammatory Responses to Intravenous Endotoxin in Normal Humans

AbstractTo determine the role of tumor necrosis factor (TNF ) in lipopolysaccharide (LPS)-induced inflammation, 12 healthy subjects received an intravenous injection with LPS (2 ng/kg) preceded by infusion of either a recombinant human dimeric TNF receptor type II-IgG fusion protein (TNFR:Fc; 6 mg/m2; n = 6) or vehicle (n = 6) from −30 minutes to directly before LPS injection. LPS elicited a transient increase in plasma TNF activity, peaking after 1.5 hours (219 ± 42 pg/mL; P < .05). Infusion of TNFR:Fc completely neutralized endogenous TNF activity. LPS administration was associated with an early activation of fibrinolysis (plasma concentrations of tissue-type plasminogen activator, plasminogen activator activity, and plasmin-α2 -antiplasmin complexes), followed by inhibition (plasma plasminogen activator inhibitor type I), changes that were completely prevented by TNFR:Fc. By contrast, TNFR:Fc did not influence LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1 + 2 and thrombin-antithrombin III complexes). TNFR:Fc strongly inhibited endothelial cell activation (plasma levels of soluble E-selectin), modestly reduced neutrophil responses (neutrophilia and plasma concentrations of elastase-α1 -antitrypsin complexes and lactoferrin), but did not affect the release of secretory phospholipase A2 or lipopolysaccharide-binding protein (P < .05). Infusion of TNFR:Fc only (without LPS) in another 6 normal subjects did not induce any inflammatory response. These data indicate that TNF is involved in only some inflammatory responses to intravenous LPS in humans.

RELT stains prominently in B-cell lymphomas and binds the hematopoietic transcription factor MDFIC

Receptor Expressed in Lymphoid Tissues (RELT) is a human tumor necrosis factor receptor superfamily member (TNFRSF) that is expressed most prominently in cells and tissues of the hematopoietic system. RELL1 and RELL2 are two homologs that physically interact with RELT and co-localize with RELT at the plasma membrane. This study sought to further elucidate the function of RELT by identifying novel protein interactions with RELT family members. The transcription factor MyoD family inhibitor domain-containing (MDFIC) was identified in a yeast two-hybrid genetic screen using RELL1 as bait. MDFIC co-localizes with RELT family members at the plasma membrane; this co-localization was most prominently observed with RELL1 and RELL2. In vitro co-immunoprecipitation (Co-IP) was utilized to demonstrate that MDFIC physically interacts with RELT, RELL1, and RELL2. Co-IP using deletion mutants of MDFIC and RELT identified regions important for physical association between MDFIC and RELT family members and a computational analysis revealed that RELT family members are highly disordered proteins. Immunohistochemistry of normal human lymph nodes revealed RELT staining that was most prominent in macrophages. Interestingly, the level of RELT staining significantly increased progressively in low and high-grade B-cell lymphomas versus normal lymph nodes. RELT co-staining with CD20 was observed in B-cell lymphomas, indicating that RELT is expressed in malignant B cells. Collectively, these results further our understanding of RELT-associated signaling pathways, the protein structure of RELT family members, and provide preliminary evidence indicating an association of RELT with B-cell lymphomas.

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

Metabolism Balance Regulation via Antagonist‐Functionalized Injectable Microsphere for Nucleus Pulposus Regeneration

AbstractAntagonist therapy represents a potential treatment for extracellular matrix (ECM) metabolic imbalance via the specific binding of inflammatory factors resulting from inflammation. However, the short half‐life of antagonist bioactivity creates challenges for their clinical application. Herein, bovine serum albumin nanoparticles (BNP) encapsulating recombinant human soluble tumor necrosis factor (TNF) receptor type II (rhsTNFRII) are grafted onto microfluidic poly(l‐lactic acid) (PLLA) porous microspheres through chemical bonds, constructing antagonist‐functionalized injectable porous microspheres (MS‐BNP) for in situ injection into the nucleus pulposus (NP), aimed at regulating the metabolic balance of ECM, thus inhibiting intervertebral disc degeneration. Several binding sites within the BNPs improve encapsulation efficiency, promote the sustained release of rhsTNFRII, and regulate ECM metabolism in the NP. Moreover, PLLA porous microspheres display excellent injectability and porosity and demonstrate efficient and uniform loading of nanoparticles through chemical grafting. By delivering MS‐BNP into the NP, a suitable environment is created in situ. Immunohistochemical analysis at 4 and 8 weeks shows that compared with other experimental groups, the expression of TNF‐α is significantly inhibited for 6.11–15.65 folds and 4.59–22.14 folds, respectively, and a significant regeneration in NP occurred. This work proposes a novel porous microsphere therapy functionalized by antagonist molecules for the treatment of ECM metabolic disorders, caused by chronic inflammatory responses.

RELL1 inhibits autophagy pathway and regulates Mycobacterium tuberculosis survival in macrophages

Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). It leads to approximately 1 million deaths annually. Receptor expressed in lymphoid tissues-like protein 1 (RELL1) is a homologous binding partner of receptor expressed in lymphoid tissues (RELT), member of the human tumor necrosis factor receptor family. Genome-wide analysis screening revealed that downregulation of RELL1 in macrophages notably reduces Mtb survival within macrophages. However, the underlying mechanism is not clear. Here, we show that RELL1 expression in macrophages significantly decreased upon Mtb infection. Mtb survival increased in RAW264.7 cells with upregulated RELL1 expression. However, the proinflammatory cytokines TNF-α and IL-6 responsible for Mtb clearance were increased. Further, RELL 1 enhanced mTOR activity and inhibited autophagy through direct interaction. Hence, the reduced autophagy may antagonize increased inflammation in RELL1 upregulated macrophages and promote Mtb survival in macrophages. Together, our results suggest that the reduction of RELL1 expression upon Mtb infection may enhance autophagy and facilitate bacterial clearance, providing a new target for Mtb treatment.

Addition of a Viral Immunomodulatory Domain to Etanercept Generates a Bifunctional Chemokine and TNF Inhibitor.

The inhibition of tumor necrosis factor (TNF) through the use of either antibodies or soluble receptors is a highly effective strategy for the clinical control of chronic inflammatory conditions such as rheumatoid arthritis. Different viruses have similarly exploited this concept by expressing a set of specifically tailored secreted TNF decoy receptors to block host inflammatory responses. Poxviruses have been shown to encode at least two distinct molecules, termed Cytokine response modifier D (CrmD) and CrmB, in which a TNF inhibitor is combined with a chemokine inhibitor on the same molecule. The ectromelia virus CrmD protein was found to be a critical determinant of virulence in vivo, being able to control local inflammation to allow further viral spread and the establishment of a lethal infection. Strikingly, both the TNF and the chemokine inhibitory domains are required for the full activity of CrmD, suggesting a model in which inhibition of TNF is supported by the concomitant blockade of a reduced set of chemokines. Inspired by this model, we reasoned that a similar strategy could be applied to modify the clinically used human TNF receptor (etanercept), producing a generation of novel, more effective therapeutic agents. Here we show the analysis of a set of fusion proteins derived from etanercept by addition of a viral chemokine-binding protein. A bifunctional inhibitor capable of binding to and blocking the activity of TNF as well as a set of chemokines is generated that is active in the prevention of arthritis in a murine disease model.

Plasmatic Extracellular Vesicles in Acute Graft-Versus-Host Disease after Haplo-Identical Allografting with Post-Transplant Cyclophosphamide

INTRODUCTION: Acute Graft-versus-Host-Disease (aGVHD) is a frequent complication where the endothelium may play a pivotal role. We recently investigated the potential role of extracellular vesicles (EVs) as novel biomarkers of aGVHD (Lia G. et al. Leukemia 2018). In this study we further investigated the correlation of plasma EVs and their content in miRNAs with the risk of developing aGVHD in the setting of post-transplant cyclophosphamide (PTCY) haploidentical-stem cell transplantation (Haplo-SCT). METHODS: Thirty-two patients who underwent a Haplo-SCT were included. Plasma samples were collected from peripheral blood at given time-points (pre-transplant, on day 0, 3, 7, 14, 21, 28, 35, 45, 60, 75 and 90 after transplant). EVs were extracted by a protamine-based precipitation method and were characterized by Nano-tracking Particle Analysis (Nanosight). EVs were then analyzed by flow-cytometry (Guava EasyCyte Flow Cytometer) with a panel of 14 antibodies (CD44, CD138, CD146, KRT18, CD120a, CD8, CD30, CD106, CD25, CD26, CD31, CD144, CD86, and CD140a). MiRNAs were extracted from EVs by miRNeasy Mini Kit (Qiagen) and retrotranscribed by miScript II RT Kit (Qiagen). Three miRNAs (miR100, miR194, miR155) were studied and quantified by qRT-PCR using the miScript SYBR Green PCR Kit (Qiagen). Concomitant plasma concentrations of human Tumor Necrosis Factor Receptor I (TNFR1) and human ST2 were also evaluated using a commercially available sandwich enzyme-linked immunosorbent assay (DualSET® ELISA R&amp;D Systems). The risk of aGVHD was evaluated by logistic regression models and Odds Ratios (ORs) were estimated as absolute levels and as proportional changes compared with pre-transplant baseline levels of each marker. Moreover, among biomarkers significantly associated with a higher risk of aGVHD, a multivariable logistic regression model using Akaike's information criteria (AIC) was estimated to define a biomarker combination. Ors were reported for 1-unit increase of standardized variables. RESULTS: AGVHD (grade II-IV) was observed in 7/32 patients (22%) at a median of 41 (range 33-90) days after transplant. Logistic regression models showed that CD146 fluorescence was associated with a significantly increased risk of acute GVHD (OR 2.93 p&amp;lt;0.001) as well as expression changes in miR100, miR155 and miR194 (OR 3.90 p&amp;lt;0.001; OR 1.84 p=0.008; OR 2.68 p&amp;lt;0.001, respectively). Concentrations of plasmatic hTNFR1 and ST2 were also confirmed to be associated with increased risk of aGVHD (OR 1.47 p=0.04; OR 1.55 p=0.05, respectively) as previously described. Of note, all biomarkers associated with risk of aGVHD showed a consensual change in signal levels before the onset of aGVHD (Figure 1). By applying a backward selection on a multivariable logistic model using the AICapproach, we found that the combination of CD146-miR100-TNFR1 with an individual AUROC of 0.858, 0.923, and 0.794, respectively, increased their discrimination ability to predict aGVHD (multivariable AUROC = 0.987). CONCLUSIONS: This study, in the setting of haplo-transplant, confirms the association of CD146, a cell adhesion molecule, and the risk of aGVHD suggesting an important role of endothelium damage in the pathogenesis of aGVHD. The association of miRNA100, miRNA155 and miRNA194, carried by EVs, and aGVHD was also significant. Interestingly, MiRNA100 was reported to regulate inflammatory neovascularization during GvHD while miR-155 plays a role in donor T cell expansion. We have also found that using three markers in combination (CD146-miR100-TNFR1) could greatly improve aGVHD predictivity. To translate our results into an in vivo model, we have recently designed preclinical mouse models to evaluate if a) antagomir (against miRNA100 and/or miRNA155) injections or b) pre-emptive treatments with endothelium protective agents such as defibrinotide or OMS721 (Anti-Masp2) may reduce the risk of aGVHD. Figure1 a) Signal variation from baseline level (preTx) of CD146 fluorescence, miR100 expression, and TNFR1 concentration before aGVHD onset. Disclosures Boccadoro: Sanofi: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

PMS13 EXPERIMENTAL STUDY OF TOPICAL TREATMENT OF RHEUMATOID ARTHRITIS WITH TNF-Α ANTAGONIST

To observe the clinical efficacy of intra-articular injection of recombinant human tumor necrosis factor receptor type II antibody fusion protein in patients with rheumatoid arthritis (RA). (1)Case selection: 60 patients with RA were randomly divided into experimental group and control group. (2) In the experimental group, 25 mg of recombinant human tumor necrosis factor receptor-antibody fusion protein was injected into each joint; in the control group, the same dose of the same drug was injected subcutaneously each time. (3) The content of MMP-3 was determined by enzyme-linked immunosorbent assay (ELISA). Clinical indicators were collected: ESR, CRP, PLT, and joint color Doppler ultrasonography. Synovial thickness and fluid depth of knee joint. After 2 weeks of treatment, ESR, CRP and PLT in the experimental group were lower than those in the control group, with no significant difference (P > 0.05). The synovial thickness and liquid depth in the experimental group were significantly lower than those in the control group (P < 0.05). After 2 weeks of treatment, the content of MMP-3 in the joint fluid of both groups decreased significantly, compared with the control group (P < 0.05). Intra-articular injection of recombinant human tumor necrosis factor receptor-antibody fusion protein can significantly reduce synovial thickness, fluid depth, and the content of MMP-3 in synovial fluid, which is an effective method for the treatment of rheumatoid arthritis.

P29 Etanercept and IBD: can we identify at risk patients?

Poster presentation Tuesday 8 October Background We present four cases of patients on etanercept treatment for juvenile idiopathic arthritis (JIA) subsequently diagnosed with inflammatory bowel disease (IBD). We hypothesise that there may be identifiable characteristics of these patients which makes them more identifiable to the physician. Etanercept is a recombinant dimer of human tumour necrosis factor (TNF) receptor proteins bound to human IgG1. It is an effective second line treatment option in JIA for those who have failed, or are intolerant to, methotrexate, and an effective first line treatment option in those who have axial disease at diagnosis. Anti-TNF treatments are effective in IBD, however etanercept in particular does not have therapeutic benefit, and in one trial there was a non-significant trend towards worsening of disease. Methods Please refer to the Results section. Results Four cases, in a 15 year old male, 11 year old female, 10 year old female and 11 year old female, have been identified at our site. The time from etanercept initiation and diagnosis of IBD was between 2 weeks and 2 years 1 month. None of the cases had gastrointestinal symptoms at JIA diagnosis. All four were ANA negative, and in the two with information, both were HLA-B27 negative. Three out of four had hip arthritis involvement. There was a family history of IBD (ulcerative colitis) in only one of the cases. Aside from non-steroidal anti-inflammatory medications, there were no other immunomodulatory medications concurrently being taken in any of the cases while on etanercept. All four were anaemic (Hb &lt; 120g/L) at diagnosis of IBD. Only one had raised ESR (43mm/hr) while all had normal CRP. Albumin was low in two (17g/L and 29g/L). Following gastroenterology review and endoscopy, two cases were diagnosed with Crohn’s disease and two with ulcerative colitis. Conclusion It remains unclear whether etanercept plays a role in the development of IBD in these patients, whether it unmasks subclinical disease, or whether there is no association and it is simply a coincidence. However, there continue to be case reports and series suggesting an association between the two. We hope to identify further cases similar to these from sources such as the UK Biologics Registry and via surveys from teams at paediatric rheumatology centres throughout the UK. Further to the identification of cases, a retrospective review of patient characteristics will be carried out. This may allow for further determination of identifiable characteristics for these patients, and therefore suggest a subgroup of JIA patients with a higher risk of developing IBD, in which case caution when considering etanercept treatment would be advised. Conflicts of Interest The authors declare no conflicts of interest.

Tumor Necrosis Factor-Alpha Antagonist Interferes With the Formation of Granulomatous Multinucleated Giant Cells: New Insights Into Mycobacterium tuberculosis Infection.

More than half of tuberculosis cases in the world are due to resuscitation of dormant Mycobacterium tuberculosis (Mtb) sequestered into cell-derived structures called granulomas. It is fairly admitted that cytokines and more particularly Tumor Necrosis Factor (TNF)-α is critical in the control of Mtb infections and that anti-TNF-α drugs constitute one of the main risk factors for reactivation of latent Mtb infection. The aim of this study was to evaluate the role of etanercept, a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human p75 TNF receptor linked to the Fc portion of human IgG1, in an in vitro model of human tuberculous granuloma. We showed that etanercept slightly delayed the formation of granuloma and reduced the generation of multinuclear giant cells (MGCs). In addition, etanercept exacerbated the expression of M1 polarization genes but also induced interleukin (IL)-10 release. In addition, our results indicated that etanercept inhibited cell fusion in an IL-10-dependent manner. Moreover, adalimumab, a human monoclonal anti-TNF-α IgG1 inhibited MGC formation in granuloma, without altering IL-10 secretion and induced macrophage apoptosis. Taken together, our data provides new insights into the role of TNF-α blockers in MGCs formation and the impact of such immunomodulatory drugs on tuberculous granuloma maturation.

HERA-GITRL activates T cells and promotes anti-tumor efficacy independent of Fc\u03b3R-binding functionality

BackgroundGlucocorticoid-induced TNFR-related protein (TNFRSF18, GITR, CD357), expressed by T cells, and its ligand (TNFSF18, GITRL), expressed by myeloid populations, provide co-stimulatory signals that boost T cell activity. Due to the important role that GITR plays in regulating immune functions, agonistic stimulation of GITR is a promising therapeutic concept. Multiple strategies to induce GITR signaling have been investigated. The limited clinical efficacy of antibody-based GITR agonists results from structural and functional characteristics of antibodies that are unsuitable for stimulating the well-defined trimeric members of the TNFRSF.MethodsTo overcome limitations of antibody-based TNFRSF agonists, we have developed HERA-GITRL, a fully human hexavalent TNF receptor agonist (HERA) targeting GITR and mimicking the natural signaling concept. HERA-GITRL is composed of a trivalent but single-chain GITRL-receptor-binding-domain (scGITRL-RBD) unit fused to an IgG1 derived silenced Fc-domain serving as dimerization scaffold. A specific mouse surrogate, mmHERA-GITRL, was also generated to examine in vivo activity in respective mouse tumor models.ResultsFor functional characterization of HERA-GITRL in vitro, human immune cells were isolated from healthy-donor blood and stimulated with anti-CD3 antibody in the presence of HERA-GITRL. Consistently, HERA-GITRL increased the activity of T cells, including proliferation and differentiation, even in the presence of regulatory T cells.In line with these findings, mmHERA-GITRL enhanced antigen-specific clonal expansion of both CD4+ (OT-II) and CD8+ (OT-I) T cells in vivo while having no effect on non-specific T cells. In addition, mmHERA-GITRL showed single-agent anti-tumor activity in two subcutaneous syngeneic colon cancer models (CT26wt and MC38-CEA). Importantly, this activity is independent of its FcγR-binding functionality, as both mmHERA-GITRL with a functional Fc- and a silenced Fc-domain showed similar tumor growth inhibition.Finally, in a direct in vitro comparison to a bivalent clinical benchmark anti-GITR antibody and a trivalent GITRL, only the hexavalent HERA-GITRL showed full biological activity independent of additional crosslinking.ConclusionIn this manuscript, we describe the development of HERA-GITRL, a true GITR agonist with a clearly defined mechanism of action. By clustering six receptor chains in a spatially well-defined manner, HERA-GITRL induces potent agonistic activity without being dependent on additional FcγR-mediated crosslinking.