A Double-Humanized Mouse Model for Studying Host Gut Microbiome-Immune Interactions in Gulf War Illness.

Unraveling the multisymptomatic Gulf War Illness (GWI) pathology and finding an effective cure have eluded researchers for decades. The chronic symptom persistence and limitations for studying the etiologies in mouse models that differ significantly from those in humans pose challenges for drug discovery and finding effective therapeutic regimens. The GWI exposome differs significantly in the study cohorts, and the above makes it difficult to recreate a model closely resembling the GWI symptom pathology. We have used a double engraftment strategy for reconstituting a human immune system coupled with human microbiome transfer to create a humanized-mouse model for GWI. Using whole-genome shotgun sequencing and blood immune cytokine enzyme linked immunosorbent assay (ELISA), we show that our double humanized mice treated with Gulf War (GW) chemicals show significantly altered gut microbiomes, similar to those reported in a Veteran cohort of GWI. The results also showed similar cytokine profiles, such as increased levels of IL-1ß, IL-6, and TNF R-1, in the double humanized model, as found previously in a human cohort. Further, a novel GWI Veteran fecal microbiota transfer was used to create a second alternative model that closely resembled the microbiome and immune-system-associated pathology of a GWI Veteran. A GWI Veteran microbiota transplant in humanized mice showed a human microbiome reconstitution and a systemic inflammatory pathology, as reflected by increases in interleukins 1ß, 6, 8 (IL-1ß, IL-6, IL-8), tumor necrosis factor receptor 1 (TNF R-1), and endotoxemia. In conclusion, though preliminary, we report a novel in vivo model with a human microbiome reconstitution and an engrafted human immune phenotype that may help to better understand gut-immune interactions in GWI.

Functional analysis of a novel G87V TNFRSF1A mutation in patients with TNF receptor-associated periodic syndrome.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease that is caused by heterozygous mutations in the TNFRSF1A gene. Although more than 150 TNFRSF1A mutations have been reported to be associated with TRAPS phenotypes only a few, such as p.Thr79Met (T79M) and cysteine mutations, have been functionally analyzed. We identified two TRAPS patients in one family harboring a novel p.Gly87Val (G87V) mutation in addition to a p.Thr90Ile (T90I) mutation in TNFRSF1A. In this study, we examined the functional features of this novel G87V mutation. In-vitro analyses using mutant TNF receptor 1 (TNF-R1)-over-expressing cells demonstrated that this mutation alters the expression and function of TNF-R1 similar to that with the previously identified pathogenic T79M mutation. Specifically, cell surface expression of the mutant TNF-R1 in transfected cells was inhibited with both G87V and T79M mutations, whereas the T90I mutation did not affect this. Moreover, peripheral blood mononuclear cells (PBMCs) from TRAPS patients harboring the G87V and T90I mutations showed increased mitochondrial reactive oxygen species (ROS). Furthermore, the effect of various Toll-like receptor (TLR) ligands on inflammatory responses was explored, revealing that PBMCs from TRAPS patients are hyper-responsive to TLR-2 and TLR-4 ligands and that interleukin (IL)-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are likely to be involved in the pathogenesis of TRAPS. These findings suggest that the newly identified G87V mutation is one of the causative mutations of TRAPS. Our findings based on unique TRAPS-associated mutations provide novel insight for clearer understanding of inflammatory responses, which would be basic findings of developing a new therapeutic and prophylactic approach to TRAPS.

Palmitoylation is required for TNF-R1 signaling.

BACKGROUND: Binding of tumor necrosis factor (TNF) to TNF-receptor 1 (TNF-R1) can induce either cell survival or cell death. The selection between these diametrically opposed effects depends on the subcellular location of TNF-R1: plasma membrane retention leads to survival, while endocytosis leads to cell death. How the respective TNF-R1 associated signaling complexes are recruited to the distinct subcellular location is not known. Here, we identify palmitoylation of TNF-R1 as a molecular mechanism to achieve signal diversification. METHODS: Human monocytic U937 cells were analyzed. Palmitoylated proteins were enriched by acyl resin assisted capture (AcylRAC) and analyzed by western blot and mass spectrometry. Palmitoylation of TNF-R1 was validated by metabolic labeling. TNF induced depalmitoylation and involvement of APT2 was analyzed by enzyme activity assays, pharmacological inhibition and shRNA mediated knock-down. TNF-R1 palmitoylation site analysis was done by mutated TNF-R1 expression in TNF-R1 knock-out cells. Apoptosis (nuclear DNA fragmentation, caspase 3 assays), NF-κB activation and TNF-R1 internalization were used as biological readouts. RESULTS: We identify dynamic S-palmitoylation as a new mechanism that controls selective TNF signaling. TNF-R1 itself is constitutively palmitoylated and depalmitoylated upon ligand binding. We identified the palmitoyl thioesterase APT2 to be involved in TNF-R1 depalmitoylation and TNF induced NF-κB activation. Mutation of the putative palmitoylation site C248 interferes with TNF-R1 localization to the plasma membrane and thus, proper signal transduction. CONCLUSIONS: Our results introduce palmitoylation as a new layer of dynamic regulation of TNF-R1 induced signal transduction at a very early step of the TNF induced signaling cascade. Understanding the underlying mechanism may allow novel therapeutic options for disease treatment in future.

Venous and arterial TNF-R1 predicts outcome and complications in acute subarachnoid hemorrhage.

BACKGROUND: There is increasing evidence for the role of inflammation in clinical outcome after subarachnoid hemorrhage (SAH). Specifically, the TNF-alfa(α) pathway seems to be relevant after SAH. Although the TNF-α main receptor, TNF-R1 is associated with aneurysm growth and rupture, its relation to prognosis is unknown. We sought to compare TNF-R1 levels in peripheral venous blood and arterial blood closer to the ruptured aneurysm to study the association of TNF-R1 blood levels with poor prognosis (modified Rankin Scale > 2 at discharge, 3 and 6 months) and complications (hydrocephalus or delayed cerebral ischemia/DCI) following SAH. METHODS: We included consecutive SAH patients admitted in the first 72 h of symptoms. Blood samples were simultaneously collected from a peripheral vein and from the main parent artery of the aneurysm. Levels of TNF-R1 were measured using enzyme-linked immunosorbent assays. RESULTS: We analyzed 58 patients. Arterial and venous levels of TNF-R1 were correlated (R = 0.706, p < 0.001). In multivariate regression analysis, venous TNF-R1 was an independent predictor of poor outcome at 6 months after adjusting by age and sex [odds ratio (OR) 11.63; 95% CI 2.09-64.7, p = 0.005] and after adjusting by Glasgow Coma Scale and Fisher scales (OR 8.74; 95% CI 1.45-52.7, p = 0.018). There was no association of TNF-R1 with DCI. A cut-off for arterial TNF-R1 of 1523.7 pg/mL had 75% sensitivity/66% specificity for the prediction of hydrocephalus. CONCLUSION: Levels of venous TNF-R1 are associated with poor outcome in SAH. A specific association was found between levels of arterial TNF-R1 and hydrocephalus. These results are consistent with the role of TNF-α pathway in SAH and need to be validated in larger cohorts.

TNF-R1 and FADD mediate UVB-Induced activation of K+ channels in corneal epithelial cells.

The goal of this study was to elucidate the role of Fas, TNF-R1, FADD and cytochrome c in UVB-induced K+ channel activation, an early step in UVB-induced apoptosis, in human corneal limbal epithelial (HCLE) cells. HCLE cells were treated with Fas, TNF-R1 or FADD siRNA and exposed to 80 or 150 mJ/cm2 UVB. K+ channel activation and loss of intracellular K+ were measured using whole-cell patch-clamp recording and ion chromatography, respectively. Cytochrome c was measured with an ELISA kit. Cells in which Fas was knocked down exhibited identical UVB-induced K+ channel activation and loss of intracellular K+ to control cells. Cells in which TNF-R1 or FADD were knocked down demonstrated reduced K+ channel activation and decreased loss of intracellular K+ following UVB, relative to control cells. Application of TNF-α, the natural ligand of TNF-R1, to HCLE cells induced K+ channel activation and loss of intracellular K+. Cytochrome c was translocated to the cytosol by 2 h after exposure to 150 mJ/cm2 UVB. However, there was no release by 10 min post-UVB. The data suggest that UVB activates TNF-R1, which in turn may activate K+ channels via FADD. This conclusion is supported by the observation that TNF-α also causes loss of intracellular K+. This signaling pathway appears to be integral to UVB-induced K+ efflux, since knockdown of TNF-R1 or FADD inhibits the UVB-induced K+ efflux. The lack of rapid cytochrome c translocation indicates cytochrome c does not play a role in UVB-induced K+ channel activation.

Correlation of higher levels of soluble TNF-R1 with a shorter survival, independent of age, in recurrent glioblastoma.

The circulating levels of soluble tumor necrosis factor receptor-1 (sTNF-R1) and sTNF-R2 are altered in numerous diseases, including several types of cancer. Correlations with the risk of progression in some cancers, as well as systemic manifestations of the disease and therapeutic side-effects, have been described. However, there is very little information on the levels of these soluble receptors in glioblastoma (GBM). Here, we report on an exploratory retrospective study of the levels of sTNF-Rs in the vascular circulation of patients with GBM. Banked samples were obtained from 112 GBM patients (66 untreated, newly-diagnosed patients and 46 with recurrent disease) from two institutions. The levels of sTNF-R1 in the plasma were significantly lower in patients with newly-diagnosed or recurrent GBM than apparently healthy individuals and correlated with the intensity of expression of TNF-R1 on the tumor-associated endothelial cells (ECs) in the corresponding biopsies. Elevated levels of sTNF-R1 in patients with recurrent, but not newly-diagnosed GBM, were significantly associated with a shorter survival, independent of age (p = 0.02) or steroid medication. In contrast, the levels of circulating sTNF-R2 were significantly higher in recurrent GBM than healthy individuals and there was no significant correlation with expression of TNF-R2 on the tumor-associated ECs or survival time. The results indicate that larger, prospective studies are warranted to determine the predictive value of the levels of sTNF-R1 in patients with recurrent GBM and the factors that regulate the levels of sTNF-Rs in the circulation in GBM patients.

The antifibrogenic effect of etanercept on development of liver cirrhosis induced by thioacetamide in rats.

Liver cirrhosis is an elevating cause of morbidity and mortality worldwide. TNF-α/TNF-R1 signal is implicated in progression of many liver diseases. This study provides histological and ultrastructural view that clarifies the effect of etanercept, a TNF-α inhibitor, on development of thioacetamide (TAA)-induced liver cirrhosis and the accompanied hemosiderosis in rats, highlighting the implication and distribution pattern of hepatic TNF-R1. Sixty male albino rats (Rattus norvegicus) were equally randomized into three groups. Group I served as the control. Liver cirrhosis was triggered in the other two groups by intraperitoneal injection of TAA twice a week for five months. Group II received TAA only, while group III subcutaneously injected with etanercept one hour before TAA, along five months. At the end of the experiment, blood was collected for biochemical analysis and livers were excised for histological, immunohistochemical, and electron microscopical preparations. Rats treated with TAA only developed hepatic cirrhosis accompanied by massive deposition of hemosiderin; strong and widespread expression of hepatic TNF-R1 in sinusoidal endothelial cells (SECs), Kupffer cells (KCs), and many hepatocytes; and frequent appearance of fibrogenic, plasma, and mast cells, at the ultrastructural level. By contrast, administration of etanercept diminished the expression of TNF-R1, attenuated the accumulation of collagen and hemosiderin, and preserved the hepatic histoarchitecture. In conclusion, TNF-α signal via TNF-R1 may be implicated in the mechanism of fibrogenesis and the associated hemosiderosis. Etanercept may provide a promising therapeutic approach not only for attenuating the progression of fibrogenesis, but also for hepatic iron overload-associated disorders.

Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1ß and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme.

Effects of moxibustion on visceral hypersensitivity and colonic inflammatory response in mice with chronic ulcerative colitis. / è‰¾ç¸å¯¹æ ¢æ€§æºƒç–¡æ€§ç»“è‚ ç‚Žå°é¼ å† è„é«˜æ•åŠç»“è‚ ç»„ç»‡ç‚Žæ€§ååº”çš„å½±å“.

OBJECTIVES: To observe the effects of moxibustion at "Zusanli" (ST36) on the expression levels of tumor necrosis factor (TNF)-α, TNF receptor 1 (TNF-R1), p38 mitogen-activated protein kinase (P38 MAPK), and transient receptor potential vanilloid 1 (TRPV1) in the colon tissue of mice with chronic ulcerative colitis (UC), so as to explore the underlying mechanisms of moxibustion in improving visceral hypersensitivity in chronic UC. METHODS: Male C57BL/6J mice were randomly divided into normal group, normal with moxibustion (NM) group, model group, and model with moxibustion (MM) group, with 10 mice in each group. The chronic UC model was established by drinking 2.5% dextran sodium sulfate for 3 cycles. Mice in the NM and MM groups received moxibustion at ST36 for 20 min, 5 days per week with a 2-day break, for a total of 4 weeks. The disease activity index (DAI) score of each group was evaluated before and after treatment. The minimum volume threshold of abdominal wall retraction reflex (AWR) was measured to observe the intestinal sensitivity of mice. The colon length was measured. The pathological changes of colon tissue were observed by HE staining. The expression of mucin in colon goblet cells was detected by periodate Scheff staining. The intestinal fibrosis was observed by Masson staining. The number of trypsin-positive cells (i.e., mast cell) and the expression level of TNF-α in colon tissue were detected by immunofluorescence staining. The expression levels of TNF-R1, P38 MAPK and TRPV1 in colon tissue were detected by immunohistochemistry. RESULTS: Compared with the normal group after treatment, the model group showed increased DAI score (P<0.001), decreased AWR minimum volume threshold (P<0.01), shortened colon length (P<0.001), significant inflammatory infiltration in the colon tissue, reduced mucin secretion (P<0.01), increased collagen fiber deposition (P<0.001), and elevated expression levels of TNF-α, TNF-R1, P38 MAPK, and TRPV1 (P<0.001, P<0.01, P<0.05). Compared with the model group, the MM group showed decreased DAI score (P<0.01), increased AWR minimum volume threshold (P<0.001), elongated colon length (P<0.001), reduced inflammatory cell infiltration, improved integrity of mucosal glandular structure, enhanced mucin secretion (P<0.01), decreased collagen fiber deposition (P<0.001), decreased number of mast cells in the colon tissue (P<0.001), and decreased expression levels of TNF-α, TNF-R1, P38 MAPK, and TRPV1 (P<0.001, P<0.01, P<0.05). There were no significant differences in the above index between the NM group and the normal group. CONCLUSIONS: Moxibustion can reduce visceral hypersensitivity, alleviate inflammatory infiltration and fibrotic damage in the colon tissue of mice with chronic UC. These effects may be associated with the down-regulation of TNF-α, TNF-R1, P38 MAPK, and TRPV1 expression in colon.

TNF-R1 Cellular Nanovesicles Loaded on the Thermosensitive F-127 Hydrogel Enhance the Repair of Scalded Skin.

Excessive inflammatory response after severe scalding is an important cause of delayed wound healing and is even life-threatening. Tumor necrosis factor α (TNF-α) is a key pro-inflammatory factor of skin trauma. Interacting with tumor necrosis factor receptor 1 (TNF-R1), TNF-α causes excessive inflammation and poor prognosis by activating NF-κB pathway. Antagonizing high levels of TNF-α is one of the therapeutic approaches for diseases associated with the overactivation of inflammatory responses. However, the available monoclonal antibodies are limited in their application due to their complex preparation process, high price, and the lack of cell targeting ability leading to systemic toxicity and side effects. In this study, by using a genetic bioengineering technique, we modified TNF-R1 on the cell membrane surface-derived nanovesicles (NVs). We confirmed that TNF-R1 NVs stably expressed TNF-R1 on the membrane surface and interacted with its ligand TNF-α. Furthermore, TNF-R1 NVs competitively antagonized the effect of TNF-α in the wound healing assay in vitro. In the scalded mouse model, TNF-R1 NVs were released continuously from the thermosensitive hydrogel Pluronic F-127, resulting in less inflammation and better wound healing. Our results revealed TNF-R1 NVs as promising cell-free therapeutic agents in alleviating TNF-α-mediated pro-inflammatory signaling and promoting wound repair.

The role of TNF receptors as mediators of chronic vasculitis in possible milder forms of the CNS tuberculosis.

INTRODUCTION: Central nervous system (CNS) tuberculosis (TB) is the most severe form of TB due to its high mortality and functional sequelae. There are several differential diagnoses for TB; and, it can also cause secondary conditions, such as vasculitis. METHODOLOGY: 155 biopsies, corresponding to 155 different patients out of 5,386 registered biopsies from 2008-2013, met the criteria of unknown etiology vasculitis and evidence of cerebral vascular disease. These were analyzed to assess the presence of central nervous system TB. The selected cases were assessed with Suzaan Marais (SM) criteria for clinical tuberculosis. After that, Ziehl-Neelsen (ZN) staining and polymerase chain reaction (PCR) were performed to amplify a fragment of the insertion sequence IS6110 of M. tuberculosis. 21 patients met the criteria for definitive tuberculosis by ZN staining and PCR, and 2 met the criteria for possible tuberculosis. Tumor necrosis factor (TNF)-α, TNF-R1, and TNF-R2 were determined by immunohistochemistry in histological sections from formalin-fixed paraffin-embedded (FF-PE) tissues in the 23 selected patients. RESULTS: Granulomatous TB was present in almost half of the cases. TNF-R1 and TNF-R2 were expressed mainly in blood vessels, histiocytes, and macrophages. TNF-R2 expression was higher than the other markers, which suggests an anti-inflammatory response against M. tuberculosis. CONCLUSIONS: The histopathological presentation of TB is not always limited to granulomas, abscesses, or meningitis; there are also clinical presentations characterized only with chronic inflammation of nervous and vascular tissue.

Describing immune factors associated with Hepatitis B surface antigen loss: A nested case-control study of a Chinese sample from Wuwei City.

Background: Hepatitis B surface antigen (HBsAg) loss is considered a functional cure for chronic hepatitis B (CHB), however, several factors influence HBsAg loss. Methods: 29 CHB patients who had achieved HBsAg loss, were selected and 58 CHB patients with persistent HBsAg were matched, according to gender and age (+/- 3 years). Logistic regression and restricted cubic spline (RCS) modelling were performed. Results: Multivariate-adjusted logistic regression, based on stepwise selection, showed that baseline HBsAg levels negatively correlated with HBsAg loss (odds ratio [OR] = 0.99, 95% confidence interval [CI] = 0.98-0.99). Interferon treatment positively related with HBsAg loss (OR = 7.99, 95%CI = 1.62-44.88). After adjusting for age, HBsAg level, ALT level, HBeAg status and interferon treatment, MMP-1 (OR = 0.66, 95%CI = 0.44-0.97), CXCL9 (OR = 0.96, 95%CI = 0.93-0.99) and TNF-R1 (OR = 0.97, 95%CI = 0.94-0.99) baseline levels all negatively correlated with HBsAg loss. Our multivariate-adjusted RCS model showed that baseline CXCL10 was associated with HBsAg loss although the relationship was "U-shaped". Conclusions: Cytokines such as MMP-1, CXCL9, CXCL10 and TNF-R1 are important factors which influence HBsAg loss. It may be possible to develop a nomogram which intercalates these factors; however, further research should consider immune processes involved in HBsAg loss.

Genotoxicity-Stimulated and CYLD-Driven Malignant Transformation.

Oxidative stress, which can cause DNA damage, can both activate TNF-R1 directly in the absence of TNF stimulation and phosphorylate c-Abl, thus promoting its cytoplasmic translocation. Persistent cytoplasmic localization of c-Abl has been associated with cellular transformation. c-Abl phosphorylates OTULIN at tyrosine 56, thereby disrupting its relationship with LUBAC. OTULIN-released LUBAC interacts with SPATA2 and is recruited to the TNF-R1sc, facilitating SPATA2-CYLD interaction. All these interactions are required for the activation of IKKß to stimulate NF-κB transcriptional activity following genotoxic stress. IKKß also induces the critical phosphorylation of CYLD at serine 568 to increase its deubiquitinating (DUB) activity required for the termination of signaling cascades. Contrary to the widespread belief that CYLD is an absolute tumor suppressor, CYLD initiates and terminates NF-κB activity by alternately using its oncoprotein and tumor suppressor activities, respectively. If IKKß fails to achieve the DUB activity-inducing phosphorylation at serine 568, CYLD would operate in a sustained mode of oncogenic activity. The resulting dysregulated NF-κB activation and other accompanying pathologies will disrupt cellular homeostasis in favor of transformation.

Roburic Acid Targets TNF to Inhibit the NF-κB Signaling Pathway and Suppress Human Colorectal Cancer Cell Growth.

Tumor necrosis factor (TNF)-stimulated nuclear factor-kappa B (NF-κB) signaling plays very crucial roles in cancer development and progression, and represents a potential target for drug discovery. Roburic acid is a newly discovered tetracyclic triterpene acid isolated from oak galls and exhibits anti-inflammatory activity. However, whether roburic acid exerts antitumor effects through inhibition of TNF-induced NF-κB signaling remains unknown. Here, we demonstrated that roburic acid bound directly to TNF with high affinity (KD = 7.066 µM), blocked the interaction between TNF and its receptor (TNF-R1), and significantly inhibited TNF-induced NF-κB activation. Roburic acid exhibited antitumor activity in numerous cancer cells and could effectively induce G0/G1 cell cycle arrest and apoptosis in colorectal cancer cells. Importantly, roburic acid inhibited the TNF-induced phosphorylation of IKKα/ß, IκBα, and p65, degradation of IκBα, nuclear translocation of p65, and NF-κB-target gene expression, including that of XIAP, Mcl-1, and Survivin, in colorectal cancer cells. Moreover, roburic acid suppressed tumor growth by blocking NF-κB signaling in a xenograft nude mouse model of colorectal cancer. Taken together, our findings showed that roburic acid directly binds to TNF with high affinity, thereby disrupting its interaction with TNF-R1 and leading to the inhibition of the NF-κB signaling pathway, both in vitro and in vivo. The results indicated that roburic acid is a novel TNF-targeting therapeutics agent in colorectal cancer as well as other cancer types.

Roles for ADAM17 in TNF-R1 Mediated Cell Death and Survival in Human U937 and Jurkat Cells.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

Intravesical Instillation of Azacitidine Suppresses Tumor Formation through TNF-R1 and TRAIL-R2 Signaling in Genotoxic Carcinogen-Induced Bladder Cancer.

Azacitidine, an inhibitor of DNA methylation, shows therapeutic effects against several malignancies by inducing apoptosis and inhibiting tumor cell proliferation. However, the anti-tumor effects of azacitidine on urinary bladder urothelial carcinoma (UBUC), especially following intravesical instillation (IVI), are not established. Here, UBUC cell lines were used to analyze the in vitro therapeutic effects of azacitidine. Potential signaling pathways were investigated by antibody arrays and Western blotting. The N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced rat UBUC model was used for in vivo quantitative analysis of tumor burden. Azacitidine significantly inhibited DNMT expression in UBUC cell lines and reduced cell viability and clonogenic activity, as determined by MTT and colony formation assays, while also inducing significant cytotoxic effects in the form of increased sub-G1 and Annexin V-PI populations (all p < 0.05). Antibody arrays confirmed the in vitro suppression of TNF-R1 and the induction of TRAIL-R2 and their downstream signaling molecules. TNF-R1 suppression reduced claspin and survivin expression, while TRAIL-R2 activation induced cytochrome C and caspase 3 expression. Rats with BBN-induced bladder cancer had a significantly reduced tumor burden and Ki67 index following IVI of azacitidine (p < 0.01). Our study provides evidence for a reduction in BBN-induced bladder cancer by IVI of azacitidine through alterations in the TRAIL-R2 and TNF-R1 signaling pathways. These findings might provide new insights for further clinical trials.

Blood Biomarkers to Predict Long-Term Mortality after Ischemic Stroke.

Stroke is a major cause of disability and death globally, and prediction of mortality represents a crucial challenge. We aimed to identify blood biomarkers measured during acute ischemic stroke that could predict long-term mortality. Nine hundred and forty-one ischemic stroke patients were prospectively recruited in the Stroke-Chip study. Post-stroke mortality was evaluated during a median 4.8-year follow-up. A 14-biomarker panel was analyzed by immunoassays in blood samples obtained at hospital admission. Biomarkers were normalized and standardized using Z-scores. Multiple Cox regression models were used to identify clinical variables and biomarkers independently associated with long-term mortality and mortality due to stroke. In the multivariate analysis, the independent predictors of long-term mortality were age, female sex, hypertension, glycemia, and baseline National Institutes of Health Stroke Scale (NIHSS) score. Independent blood biomarkers predictive of long-term mortality were endostatin > quartile 2, tumor necrosis factor receptor-1 (TNF-R1) > quartile 2, and interleukin (IL)-6 > quartile 2. The risk of mortality when these three biomarkers were combined increased up to 69%. The addition of the biomarkers to clinical predictors improved the discrimination (integrative discriminative improvement (IDI) 0.022 (0.007-0.048), p < 0.001). Moreover, endostatin > quartile 3 was an independent predictor of mortality due to stroke. Altogether, endostatin, TNF-R1, and IL-6 circulating levels may aid in long-term mortality prediction after stroke.

Regulation of Death Receptor Signaling by S-Palmitoylation and Detergent-Resistant Membrane Micro Domains-Greasing the Gears of Extrinsic Cell Death Induction, Survival, and Inflammation.

Death-receptor-mediated signaling results in either cell death or survival. Such opposite signaling cascades emanate from receptor-associated signaling complexes, which are often formed in different subcellular locations. The proteins involved are frequently post-translationally modified (PTM) by ubiquitination, phosphorylation, or glycosylation to allow proper spatio-temporal regulation/recruitment of these signaling complexes in a defined cellular compartment. During the last couple of years, increasing attention has been paid to the reversible cysteine-centered PTM S-palmitoylation. This PTM regulates the hydrophobicity of soluble and membrane proteins and modulates protein:protein interaction and their interaction with distinct membrane micro-domains (i.e., lipid rafts). We conclude with which functional and mechanistic roles for S-palmitoylation as well as different forms of membrane micro-domains in death-receptor-mediated signal transduction were unraveled in the last two decades.

Computational Analysis of Domains Vulnerable to HPV-16 E6 Oncoprotein and Corresponding Hot Spot Residues.

BACKGROUND: Human Papilloma Virus (HPV) is the primary cause of cancers in cervix, head and neck regions. Oncoprotein E6 of HPV-16, after infecting human body, alters host protein- protein interaction networks. E6 interacts with several proteins, causing the infection to progress into cervical cancer. The molecular basis for these interactions is the presence of short linear peptide motifs on E6 identical to those on human proteins. METHODS: Motifs of LXXLL and E/DLLL/V-G after identification on E6, were analyzed for their dynamic fluctuations by use of elastic network models. Correlation analysis of amino acid residues of E6 was also performed in specific regions of motifs. RESULTS: Arginine, Leucine, Glutamine, Threonine and Glutamic acid have been identified as hot spot residues of E6 which can subsequently provide a platform for drug designing and understanding of pathogenesis of cervical cancer. These amino acids play a significant role in stabilizing interactions with host proteins, ultimately causing infections and cancers. CONCLUSION: Our study validates the role of linear binding motifs of E6 of HPV in interacting with these proteins as an important event in the propagation of HPV in human cells and its transformation into cervical cancer. The study further predicts the domains of protein kinase and armadillo as part of the regions involved in the interaction of E6AP, Paxillin and TNF R1, with viral E6.

An elevated polyclonal free light chain level reflects a strong interferon signature in patients with systemic autoimmune diseases.

High amount of polyclonal free light chains (FLC) are reported in systemic autoimmune diseases (SAD) and we took advantage of the PRECISESADS study to better characterize them. Serum FLC levels were explored in 1979 patients with SAD (RA, SLE, SjS, Scl, APS, UCTD, MCTD) and 614 healthy controls. Information regarding clinical parameters, disease activity, medications, autoantibodies (Ab) and the interferon α and/or γ scores were recorded. Among SAD patients, 28.4% had raised total FLC (from 12% in RA to 30% in SLE and APS) with a normal kappa/lambda ratio. Total FLC levels were significantly higher in SAD with inflammation, active disease in SLE and SjS, and an impaired pulmonary functional capacity in SSc, while independent from kidney impairment, infection, cancer and treatment. Total FLC concentrations were positively correlated among the 10/17 (58.8%) autoantibodies (Ab) tested with anti-RNA binding protein Ab (SSB, SSA-52/60 kDa, Sm, U1-RNP), anti-dsDNA/nucleosome Ab, rheumatoid factor and negatively correlated with complement fractions C3/C4. Finally, examination of interferon (IFN) expression as a potential driver of FLC overexpression was tested showing an elevated level of total FLC among patients with a high IFNα and IFNγ Kirou's score, a strong IFN modular score, and the detection in the sera of B-cell IFN dependent factors, such as TNF-R1/TNFRSF1A and CXCL10/IP10. In conclusion, an elevated level of FLC, in association with a strong IFN signature, defines a subgroup of SAD patients, including those without renal affectation, characterized by increased disease activity, autoreactivity, and complement reduction.