Soluble Tumor Necrosis Factor-alpha Research Articles

The dose-response effect of aerobic exercise on inflammation in colon cancer survivors.

Physical activity after surgical resection for colon cancer is associated with significantly longer disease-free survival. Inflammation is hypothesized to mediate the association between physical activity and disease-free survival in colon cancer. In this exploratory analysis of a randomized dose-response trial, 39 colon cancer survivors who completed standard therapy were stratified by cancer stage and randomized in a 1:1:1 ratio to one of three treatment groups for 24 weeks of usual-care control, 150 min/wk of moderate-intensity aerobic exercise (low-dose), or 300 min/wk of moderate-intensity aerobic exercise (high-dose). Inflammation outcomes included high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL6), and soluble tumor necrosis factor-alpha receptor 2 (sTNFαR2). Mixed models for repeated measures were used to test the hypothesis that exercise was associated with dose-response reductions in inflammation; exploratory analyses examined treatment effects by cancer stage. In the overall population, aerobic exercise was not associated with dose-response reductions in hs-CRP, IL6, or sTNFαR2. Cancer stage modified the association between randomized group and hs-CRP (P=0.022) and IL6 (P<0.001) but not sTNFαR2 (P=0.39). In stage I-II disease, compared to control, exercise was not associated with inflammation outcomes. In stage III disease, compared to control, low-dose exercise reduced hs-CRP: -35.4% (95% CI: -70.1, -0.7) and IL6: -29.6% (95% CI: -58.4, -0.8) but not sTNFαR2: 2.7% (95% CI: sTNFαR2: -15.7, 21.1); high-dose exercise was not associated with inflammation outcomes in stage III disease. This exploratory analysis offers preliminary data to support the hypothesis that inflammation may mediate the association between physical activity and disease-free survival in colon cancer. clinicaltrials.gov, identifier NCT02250053.

Zinc Deficiency And sTNF-RII Are Associated With Worse COVID-19 Outcomes

BackgroundZinc (Zn) is known for its substantial involvement in the immune response as an antioxidant and anti-inflammatory agent. Zn plasma levels’ clinical significance in coronavirus disease (COVID) diagnosis is not yet fully established. ObjectiveWe assessed the association between Zn deficiency, gut integrity, inflammation, and COVID-19 outcomes. MethodsA prospective observational cohort in which plasma Zn, soluble tumor necrosis factor alpha receptor II (sTNF-RII) intestinal fatty-acid binding protein (IFABP; marker of intestinal integrity), and zonulin levels (intestinal permeability) were collected from participants during the acute phase of a confirmed COVID-19 diagnosis. Zn was modeled as continuous and binary, categorized as Zn deficiency (Zn < 75 μg/dL) and Zn sufficiency (Zn ≥ 75 μg/dL). COVID-19 outcomes were classified according to the World Health Organization clinical progression scale. We used cumulative probit regression to assess if suboptimal Zn levels, gut, and inflammatory markers increase the likelihood of worse COVID-19 outcomes. ResultsZn deficiency was independently associated with 63% higher predicted odds of worse COVID outcomes. Increases in sTNF-RII {unadjusted odds ratio (uOR): 3.43 [95% confidence interval (CI): 2.02, 5.82]} and zonulin [uOR: 1.83 (95% CI: 1.21, 2.76)] levels were associated with greater odds of worse COVID outcomes. IFABP was not associated with worse COVID outcomes [uOR: 1.12 (95% CI: 0.82, 1.53)] or acute Zn deficiency [uOR: 1.35 (95% CI: 0.79, 2.35)]. The adjusted predicted odds of worse COVID outcomes are 3-fold higher (P = 0.04) for every one-unit decrease in Zn and is more than 2 times greater odds of COVID severity (P = 0.01) for every 1-unit increase in sTNF-RII. ConclusionZn deficiency and inflammation were independently associated with greater odds of worse COVID outcomes.

Extracorporeal removal of soluble tumor necrosis factor receptors for the treatment of advanced refractory solid tumors.

e14593 Background: Tumor necrosis factor-alpha (TNF) has well-established anti-tumor effects including cytotoxicity against tumor vasculature. Past use of recombinant TNF to treat solid tumors resulted in unacceptable toxicities when given systemically but is successfully used in localized limb perfusion to treat melanoma and sarcomas. An alternative to promote endogenous TNF-mediated anticancer activity involves therapeutic apheresis to reduce the high concentrations of soluble TNF receptors (sTNF-Rs) produced by tumors, which sequester TNF and inhibit its tumoricidal effects. Methods: The LW-02 Column contains recombinant single-chain TNF, a capture ligand that selectively removes sTNF-Rs from plasma, covalently linked to a bead matrix. LW-02 Column Immunopheresis was evaluated in heavily pretreated patients with metastatic melanoma, renal cell carcinoma or triple negative breast cancer [NCT04142931]. LW-02 Column Immunopheresis was performed 3x/week, processing up to two plasma volumes for approximately 12 weeks as monotherapy (Cohort A, n = 6) or combined with nivolumab (Cohort B, n = 5). The study evaluated column performance, safety, clinical efficacy, and immunological biomarkers in the tumor microenvironment and blood. Results: The LW-02 Column effectively reduced sTNF-R plasma concentrations with capture efficiencies of 95.6% and 82.8% for sTNF-R1 and sTNF-R2, respectively, at the 30-minute procedural time point. At the data cut-off (Sept 30, 2022), median overall survival (OS) was 34.7 and 44.4 weeks for all patients (n = 11) and patients treated for ≥4 weeks (n = 8) (the latter group was prespecified in the protocol), respectively. For Cohorts A and B, median OS was 34.7 weeks and 45.4 weeks, respectively, for patients treated for ≥4 weeks. Evaluation of safety showed that only 2 adverse events (AEs) of 95 total AEs (including 7 SAEs) were deemed possibly related to LW-02 Column treatment. Immunohistochemical evaluation of tumor biopsies obtained 10-14 weeks after initiating LW-02 Column Immunopheresis revealed increased tumor-infiltrating lymphocytes (CD8+, HLA-DR+, and/or PD-1+ cells; 5/5 patients examined), and a reduced presence of tumor-associated macrophages (CD68+; 4/5 patients) compared to pretreatment biopsies. Preliminary immune profiling of peripheral blood mononuclear cells (5 patients) revealed increased expression levels of transmembrane TNF-R1 and TNF-R2, suggestive of increased cellular TNF responsiveness, and increased expression of activation molecules HLA-DR and CD86 after 4 weeks of LW-02 Column monotherapy compared to pretreatment levels. Conclusions: LW-02 Column Immunopheresis achieved safe and effective depletion of sTNF-Rs from plasma of patients with solid tumors. Further studies will assess the clinical benefit profile of the LW-02 Column for resensitizing tumors to respond to immunotherapy. Clinical trial information: NCT04142931 .

Associations between circulating cell-free mitochondrial DNA, inflammatory markers, and cognitive and physical outcomes in community dwelling older adults

BackgroundDementia and frailty are common age-related syndromes often linked to chronic inflammation. Identifying the biological factors and pathways that contribute to chronic inflammation is crucial for developing new therapeutic targets. Circulating cell-free mitochondrial DNA (ccf-mtDNA) has been proposed as an immune stimulator and potential predictor of mortality in acute illnesses. Dementia and frailty are both associated with mitochondrial dysfunction, impaired cellular energetics, and cell death. The size and abundance of ccf-mtDNA fragments may indicate the mechanism of cell death: long fragments typically result from necrosis, while short fragments arise from apoptosis. We hypothesize that increased levels of necrosis-associated long ccf-mtDNA fragments and inflammatory markers in serum are linked to declines in cognitive and physical function, as well as increased mortality risk.ResultsOur study of 672 community-dwelling older adults revealed that inflammatory markers (C-Reactive Protein, soluble tumor necrosis factor alpha, tumor necrosis factor alpha receptor 1 [sTNFR1], and interleukin-6 [IL-6]) positively correlated with ccf-mtDNA levels in serum. Although cross-sectional analysis revealed no significant associations between short and long ccf-mtDNA fragments, longitudinal analysis demonstrated a connection between higher long ccf-mtDNA fragments (necrosis-associated) and worsening composite gait scores over time. Additionally, increased mortality risk was observed only in individuals with elevated sTNFR1 levels.ConclusionIn a community dwelling cohort of older adults, there are cross-sectional and longitudinal associations between ccf-mtDNA and sTNFR1 with impaired physical and cognitive function and increased hazard of death. This work suggests a role for long ccf-mtDNA as a blood-based marker predictive of future physical decline.

Levels of peri-implant sulcular fluid levels of soluble urokinase plasminogen activator receptor and TNF-α among cigarette smokers and non-smokers with peri-implantitis.

Soluble urokinase plasminogen activator receptor (suPAR) and tumor necrosis factor-alpha (TNF-α) are inflammatory biomarkers. No studies have yet assessed the suPAR levels in relation with TNF-α in the peri-implant sulcular fluid (PISF) among cigarette smokers and non-smokers with peri-implantitis. The aim was to evaluate PISF levels of suPAR, and TNF-α among cigarette smokers and non-smokers with and without peri-implantitis. Sixty male patients with peri-implantitis were included. There were 20 cigarette smokers and 20 and non-smokers with peri-implantitis (Groups 1 and 2), and 20 non-smokers without peri-implantitis (Group 3). Demographic data and information related to cigarette smoking was recorded. Peri-implant clinicoradiographic parameters (plaque index [PI], gingival index [GI], probing depth [PD] and crestal bone loss [CBL]) were assessed. The PISF samples were collected and levels of suPAR and TNF-α were measured. Sample-size estimation was performed and all parameters were statistically assessed. Level of significance was set at P< 0.05. Sixty individuals were included in Groups 1, 2 and 3 (20 in each). Peri-implant PI (P< 0.01), PD (P< 0.01) and mesial (P< 0.01) and distal (P< 0.01) CBL were significantly higher in Group 1 than in Groups 2 and 3. The PISF volume (P< 0.01) and suPAR (P< 0.01) and TNF-α levels (P< 0.01) were significantly higher in Groups 1 and 2 than in Group 3. There was no difference in PISF volume and suPAR and TNF-α levels between patients in Groups 1 and 2. In Group 2, there was a statistically significant correlation between peri-implant PD and PISF suPAR and TNF-α levels (P< 0.01). The suPAR and TNF-α levels are expressed in high concentrations in the PISF of smokers and non-smokers with peri-implantitis compared with non-smokers without peri-implantitis. In non-smokers, PISF suPAR and TNF-α levels are correlated with peri-implant PD.

Discovery of novel targets in a complex regional pain syndrome mouse model by transcriptomics: TNF and JAK-STAT pathways

Complex Regional Pain Syndrome (CRPS) represents severe chronic pain, hypersensitivity, and inflammation induced by sensory-immune-vascular interactions after a small injury. Since the therapy is unsatisfactory, there is a great need to identify novel drug targets. Unbiased transcriptomic analysis of the dorsal root ganglia (DRG) was performed in a passive transfer-trauma mouse model, and the predicted pathways were confirmed by pharmacological interventions. In the unilateral L3–5 DRGs 125 genes were differentially expressed in response to plantar incision and injecting IgG of CRPS patients. These are related to inflammatory and immune responses, cytokines, chemokines and neuropeptides. Pathway analysis revealed the involvement of Tumor Necrosis Factor (TNF) and Janus kinase (JAK-STAT) signaling. The relevance of these pathways was proven by abolished CRPS IgG-induced hyperalgesia and reduced microglia and astrocyte markers in pain-associated central nervous system regions after treatment with the soluble TNF alpha receptor etanercept or JAK inhibitor tofacitinib. These results provide the first evidence for CRPS-related neuroinflammation and abnormal cytokine signaling at the level of the primary sensory neurons in a translational mouse model and suggest that etanercept and tofacitinib might have drug repositioning potentials for CRPS-related pain.

Candidate biomarkers of physical frailty in heart failure: an exploratory cross-sectional study.

Physical frailty is highly prevalent and predictive of worse outcomes in heart failure (HF). Candidate biomarker analysis may help in understanding the mechanisms underlying physical frailty in HF. We aimed to identify candidate biomarkers associated with physical frailty in HF using a multimarker strategy of distinct pathophysiological processes. We collected data and plasma samples from 113 adults with New York Heart Association Functional Class I-IV HF. Physical frailty was measured with the Frailty Phenotype Criteria. Plasma biomarkers included: N-terminal pro-B-type natriuretic peptide, norepinephrine, dihydroxyphenylglycol, soluble tumour necrosis factor alpha receptor-1, adiponectin, insulin, glucose, insulin-like growth factor-1 (IGF-1), and myostatin. Comparative statistics and multivariate linear regression were used to test group differences and associations. The average age was 63.5 ± 15.7 years, half were women (48%), and most had a non-ischaemic aetiology of HF (73%). Physical frailty was identified in 42% and associated with female sex, higher body mass index and percent body fat, more comorbidities, and HF with preserved ejection fraction. Adjusting for Seattle HF Model projected survival score, comorbidities, body composition, and sex, physical frailty was associated with significantly lower plasma adiponectin [β ± standard error (SE) -0.28 ± 0.14, P = 0.047], IGF-1 (β ± SE -0.21 ± 0.10, P = 0.032), and myostatin (β ± SE -0.22 ± 0.09, P = 0.011). In sex-stratified analyses, IGF-1 and myostatin were significantly associated with physical frailty in men but not women. We identified biomarkers involved in adipose tissue and skeletal muscle development, maintenance, and function that were associated with physical frailty in HF.

The acute effect of a laboratory shame induction protocol on endothelial function in young, healthy adults.

What is the central question of this study? Shame is a form of social stress that involves internalizing social devaluations imposed by others. The aim of this study was to determine, for the first time, how acutely experienced shame impacts endothelial function. What is the main finding and its importance? Brachial artery flow-mediated dilatation, an index of endothelial function, was impaired after an intervention that acutely increased self-reported shame. This occurred without increases in cortisol or tumor necrosis factor alpha receptor binding. Frequent or prolonged shame-induced endothelial dysfunction could have important cardiovascular consequences. The objective of this study was to examine the impact of a shame induction protocol on endothelial function. Fifteen participants (n=7 men, n=8 women) completed both a written shame induction protocol and a control protocol on two different experimental days. Pre- and post-protocol we assessed: (1) endothelial function and arterial shear rate via a standard brachial artery reactive hyperaemia flow-mediated dilatation (FMD) test across two post-intervention time points (15 and 35min post); (2) perceived shame via the experiential shame scale (ESS); and (3) cortisol and soluble tumor necrosis factor alpha receptor (sTNFαRII) through oral fluid analysis. Shame increased after the shame induction protocol (pre, 2.9±0.6 vs. post, 3.7±0.5, P<0.001) but not the control protocol (pre, 3.0±0.5 vs. post, 2.8±0.5, P=0.15; protocol by time interaction, P<0.001). When all three time points were included in the analysis, %FMD did not change over time. Considering only the lowest post time point, %FMD decreased significantly in response to the shame protocol (pre, 4.8±1.9 vs. post, 3.2±1.6, P<0.001) but not the control protocol (pre, 4.2±1.8 vs. post, 3.8±1.5, P=0.45; protocol by time interaction, P=0.035). Covariation of the shear rate stimulus for FMD did not alter the FMD results. When including both the control and shame protocols, but not the shame protocol alone, increased shame was significantly associated with decreased FMD (r=-0.37, P<0.046). There were no significant time by protocol interaction effects for cortisol or sTNFαRII. In conclusion, temporary increases in shame might cause transient endothelial dysfunction which, if chronically repeated, could manifest as reduced vasoprotection against atherosclerosis.

MO640: Treatment with Sodium–Glucose Cotransporter 2 Inhibitors Increases Klotho in Type 2 Diabetic Patients

Abstract BACKGROUND AND AIMS Klotho (KL) is a transmembrane protein mainly expressed by the tubular cells of the kidneys, with an additional soluble form that is present in blood and urine. Preserved KL has been related to physiological protection of the kidneys. Sodium–glucose cotransporter type-2 inhibitors (SGLT2i) are a new class of anti-diabetic drugs with important renal benefits. The aim of the present study was to analyze the effect of treatment with SGLT2i on soluble KL concentrations as well as on mRNA expression levels of the KL gene in renal tubular cells. METHOD Fifty-seven patients (30 males and 27 females; mean age 61 ± 5 years) with type 2 diabetes and chronic kidney disease stage G2-A2, all treated with metformin and blockers of the renin–angiotensin system, were included in the study. All patients were in need of intensified anti-diabetic therapy at the discretion of their physicians in accordance with the routine clinical practice. Forty-five received SGLT2i during 3 months (15 empagliflozin, 15 canagliflozin and 15 dapagliflozin), and the evolution was compared with a group of 12 patients matched by age, sex and stage of CKD who received DPP4 inhibitors (control group). Serum and urine levels of soluble KL and tumor necrosis factor-alpha (TNFa) were determined by ELISA. In addition, the effect on mRNA expression levels of KL was assessed in vitro in renal tubular cells cultures. RESULTS Baseline values of serum and urinary KL and TNFa were similar in both groups. Urinary KL was inversely correlated with albuminuria (r = −0.45, P &amp;lt; 0.001) and urine TNFa excretion (r = −0.40, P &amp;lt; 0.01). At the end of the study, there was a similar improvement in metabolic control in both groups; however, only patients treated with SGLT2i showed a significant reduction in albuminuria and urinary TNFa (P &amp;lt; 0.01). Serum and urinary KL increased by 5.2% and 38.9%, respectively, only in patients receiving SGLT2i (P &amp;lt; 0.001 versus baseline), without changes in controls (P &amp;lt; 0.01 between groups). There were no differences among the three drugs in the SGLT2i group. The reduction in albuminuria (β = −0.35, P &amp;lt; 0.01) and urinary TNFa (β = −0.57, P &amp;lt; 0.001) were independently associated with the increase in urine KL, while the change in albuminuria was related to the increase in serum KL (β = −0.39, P &amp;lt; 0.05). Cultured renal tubular cells under high glucose, inflammatory stimulus with TWEAK or serum bovine albumin decreased Klotho mRNA and protein expression within 24 h, an effect that was prevented by all the SGLT2i when added to the cultures. CONCLUSION In type 2 diabetic patients, treatment with SGLT2i induces a reduction in albuminuria and modulates inflammation (as reflected by a decrease in the urinary excretion of TNFa), which is associated with a significant increase in soluble KL concentrations. In addition, SGLT2i prevent the reduction in protein and mRNA expression levels of the KL gene in renal tubular cells. The preservation of KL by SGLT2i may be an important mechanism of renal protection.

Effects of aldosterone on the human placenta: Insights from placental perfusion studies

IntroductionIn pregnancy, aldosterone is linked to maternal plasma volume expansion, improved fetal and placental growth/angiogenesis and reduced maternal blood pressure. Aldosterone levels are low in women with pre-eclampsia. Given the placental growth properties of aldosterone in pregnancy, we hypothesised that increased aldosterone improves placental function ex vivo. We applied aldosterone in the dual human placenta perfusion model and analysed specific regulatory markers. MethodsA single cotyledon was perfused using a trimodal perfusion setup consisting of a control phase (CP; basic perfusion medium (BPM) alone) and two consecutive experimental phases (EP1/EP2; BPM supplemented with 1.5 x 10−9M and 1.5 x 10−7M aldosterone, respectively). CP and EP1/EP2 were conducted in closed circuits lasting 2 h each. Quality/time control perfusions using BPM alone were performed for 360 min to distinguish time-dependent effects from aldosterone-related effects. Perfusates were assessed for control parameters (pH/pO2/pCO2/glucose/lactate/creatinine/antipyrine). Maternal perfusates were analysed for placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α) using ELISAs. mRNA expression of abovementioned factors was measured by qPCR in post-perfusion tissue. ResultsData from quality/time control perfusions indicated that TNF-α and IL-10 release continuously increased over time. Contrary, in the trimodal perfusion setup the application of aldosterone decreased TNF-α secretion (P < 0.05, EP1/EP2 vs CP, 120 min) and increased PlGF release (P < 0.05, EP1 vs CP, 90/120 min) into the maternal perfusates. mRNA expression followed similar trends, but did not reach significance. DiscussionOur ex vivo placental perfusion data suggest that increasing aldosterone promotes anti-inflammatory and pro-angiogenic factors, which could positively contribute to healthy pregnancy outcomes.

Improved prediction of radiation pneumonitis by combining biological and radiobiological parameters using a data-driven Bayesian network analysis

Grade 2 and higher radiation pneumonitis (RP2) is a potentially fatal toxicity that limits efficacy of radiation therapy (RT). We wished to identify a combined biomarker signature of circulating miRNAs and cytokines which, along with radiobiological and clinical parameters, may better predict a targetable RP2 pathway. In a prospective clinical trial of response-adapted RT for patients (n = 39) with locally advanced non-small cell lung cancer, we analyzed patients’ plasma, collected pre- and during RT, for microRNAs (miRNAs) and cytokines using array and multiplex enzyme linked immunosorbent assay (ELISA), respectively. Interactions between candidate biomarkers, radiobiological, and clinical parameters were analyzed using data-driven Bayesian network (DD-BN) analysis. We identified alterations in specific miRNAs (miR-532, -99b and -495, let-7c, -451 and -139-3p) correlating with lung toxicity. High levels of soluble tumor necrosis factor alpha receptor 1 (sTNFR1) were detected in a majority of lung cancer patients. However, among RP patients, within 2 weeks of RT initiation, we noted a trend of temporary decline in sTNFR1 (a physiological scavenger of TNFα) and ADAM17 (a shedding protease that cleaves both membrane-bound TNFα and TNFR1) levels. Cytokine signature identified activation of inflammatory pathway. Using DD-BN we combined miRNA and cytokine data along with generalized equivalent uniform dose (gEUD) to identify pathways with better accuracy of predicting RP2 as compared to either miRNA or cytokines alone. This signature suggests that activation of the TNFα-NFκB inflammatory pathway plays a key role in RP which could be specifically ameliorated by etanercept rather than current therapy of non-specific leukotoxic corticosteroids.

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha.

Simple SummaryAs a result of increasing demand for the pleiotropic cytokine TNF-α, recombinant human TNF-α protein with appropriate bioactivities was produced in several heterologous in vivo expression systems. While in vivo expression of this cytokine is laborious and lengthy, cell-free or in vitro expression system has the benefits of speed, simplicity, flexibility, focus of all the system energy on target protein synthesis alone, besides high soluble and functional protein yield. Therefore, we employed and optimized an E. coli-based cell-free system for the first time to express recombinant human TNF-α. Our findings revealed that cell-free expression system can be an alternative platform for producing soluble and functionally active recombinant TNF-α with a yield of 390 µg/mL in only 2 h at a temperature of 40 °C for further research and clinical trials.Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

Abstract 10895: Biomarkers of Physical Frailty in Heart Failure

Introduction: Physical frailty is highly prevalent and associated with worse outcomes among adults with heart failure (HF). However, the biological mechanisms underlying physical frailty in HF are poorly understood. The aim was to identify biomarkers associated with physical frailty in HF using a multimarker strategy to reflect multiple physiological processes. Hypothesis: We hypothesized that biomarkers of neurohormonal activation, inflammation, insulin resistance, and adipose and skeletal muscle dysfunction would differentiate physically frail from non-physically frail adults with HF. Methods: We collected data and plasma samples from adults with New York Heart Association Functional Class I-IV HF. Physical frailty was measured with the Frailty Phenotype Criteria. Plasma biomarkers assayed were: N-terminal pro-B-type natriuretic peptide, norepinephrine, dihydroxyphenolglycol, soluble tumor necrosis factor alpha receptor 1, adiponectin, insulin, glucose, insulin-like growth factor-1, and myostatin. Comparative statistics and multivariate linear regression were used. Results: The average age of the participants (n = 113) was 63.5±15.7 years, half (48%) were women, and most had a non-ischemic etiology of HF (73%). Physical frailty was identified in 42% of the sample and associated with female gender, higher body mass index, more comorbidities, and HF with preserved ejection fraction. After adjusting for Seattle HF Model projected survival scores, comorbidities, body mass index, and gender, those physically frail had significantly lower plasma adiponectin, lower insulin-like growth factor-1, and lower myostatin compared with those non-physically frail (all p &lt; 0.05). There were no significant differences in other biomarkers between physical frailty groups. Conclusions: Biomarkers involved in adipose tissue and skeletal muscle development, maintenance, and function were significantly different between those physically frail and non-physically frail. Physical frailty in HF may be, in part, underpinned by dysfunctional peripheral processes involving the adipose tissue and skeletal muscle.

LncRNA NEAT1 Promote Inflammatory Responses in Coronary Slow Flow Through Regulating miR-148b-3p/ICAM-1 Axis.

BackgroundCoronary slow flow (CSF) is an angiographic phenomenon characterized by delayed coronary opacification with normal or near-normal epicardial coronary arteries. The pathogenesis of CSF is closely related to inflammatory response. Accumulating evidence shows that long non-coding RNAs (lncRNAs) play an important role in cardiovascular disease. However, the mechanism underlying the influence of the lncRNA nuclear enriched abundant transcripts 1 (NEAT1) on CSF is still unknown.Patients and MethodsForty CSF patients and forty control subjects were included in the study and underwent coronary angiography, Seattle angina questionnaire (SAQ) and echocardiography. The plasma levels of the inflammatory factors soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were determined by ELISA. The expression levels of NEAT1, miR-148b-3p and ICAM-1 in cells were measured by qRT-PCR or Western blotting. Cell proliferation was measured by 5‐Ethynyl‐2ʹ‐deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by apoptosis assay. The relationship between NEAT1 and miR-148b-3p was verified by luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay and avidin-biotin pull-down assay. The relationship between ICAM-1 and miR-148b-3p was verified by luciferase reporter gene assay and avidin-biotin pull-down assay.ResultsThis study showed that plasma sICAM-1, miR-148b-3p, and NEAT1 as independent predictors of a CSF diagnosis. Furthermore, plasma NEAT1 level showed superior diagnostic ability for CSF compared with sICAM-1 and miR-148b-3p. It was also shown that high expression of NEAT1 in oxygen-glucose deprivation (OGD)-treated human umbilical vein endothelial cells (HUVECs) functions as a competing endogenous RNA (ceRNA). By specifically binding miR-148b-3p, it weakened the negative regulatory effects of miR-148b-3p on the ICAM-1 target gene leading to upregulated expression of ICAM-1. This interaction was also shown to inhibit HUVEC proliferation and enhance apoptosis.ConclusionThis study demonstrated for the first time the important mechanism of action of the NEAT1/miR-148b-3p/ICAM-1 axis in the progression of CSF disease, and indicated the potential of NEAT1, miR-148b-3p, and ICAM-1 as a new target for the diagnosis and treatment of CSF.

Lipoprotein (a) and coronary artery calcification: prospective study assessing interactions with other risk factors

BackgroundElevated plasma lipoprotein (a) [Lp(a)] and coronary artery calcification (CAC) are established cardiovascular risk factors that correlate with each other. We hypothesized that other cardiovascular risk factors could affect their relationship. MethodsWe tested for interactions of 24 study variables related to dyslipidemia, diabetes, insulin resistance, hypertension, inflammation and coagulation with baseline Lp(a) on change in CAC volume and density over 9.5 years in 5975 Multi-Ethnic Study of Atherosclerosis (MESA) participants, free of apparent cardiovascular disease at baseline. ResultsElevated Lp(a) was associated with larger absolute increase in CAC volume (3.21 and 4.45 mm3/year higher for Lp(a) ≥30 versus <30 mg/dL, and Lp(a) ≥50 versus <50 mg/dL, respectively), but not relative change in CAC volume. No association was found with change in CAC density when assessing continuous ln-transformed Lp(a). The association between elevated Lp(a) (≥30 mg/dL) and absolute change in CAC volume was greater in participants with higher circulating levels of interleukin-2 soluble receptor α, soluble tumor necrosis factor alpha receptor 1 and fibrinogen (15.33, 11.81 and 7.02 mm3/year in quartile 4, compared to −3.44, −0.59 and 1.91 mm3/year in quartile 1, respectively). No significant interaction was found for other study variables. Similar interactions were seen when assessing Lp(a) levels ≥50 mg/dL. ConclusionsElevated Lp(a) was associated with an absolute increase in CAC volume, especially in participants with higher levels of selected markers of inflammation and coagulation. These results suggest Lp(a) as a potential biomarker for CAC volume progression.

Periodontal Clinicoradiographic Status and Whole Saliva Soluble Urokinase Plasminogen Activation Receptor and Tumor Necrosis Factor Alpha Levels in Type-2 Diabetic and Non-diabetic Individuals.

The authors hypothesise that whole saliva soluble-urokinase-type plasminogen-activator receptor (suPAR) and tumor necrosis factor-alpha (TNF-α) levels are higher in patients with poorly-controlled than well-controlled type-2 diabetes mellitus (DM) and non-diabetic controls. The aim was to assess the periodontal clinicoradiographic status and whole-salivary suPAR and TNF-α levels in type-2 diabetic and non-diabetic individuals. Patients with and without type-2 DM were included. In all patients, hemoglobin A1c (HbA1c) levels were measured. Participants were divided into 4 groups. Group 1: patients with poorly controlled type-2 DM; group 2: patients with well-controlled type-2 DM; group 3: non-diabetic patients with periodontitis; group 4: non-diabetic patients without periodontitis. Clinicoradiographic periodontal parameters (plaque index [PI], gingival index [GI], clinical attachment loss [AL], probing depth [PD] and mesial and distal marginal bone loss [MBL]) were measured. The whole saliva total protein concentration (TPC) and suPAR as well as TNF-α levels were measured. The level of statistical significance was set at p < 0.01. One hundred patients (25 patients per group) were included. Scores of PI (p < 0.01), GI (p < 0.01), clinical AL (p < 0.01), PD (p < 0.01), number of missing teeth and mesial (p < 0.01) and distal (p < 0.01) MBL were statistically significantly higher in group 1 than in groups 2-4. Scores of PI, GI, clinical AL, PD, mesial and distal MBL, and numbers of missing teeth were higher in group 3 (p < 0.01) than in groups 2 and 4. The whole saliva TPC, suPAR and TNF-α levels were statistically significantly higher among patients in group 1 (p < 0.01) than in groups 2-4. Patients with poorly-controlled type-2 DM presented with poorer clinicoradiographic periodontal status and increased whole saliva levels of suPAR, TNF-α and TPC compared with patients with well-controlled type-2 DM and non-diabetic individuals.

Pharmacological Inhibition of Soluble Tumor Necrosis Factor-Alpha Two Weeks after High Thoracic Spinal Cord Injury Does Not Affect Sympathetic Hyperreflexia.

After a severe, high-level spinal cord injury (SCI), plasticity to intraspinal circuits below injury results in heightened spinal sympathetic reflex activity and detrimentally impacts peripheral organ systems. Such sympathetic hyperreflexia is immediately apparent as an episode of autonomic dysreflexia (AD), a life-threatening condition characterized by sudden hypertension and reflexive bradycardia following below-level sensory inputs; for example, pressure sores or impacted fecal matter. Over time, plasticity within the spinal sympathetic reflex (SSR) circuit contributes to the progressive intensification of AD events, as the frequency and severity of AD events increase greatly beginning ∼2 weeks post-injury (wpi). The neuroimmune system has been implicated in driving sympathetic hyperreflexia, as inhibition of the cytokine soluble tumor necrosis factor-alpha (sTNFα) using the biological mimetic XPro1595 beginning within days post-SCI has been shown to attenuate the development of AD. Here, we sought to further understand the effective therapeutic time window of XPro1595 to diminish sympathetic hyperreflexia, as indicated by AD. We delayed the commencement of continuous intrathecal administration of XPro1595 until 2 weeks after a complete, thoracic level 3 injury in adult rats. We examined the severity of colorectal distension-induced AD biweekly. We found that initiation of sTNFα inhibition at 2 wpi does not attenuate the severity or intensification of sympathetic hyperreflexia compared with saline-treated controls. Coupled with previous data from our group, these findings suggest that central sTNFα signaling must be targeted prior to 2 weeks post-SCI in order to decrease sympathetic hyperreflexia.

P1008THERAPY WITH SODIUM-GLUCOSE COTRANSPORTER TYPE 2 INHIBITORS INCREASES KLOTHO IN TYPE 2 DIABETIC PATIENTS

Abstract Background and Aims Klotho (KL) is a transmembrane protein that is expressed the highest in the tubular cells of the kidneys. In addition to membrane KL, a secreted form of this protein that is present in blood and urine is generated by ectodomain shedding of membrane KL from the cell surface or from the KL gene through alternative splicing. Preserved KL expression has been related to physiological protection in the human kidneys. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are a new class of antidiabetic drugs with important renal benefits. The aim of the present proof-of-concept study was to analyze if treatment with SGLT2i is associated with changes in soluble KL concentrations as well as with modifications in mRNA expression levels of the KL gene in renal tubular cells. Method Thirty-four patients (18 males and 16 males; mean age 61±5 years) with type 2 diabetes (mean diabetes time higher than 10 years) and CKD G2-A2, all treated with metformin and blockers of the renin-angiotensin system during more than one year, were included in the study. Twenty-four of them received SGLT2i during 6 months (8 empagliflozin, 8 canagliflozin, 8 dapagliflozin), and their data and evolution were compared with a group of 10 patients matched by age, sex and stage of CKD who received sulfonylureas or DPP4 inhibitors. Serum and urine levels of soluble KL and tumor necrosis factor-alpha (TNFa) were determined by ELISA. In addition, the effect of SGLT2i on mRNA expression levels of KL was assessed in vitro in renal tubular cells cultures. Results Baseline median values of serum and urinary KL and TNFa were similar in both groups. Urinary KL was inversely correlated with albuminuria and TNFa excretion (r = -0.39 and r = -0.37, respectively, P&amp;lt;0.05). There were no relationship between the serum and urinary levels of these molecules, suggesting their renal origin. At the end of the study, there was a similar improvement in metabolic control, with a reduction in blood pressure significantly higher in patients treated with SGLT2i. Estimated glomerular filtration rate and albuminuria decreased by 5.8% and 17%, respectively, in subjects receiving SGLT2i (P&amp;lt;0.0001), without changes in the control group. In patients under SGLT2i therapy there was a 21% significant reduction in urinary TNFa with a concomitant 41% increase in urinary KL (P&amp;lt;0.001); there was also a small but significant rise in serum KL (P&amp;lt;0.01 vs baseline). Partial correlation analysis showed that the changes in the urinary excretion of albumin and TNFa were associated with the variation in urinary KL after controlling for other variables (r = -0.50, P&amp;lt;0.01, and r = -0.41, P&amp;lt;0.05, respectively). Finally, renal tubular cells cultured with dapagliflozin showed a significant and dose-dependent increase of mRNA expression levels of KL. Conclusion Treatment with SGLT2i induces a reduction in albuminuria and modulates inflammation (as reflected by a decrease in the urinary excretion of TNFa), which is associated with a significant increase in soluble KL concentrations as well as mRNA expression levels of the KL gene. The preservation of KL by SGLT2i may be an important mechanism of renal protection in type 2 diabetes.

Plasma levels of soluble TNF receptors are associated with cardiac function in patients with Chagas heart disease

BackgroundThe soluble receptors tumor necrosis factor-alpha (sTNFRs) can lead to an increase in the expression of tumor necrosis factor, increasing its detrimental to systemic inflammatory activation in Chagas cardiomyopathy (ChC). However, the correlation between sTNFRs levels, echocardiographic, and functional levels in patients with ChC remains unknown. This study aimed to verify the correlation between the plasma sTNFRs levels, echocardiographic, and NYHA functional levels in patients with ChC. MethodsSixty-four patients with ChD (54 ± 2 years, 44% males, NYHA I-II) were evaluated by anamnesis protocol, echocardiography, and plasma sTNFR1 and sTNFR2 measurement. Linear regression analysis and Student's t-test were used as appropriate. ResultsHigher plasma sTNFR1 and sTNFR2 levels were associate with worse systolic function (R2 = 0.10; p = 0.008 and R2 = 0.44; p < 0.001) and cardiac dilation (R2 = 0.13; p = 0.002 and R2 = 0.43; p < 0.001). Patients with systolic dysfunction and cardiac dilatation had higher sTNFRs levels (p < 0.001). There were no significant differences among NYHA functional classes for both sTNFRs. ConclusionPlasma sTNFR1 and sTNFR2 levels are associated with greater cardiac dilation and poor systolic function in ChC patients.

Association of peripheral inflammatory markers with connectivity in large-scale functional brain networks of non-demented older adults

BackgroundSystemic inflammation has emerged as a risk factor for cognitive decline and Alzheimer’s disease, but inflammation’s effect on distributed brain networks is unclear. We examined the relationship between peripheral inflammatory markers and subsequent functional connectivity within five large-scale cognitive networks and evaluated the modifying role of cortical amyloid and APOE ε4 status. MethodsBlood levels of soluble tumor necrosis factor-alpha receptor-1 and interleukin 6 were assessed in 176 participants (at baseline mean age: 65 (SD 9) years; 63% women; 85% cognitively normal, 15% mild cognitive impairment (MCI)) and were combined to derive an Inflammatory Index. Approximately six years later, participants underwent resting-state functional magnetic resonance imaging to quantify functional connectivity; a subset of 137 participants also underwent 11C Pittsburgh compound-B (PiB) PET imaging to assess cortical amyloid burden. ResultsUsing linear regression models adjusted for demographic characteristics and cardiovascular risk factors, a higher Inflammatory Index was associated with lower connectivity within the Default Mode (β = −0.013; 95% CI: −0.023, −0.003) and the Dorsal Attention Networks (β = −0.017; 95% CI: −0.028, −0.006). The strength of these associations did not vary by amyloid status (positive/negative). However, there was a significant interaction between Inflammatory Index and APOE ε4 status, whereby ε4-positive participants with a higher Inflammatory Index demonstrated lower connectivity. Inflammatory Index was unrelated to connectivity within other large-scale cognitive networks (Control, Limbic, and Salience/Ventral Attention networks). ConclusionPeripheral pro-inflammatory signaling in older adults without dementia, especially among APOE ε4-positive individuals, is associated with altered connectivity within two large-scale cognitive networks.