Citations

Abstract Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes the generation of memory populations following acute infections. However, data suggest that P2RX7 may limit the efficacy of antitumor responses. Herein, we show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T-cell adoptive transfer model. Tumor-specific P2rx7–/– CD8+ T cells exhibited impaired mitochondrial maintenance and function but did not display signs of overt exhaustion early in the antitumor response. However, as the tumor burden increased, the relative frequency of P2RX7-deficient CD8+ T cells declined within the tumor; this correlated with reduced proliferation, increased apoptosis, and mitochondrial dysfunction. Extending these studies, we found that the transient in vitro stimulation of P2RX7 using the ATP analogue BzATP led to enhanced B16 melanoma control by CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and underscore the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy.

AbstractAlzheimer's disease (AD) is a pervasive neurodegeneration disease with high heritability. In this study, we employed CRISPR‐Cas9‐engineered technology to investigate the effects of a rare mutation (rs144662445) in the A kinase anchoring protein 9 (AKAP9) gene, which is associated with AD in African Americans (AA), on tau pathology and the tau interactome in SH‐SY5Y P301L neuron‐like cells. The mutation significantly increased the level of phosphorylated tau, specifically at the site Ser396/Ser404. Moreover, analyses of the tau interactome measured by affinity purification‐mass spectrometry revealed that differentially expressed tau‐interacting proteins in AKAP9 mutant cells were associated with RNA translation, RNA localization and oxidative activity, recapitulating the tau interactome signature previously reported with human AD brain samples. Importantly, these results were further validated by functional studies showing a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared with wild type cells in a tau‐dependent manner, which are mirrored with pathological phenotype frequently seen in AD. Our results demonstrated specific effects of rs14462445 on mis‐processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non–Small Cell Lung Cancer (NSCLC) provide recommended management for patients with NSCLC, including diagnosis, primary treatment, surveillance for relapse, and subsequent treatment. Patients with metastatic lung cancer who are eligible for targeted therapies or immunotherapies are now surviving longer. This selection from the NCCN Guidelines for NSCLC focuses on targeted therapies for patients with metastatic NSCLC and actionable mutations.

Background: There are good data to support using a single high-sensitivity cardiac troponin T (hs-cTnT) below the limit of detection of 5 ng/L to exclude acute myocardial infarction. Per the US Food and Drug Administration, hs-cTnT can only report to the limit of quantitation of 6 ng/L, a threshold for which there are limited data. Our goal was to determine whether a single hs-cTnT below the limit of quantitation of 6 ng/L is a safe strategy to identify patients at low risk for acute myocardial injury and infarction. Methods: The efficacy (proportion identified as low risk based on baseline hs-cTnT<6 ng/L) of identifying low-risk patients was examined in a multicenter (n=22 sites) US cohort study of emergency department patients undergoing at least 1 hs-cTnT (CV Data Mart Biomarker cohort). We then determined the performance of a single hs-cTnT<6 ng/L (biomarker alone) to exclude acute myocardial injury (subsequent hs-cTnT >99th percentile in those with an initial hs-cTnT<6 ng/L). The clinically intended rule-out strategy combining a nonischemic ECG with a baseline hs-cTnT<6 ng/L was subsequently tested in an adjudicated cohort in which the diagnostic performance for ruling out acute myocardial infarction and safety (myocardial infarction or death at 30 days) were evaluated. Results: A total of 85 610 patients were evaluated in the CV Data Mart Biomarker cohort, among which 24 646 (29%) had a baseline hs-cTnT<6 ng/L. Women were more likely than men to have hs-cTnT<6 ng/L (38% versus 20%, P <0.0001). Among 11 962 patients with baseline hs-cTnT<6 ng/L and serial measurements, only 1.2% developed acute myocardial injury, resulting in a negative predictive value of 98.8% (95% CI, 98.6–99.0) and sensitivity of 99.6% (95% CI, 99.5–99.6). In the adjudicated cohort, a nonischemic ECG with hs-cTnT<6 ng/L identified 33% of patients (610/1849) as low risk and resulted in a negative predictive value and sensitivity of 100% and a 30-day rate of 0.2% for myocardial infarction or death. Conclusions: A single hs-cTnT below the limit of quantitation of 6 ng/L is a safe and rapid method to identify a substantial number of patients at very low risk for acute myocardial injury and infarction.

ABSTRACT Background Penile length loss and erectile dysfunction (ED) are common post-prostatectomy. Recent results from a randomized, controlled study (RCT) of RestoreX penile traction therapy (RxPTT) demonstrated preserved erectile function and penile length improvements in men post-prostatectomy. Objective To report outcomes of the open label phase (6-9 month time point) of the previously noted RCT comparing efficacy of RxPTT to control in men post-prostatectomy. Methods A randomized, controlled trial (NCT03500419) has been completed of 82 men post-prostatectomy. Men were randomized 1-month post-prostatectomy to no therapy (control, n=27) or RxPTT (n=55) for 5 months. Participants then entered an open label phase for 3 months. Assessments included stretched penile length (corona), standardized (International Index of Erectile Function [IIEF]), and non-standardized questionnaires. Comparisons were performed of men who utilized traction at least 15 minutes daily for both phases to men who were originally control and continued with no treatment (as-treated analysis). Analyses were also performed of men who crossed over from control to &gt;15 min daily (crossover to treatment) and those who were originally in the treatment group and continued with &gt;15 min daily use during the open label phase (ongoing treatment). Results Of the 82 men enrolled (mean age 58.6), 55 had 6-month, and 42 had 9-month data available to review. Baseline characteristics (IIEF scores, penile length) were similar between cohorts. In the as-treated analysis (no treatment n=7, RxPTT x 8 months n=12, respectively), differences were as noted: IIEF-EFD (-8 vs -0.5, p=0.19), IIEF-OFD (-3 vs -2, p=0.86), IIEF-SD (-1 vs 0.5, p=0.09), IIEF-ISD (-3 vs -0.5, p=0.31), IIEF-OSD (-2 vs 0, p=0.29), penile length (-0.1 vs +1.8 cm, p&lt;0.01), PDE5 use (86% vs 75%, p=0.57), intracavernosal injection use (86% vs 17%, p&lt;0.01). Comparing men who did not use therapy at any time (n=7) to those who crossed over to RxPTT during the open label phase (n=8) demonstrated the following results: IIEF-EFD (-8 vs -6, p=0.49), IIEF-OFD (-3 vs -1, p=0.90), IIEF-SD (-1 vs 0, p=0.55), IIEF-ISD (-3 vs -1, p=0.45), IIEF-OSD (-2 vs -1, p=0.51), penile length (-0.1 vs +0.69 cm, p=0.39). Men who crossed over from RxPTT to no treatment (n=10) had relatively preserved improvements compared to those who continued with RxPTT during the open label phase (n=12) compared to baseline, respectively: IIEF-EFD (0 vs -0.5, p=0.95), IIEF-OFD (-3.5 vs -2, p=0.97), IIEF-SD (0 vs 0.5, p=0.18), IIEF-ISD (0 vs -0.5, p=0.87), IIEF-OSD (0 vs 0, p=0.66), penile length (+1.8 vs +1.8, p=0.99). Conclusions Although sample size and statistical power are limited, results from the open label phase confirm earlier findings of preserved sexual function and penile length in men treated with RxPTT compared to controls. Men who crossed over from controls to treatment experience benefits, although not as significant as those who were treated beginning at 1 month post-prostatectomy. Men who stopped therapy after 6 months demonstrated stable improvements from baseline, suggesting a preserved effect of RxPTT even after discontinuing use. If validated with external series, these findings would represent a significant advancement in the prevention of sexual dysfunction post-prostatectomy. Disclosure Work supported by industry: no.

Objective To determine the harms of proprotein convertase subtilisin/kexin 9 (PCSK9) inhibitors in people who need lipid-lowering therapy. Methods This systematic review included randomised controlled trials that compared PCSK9 inhibitors with placebo, standard care or active lipid-lowering comparators in people who need lipid-lowering therapy with the follow-up duration of at least 24 weeks. We summarised the relative effects for potential harms from PCSK9 inhibitors using random-effect pairwise meta-analyses and assessed the certainty of evidence using GRADE (Grading of Recommendation Assessment, Development and Evaluation) for each outcome. Results We included 32 trials with 65 861 participants (with the median follow-up duration of 40 weeks, ranging from 24 to 146 weeks). The meta-analysis showed an incidence of injection-site reaction leading to discontinuation (absolute incidence of 15 events (95% CI 11 to 20) per 1000 persons in a 5-year time frame, high certainty evidence). PCSK9 inhibitors do not increase the risk of new-onset diabetes mellitus, neurocognitive events, cataracts or gastrointestinal haemorrhage with high certainty evidence. PCSK9 inhibitors probably do not increase the risks of myalgia or muscular pain leading to discontinuation or any adverse events leading to discontinuation with moderate evidence certainty. Given very limited evidence, PCSK9 inhibitors might not increase influenza-like symptoms leading to discontinuation (risk ratio 1.5; 95% CI 0.06 to 36.58). We did not identify credible subgroup analyses results, including shorter versus longer follow-up duration of trials. Conclusions PCSK9 inhibitors slightly increase the risk of severe injection-site reaction but not cataracts, gastrointestinal haemorrhage, neurocognitive events, new-onset diabetes or severe myalgia or muscular pain.

Irritable bowel syndrome is a globally prevalent disorder categorized into constipation, diarrhea or mixed (IBS‐C, IBD‐D, IBS‐M) based on the predominant stool form/frequency. While the gut microbiome is recognized as an important factor underlying the pathophysiology of IBS, the specific mechanisms remain an area of active investigation. In our recent longitudinal multi‐omics study in IBS patients, we found bacteria‐derived hypoxanthine was significantly decreased in stool from IBS‐C patients. The aim of this study was to determine if IBS‐C relevant molecular pathways are affected by hypoxanthine. We hypothesized that hypoxanthine acts on enterochromaffin (EC) cells to stimulate serotonin (5‐HT) release, where 5‐HT is an important determinant of gastrointestinal (GI) transit time. We used Ca 2+ imaging in QGP cells (EC model), T84 cells (colonocyte model), primary cells and organoids from NeuroD1‐cre;GCaMP5‐tdTomato mice [NeuroD1 is a marker for enteroendocrine cells (EECs) which express calcium indicator GCaMP in these mice] in the absence or presence of receptor antagonists, and in ex vivo colon preparations with intact innervation from E2aCre:GCaMP6s mice (GCaMP is expressed in neuronal and non‐neuronal cells), to test the effect of hypoxanthine. The physiologic relevance of hypoxanthine signaling was assessed by measuring whole gut transit time. We found hypoxanthine increased Ca 2+ influx in QGP cells [using Cal520 (qualitative) and Fura‐2AM (quantitative)], primary EECs and EECs within organoids (previous research showed that the majority of these EECs are EC cells), but not in T84 cells. Hypoxanthine‐evoked Ca 2+ response was absent in Ca 2+ ‐free medium (n=6‐8, t‐test, P <0.0001), inhibited by G αi/o ‐GPCR antagonist (pertussis toxin; n=8‐14, t‐test, P <0.01)), Transient Receptor Potential Cation Channel Subfamily C Member 4 (TRPC4) antagonist (ML204; n=14‐15, t‐test, P <0.0001) and TRPC4 siRNA (n=5‐21, t‐test, P <0.01), and was accompanied by increased 5‐HT release (measured using 5‐HT biosensor). In ex vivo experiments, luminal application of hypoxanthine activated a subset of epithelial cells (likely EC cells) followed by subsets of neurons (latency to response onset, n=4, Mann‐Whitney, P <0.0001), as well as movement of the imaging field, which indicates smooth muscle contraction/relaxation. Our in vitro findings were confirmed by in vivo experiments showing that germ‐free mice colonized with hypoxanthine‐producing bacteria had faster whole gut transit and higher stool 5‐HT level compared to hypoxanthine‐consuming bacteria (n=7‐8, t‐test, P <0.05). In summary, hypoxanthine increases Ca 2+ influx specifically in EC cells by activating a signaling cascade involving a G αi/o ‐GPCR and TRPC4 channels, which results in 5‐HT release. The delay in activation of myenteric neurons and motor activity suggests that EC cell 5‐HT release likely induces neurogenic motor patterns and may underlie the shorter GI transit time seen with increased bacterial hypoxanthine production. While the current empirically designed probiotics have proven ineffective in IBS, our findings will allow development of novel mechanism‐based probiotic therapies for IBS‐C using hypoxanthine‐producing bacteria.

Background Extracellular DNA traps (ETs) have important implications in both thrombosis and thrombolysis. Thus, developing benchtop thrombus analogs that recapitulate clinical ETs is potentially of great value for preclinical development and testing of thrombolytic agents and thrombectomy devices. In this study, we aimed to develop ETs-rich thrombus analogs for preclinical testing. Methods Red blood cell (RBC)-rich, fibrin-rich, and platelet-rich thrombus analogs were created using human whole blood, platelet-poor plasma, and platelet-rich plasma obtained from the blood bank following institutional approval. Peripheral blood mononuclear cells (9.9×106 cells/mL) isolated from human whole blood and lipopolysaccharide (1 µg/mL) were added to induce ETs. Histochemical, immunohistochemistry and immunofluorescence were used to identify thrombus components and ETs. Scanning electronic microscopy was used to investigate the ultrastructure of the thrombus analogs. The thrombus compositions, morphologic features of ETs and citrullinated histone H3 (H3Cit) expression were compared with those of thrombi retrieved from patients by thrombectomy. Results ETs-rich thrombus analogs were more compacted th-an the ETs-poor thrombus analogs. ETs were identified in both ETs-rich thrombus analogs and patient thrombi showing morphologic features including nuclear lobulation, nuclear swelling, diffused chromatin within cytoplasm, DNA/chromatin extending intracellularly and extracellularly, and extracellular chromatin patches and bundles. In the ETs-poor thrombus analogs, ETs were not observed and H3Cit expression was absent to minimal. The compositions and H3Cit expression in the ETs-rich thrombus analogs fell in the range of patient thrombi. Conclusions ETs-rich thrombus analogs can be consistently created in vitro and may benefit the preclinical development and testing of new thrombolytic agents and thrombectomy devices.

ABSTRACT Introduction Penile length loss and erectile dysfunction (ED) are common post-prostatectomy. Recent results from a randomized, controlled study (RCT) of RestoreX penile traction therapy (RxPTT) demonstrated preserved erectile function and penile length improvements in men post-prostatectomy. Objective To report outcomes of the open label phase (6-9 month time point) of the previously noted RCT comparing efficacy of RxPTT to control in men post-prostatectomy. Methods A randomized, controlled trial (NCT03500419) has been completed of 82 men post-prostatectomy. Men were randomized 1-month post-prostatectomy to no therapy (control, n=27) or RxPTT (n=55) for 5 months. Participants then entered an open label phase for 3 months. Assessments included stretched penile length (corona), standardized (International Index of Erectile Function [IIEF]), and non-standardized questionnaires. Comparisons were performed of men who utilized traction at least 15 minutes daily for both phases to men who were originally control and continued with no treatment (as-treated analysis). Analyses were also performed of men who crossed over from control to &gt;15 min daily (crossover to treatment) and those who were originally in the treatment group and continued with &gt;15 min daily use during the open label phase (ongoing treatment). Results Of the 82 men enrolled (mean age 58.6), 55 had 6-month, and 42 had 9-month data available to review. Baseline characteristics (IIEF scores, penile length) were similar between cohorts. In the as-treated analysis (no treatment n=7, RxPTT x 8 months n=12, respectively), differences were as noted: IIEF-EFD (-8 vs -0.5, p=0.19), IIEF-OFD (-3 vs -2, p=0.86), IIEF-SD (-1 vs 0.5, p=0.09), IIEF-ISD (-3 vs -0.5, p=0.31), IIEF-OSD (-2 vs 0, p=0.29), penile length (-0.1 vs +1.8 cm, p&lt;0.01), PDE5 use (86% vs 75%, p=0.57), intracavernosal injection use (86% vs 17%, p&lt;0.01). Comparing men who did not use therapy at any time (n=7) to those who crossed over to RxPTT during the open label phase (n=8) demonstrated the following results: IIEF-EFD (-8 vs -6, p=0.49), IIEF-OFD (-3 vs -1, p=0.90), IIEF-SD (-1 vs 0, p=0.55), IIEF-ISD (-3 vs -1, p=0.45), IIEF-OSD (-2 vs -1, p=0.51), penile length (-0.1 vs +0.69 cm, p=0.39). Men who crossed over from RxPTT to no treatment (n=10) had relatively preserved improvements compared to those who continued with RxPTT during the open label phase (n=12) compared to baseline, respectively: IIEF-EFD (0 vs -0.5, p=0.95), IIEF-OFD (-3.5 vs -2, p=0.97), IIEF-SD (0 vs 0.5, p=0.18), IIEF-ISD (0 vs -0.5, p=0.87), IIEF-OSD (0 vs 0, p=0.66), penile length (+1.8 vs +1.8, p=0.99). Conclusions Although sample size and statistical power are limited, results from the open label phase confirm earlier findings of preserved sexual function and penile length in men treated with RxPTT compared to controls. Men who crossed over from controls to treatment experience benefits, although not as significant as those who were treated beginning at 1 month post-prostatectomy. Men who stopped therapy after 6 months demonstrated stable improvements from baseline, suggesting a preserved effect of RxPTT even after discontinuing use. If validated with external series, these findings would represent a significant advancement in the prevention of sexual dysfunction post-prostatectomy. Disclosure No

ABSTRACT Introduction Erectile dysfunction (ED) is a known precursor and sequelae to diagnosed cardiovascular disease (CVD). ED AUA guidelines recommend evaluating standard CV risk factors including serum lipids and penile doppler to help stratify CV risk, but few patients are referred for further evaluation. Plasma ceramides are metabolic byproducts that are novel predictors of adverse cardiovascular events resulting from unstable atherosclerotic plaque. CVD risk conferred by ceramide levels has been shown to be independent of conventional risk factors such as LDL and hsCRP levels. Thus, Plasma MI-Heart Ceramide is a new test that useful for evaluating shorter term major adverse cardiovascular events (MACE) in the next 1 to 5 years. Objective We sought to evaluate the utility of ceramide levels in ED patients Methods We initiated ceramide testing in all patients with ED in late 2019 (in addition to lipids and Hgba1c). We queried our electronic medical record for ceramide levels and men with a diagnosis of ED. Ceramide levels stratified patients in normal, moderate, high, and very high risk. Twenty eight patients additionally had penile duplex ultrasound (DUS). Left and right peak systolic velocity values were averaged and a value of &lt;25 cm/s was defined as penile arterial insufficiency. Results 245 men with ED and ceramide levels were analyzed. 44% (n=107) were normal, 42% (n=103) were moderate risk, 12% (n=29) were high risk and 2% (n=6) were very high risk. As result of their ceramide and office evaluation, 42% (15/35) of high/very high risk patients were referred for cardiac evaluation. Comparing ceramide scores in men with penile arterial insufficiency (16.7 cm/sec) vs patients without (43.2 cm/sec), we found no significant difference (p=1). Similarly, when comparing DUS results between ceramide risk categories, we found no significant differences (p=1). Conclusions One in 7 men presenting to an ED clinic had a high or very high ceramide risk. DUS results did not correlate with ceramide risk. Patients undergoing DUS (even without arterial insufficiency) may benefit from additional ceramide testing and possible cardiac referral. Further study is warranted to optimize utility of ceramide in men with ED and potentially decrease peri-procedural IPP cardiac morbidity. Disclosure No