CD73 contributes to the pathogenesis of fusion-negative rhabdomyosarcoma through the purinergic signaling pathway.

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and adolescents. Fusion-negative RMS (FN-RMS) accounts for more than 80% of all RMS cases. The long-term event-free survival rate for patients with high-grade FN-RMS is below 30%, highlighting the need for improved therapeutic strategies. CD73 is a 5' ectonucleotidase that hydrolyzes AMP to adenosine and regulates the purinergic signaling pathway. We found that CD73 is elevated in FN-RMS tumors that express high levels of TWIST2. While high expression of CD73 contributes to the pathogenesis of multiple cancers, its role in FN-RMS has not been investigated. We found that CD73 knockdown decreased FN-RMS cell growth while up-regulating the myogenic differentiation program. Moreover, mutation of the catalytic residues of CD73 rendered the protein enzymatically inactive and abolished its ability to stimulate FN-RMS growth. Overexpression of wildtype CD73, but not the catalytically inactive mutant, in CD73 knockdown FN-RMS cells restored their growth capacity. Likewise, treatment with an adenosine receptor A2A-B agonist partially rescued FN-RMS cell proliferation and bypassed the CD73 knockdown defective growth phenotype. These results demonstrate that the catalytic activity of CD73 contributes to the pathogenic growth of FN-RMS through the activation of the purinergic signaling pathway. Therefore, targeting CD73 and the purinergic signaling pathway represents a potential therapeutic approach for FN-RMS patients.

Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model.

Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.

Dynamic remodeling of host membranes by self-organizing bacterial effectors.

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.

A Bacterial Effector Mimics a Host HSP90 Client to Undermine Immunity.

The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity.

Protein AMPylation by an Evolutionarily Conserved Pseudokinase.

Approximately 10% of human protein kinases are believed to be inactive and named pseudokinases because they lack residues required for catalysis. Here, we show that the highly conserved pseudokinase selenoprotein-O (SelO) transfers AMP from ATP to Ser, Thr, and Tyr residues on protein substrates (AMPylation), uncovering a previously unrecognized activity for a member of the protein kinase superfamily. The crystal structure of a SelO homolog reveals a protein kinase-like fold with ATP flipped in the active site, thus providing a structural basis for catalysis. SelO pseudokinases localize to the mitochondria and AMPylate proteins involved in redox homeostasis. Consequently, SelO activity is necessary for the proper cellular response to oxidative stress. Our results suggest that AMPylation may be a more widespread post-translational modification than previously appreciated and that pseudokinases should be analyzed for alternative transferase activities.

Progression of coronary calcium and incident coronary heart disease events: MESA (Multi-Ethnic Study of Atherosclerosis).

OBJECTIVES: The study examined whether progression of coronary artery calcium (CAC) is a predictor of future coronary heart disease (CHD) events. BACKGROUND: CAC predicts CHD events and serial measurement of CAC has been proposed to evaluate atherosclerosis progression. METHODS: We studied 6,778 persons (52.8% female) aged 45 to 84 years from the MESA (Multi-Ethnic Study of Atherosclerosis) study. A total of 5,682 persons had baseline and follow-up CAC scans approximately 2.5 ± 0.8 years apart; multiple imputation was used to account for the remainder (n = 1,096) missing follow-up scans. Median follow-up duration from the baseline was 7.6 (max = 9.0) years. CAC change was assessed by absolute change between baseline and follow-up CAC. Cox proportional hazards regression providing hazard ratios (HRs) examined the relation of change in CAC with CHD events, adjusting for age, gender, ethnicity, baseline calcium score, and other risk factors. RESULTS: A total of 343 and 206 hard CHD events occurred. The annual change in CAC averaged 24.9 ± 65.3 Agatston units. Among persons without CAC at baseline (n = 3,396), a 5-unit annual change in CAC was associated with an adjusted HR (95% Confidence Interval) of 1.4 (1.0 to 1.9) for total and 1.5 (1.1 to 2.1) for hard CHD. Among those with CAC >0 at baseline, HRs (per 100 unit annual change) were 1.2 (1.1 to 1.4) and 1.3 (1.1 to 1.5), respectively. Among participants with baseline CAC, those with annual progression of ≥300 units had adjusted HRs of 3.8 (1.5 to 9.6) for total and 6.3 (1.9 to 21.5) for hard CHD compared to those without progression. CONCLUSIONS: Progression of CAC is associated with an increased risk for future hard and total CHD events.

Abdominal aortic calcium and multi-site atherosclerosis: the Multiethnic Study of Atherosclerosis.

BACKGROUND: Abdominal aortic calcification (AAC) is a measure of subclinical cardiovascular disease (CVD). Data are limited regarding its relation to other measures of atherosclerosis. METHODS: Among 1812 subjects (49% female, 21% black, 14% Chinese, and 25% Hispanic) within the population-based Multiethnic Study of Atherosclerosis, we examined the cross-sectional relation of AAC with coronary artery calcium (CAC), ankle brachial index (ABI), and carotid intimal medial thickness (CIMT), as well as multiple measures of subclinical CVD. RESULTS: AAC prevalence ranged from 34% in those aged 45-54 to 94% in those aged 75-84 (p < 0.0001), was highest in Caucasians (79%) and lowest in blacks (62%) (p < 0.0001). CAC prevalence, mean maximum CIMT ≥ 1mm, and ABI < 0.9 was greater in those with vs. without AAC: CAC 60% vs. 16%, CIMT 38% vs. 7%, and ABI 5% vs. 1% for women and CAC 80% vs. 37%, CIMT 43% vs. 16%, and ABI 4% vs. 2% for men (p < 0.01 for all except p < 0.05 for ABI in men). The substantially greater prevalence for CAC in men compared to women all ages is not seen for AAC. By age 65, 97% of men and 91% of women have AAC, CAC, increased CIMT, and/or low ABI. The presence of multi-site atherosclerosis (≥ 3 of the above) ranged from 20% in women to 30% in men (p < 0.001), was highest in Caucasians (28%) and lowest in Chinese (16%) and ranged from 5% in those aged 45-54 to 53% in those aged 75-84 (p < 0.01 to p < 0.001). Finally, increased AAC was associated with 2-3-old relative risks for the presence of increased CIMT, low ABI, or CAC. CONCLUSIONS: AAC is associated with an increased likelihood of other vascular atherosclerosis. Its additive prognostic value to these other measures is of further interest.

Risk factor control and adherence to treatment in patients with coronary heart disease in the Republic of Srpska, Bosnia and Herzegovina in 2005-2006.

INTRODUCTION: European treatment guidelines in persons with known coronary heart disease (CHD) focus on adherence to antiplatelet therapy, ß-blockers, ACE/ARBs, and lipid-lowering agents, with goals for blood pressure (BP) of < 140/90 mm Hg and LDL cholesterol of < 3.0 mmol/l. Data on adherence to these measures in Eastern Europe are limited. MATERIAL AND METHODS: The Third Republic of Srpska, Bosnia and Herzegovina, Coronary Prevention Study (ROSCOPS III) was conducted in 2005-2006 at 10 primary heath care centres in 601 patients (36% female, mean age 55 years) with CHD including acute myocardial infarction or ischaemia, coronary artery bypass graft, or angioplasty who were examined and interviewed at least 6 months after the event. We examined the proportion of subjects on recommended treatments and at goal for BP, LDL-C, and non-smoking. RESULTS: The proportion of subjects on recommended treatments included 61% for ß-blockers, 79% for ACE/ARBs, 63% for lipid-lowering agents and 74% for antiplatelet therapy. Only 30% of subjects were on all four of these treatments. 59% of subjects had BP at goal of < 140/90 mm Hg and 33% were controlled to < 130/80 mm Hg, 41% for LDL-C, and 88% were non-smokers. Improvements were seen in lipid-lowering and ACE/ARB drug use and non-smoking status from an earlier survey (ROSCOPS II) in 2002-2003. CONCLUSIONS: Our data show, despite improvement over recent years, that many persons with CHD in the Republic of Srpska, Bosnia and Herzegovina are neither on recommended treatments nor at target for BP and/or LDL-C. Improved efforts targeted at both physicians and patients to address these issues are needed.

Combined association of lipids and blood pressure in relation to incident cardiovascular disease in the elderly: the cardiovascular health study.

BACKGROUND: Hypertension and dyslipidemia are highly prevalent in the elderly. We studied the combined impact of both conditions on cardiovascular disease (CVD) events. METHODS: We studied 4,311 participants aged 65-98 (61.2% female) from the Cardiovascular Health Study (CHS), a longitudinal epidemiologic study, with no prior CVD. We evaluated the relation of low-density lipoprotein (LDL), high-density lipoprotein (HDL), or non-HDL-cholesterol combined with blood pressure (BP) categories to incident CVD-including coronary heart disease (CHD) (angina, myocardial infarction (MI), angioplasty, coronary bypass surgery, or CHD death), stroke, claudication, and CVD death over 15 years. RESULTS: CVD incidence (per 1,000 person years) ranged from 38.4 when BP <120/80 mm Hg and LDL-C <100 mg/dl to 94.8 when BP >or=160/100 mm Hg and LDL-C >or=160 mg/dl, and from 28.9 when BP <120/80 mm Hg and HDL >60 mg/dl to 87.1 for a BP >or=160/100 and HDL-C <40 mg/dl. Compared with those with BP <120/80 mm Hg with either LDL-C <100 mg/dl or HDL-C >60 mg/dl, hazard ratios (HRs) for CVD events were 2.1 when BP >or=160/100 mm Hg and LDL-C >or=160 mg/dl and 2.1 when BP >or=160/100 and HDL-C <40 mg/dl (all P < 0.01), with similar results for non-HDL-C. Elevated BP was associated with increased risk across all lipid levels. Increased LDL-C added risk mainly when BP <140/90 mm Hg, but lower HDL-C also predicted CVD in those with higher BP. CONCLUSION: Increased BP confers increased risks for CVD in elderly persons across all lipid levels. Although increased LDL-C added risk mainly when BP <140/90 mm Hg, low HDL-C added risk also in those with hypertension. These results document the importance of combined hypertension and dyslipidemia.

Cardiovascular comorbidities and blood pressure control in stroke survivors.

OBJECTIVE: To examine the association of cardiovascular(CV) comorbidities with the likelihood of being a stroke survivor and to determine the prevalence, treatment, and control of hypertension in this population. METHODS: In the National Health and Nutrition Examination Survey from 1999 to 2004, 495 stroke survivors, aged 20 years or older, were characterized by CV risk factors and comorbidities. Hypertension prevalence, treatment,and control rates were determined and logistic regression was performed to examine the likelihood of stroke according to the presence of comorbidities. RESULTS: Of the stroke survivors, 59.4% were women, 57.1%were at least 65 years of age, 66.2% were overweight/obese, 25.1% were current smokers, 75.1% had hypertension, and 79.3% had additional comorbidities,including diabetes mellitus (24.7%), coronary artery disease(28.8%), chronic kidney disease (25.3%), heart failure(16.5%), and peripheral arterial disease (10.9%). The odds(and 95% confidence intervals) of prevalent stroke were 2.2(1.5-3.2), 5.0 (3.2-7.8), 4.1 (2.1-7.8), and 10.0 (4.8-20.9)with one, two, three, and four or more comorbidities,respectively. Of these high-risk stroke survivors with comorbidities and hypertension, 18% were not receiving antihypertensive therapy; of those receiving therapy, 55.3%did not meet a systolic blood pressure goal of less than 140 mmHg and, on average, were 20 mmHg above this target. CONCLUSION: A high percentage of stroke survivors,projected to 4.98 million adults in the USA have multiple CV risk factors, numerous comorbidities, and poor control of hypertension, placing them at increased risk for further complications. Therefore, increased efforts must be made to reduce overall global risk in these high-risk persons.

Association of FVC and total mortality in US adults with metabolic syndrome and diabetes.

BACKGROUND: Reduced pulmonary function is an independent predictor of metabolic syndrome (MetS) and diabetes mellitus (DM), conditions associated with increased mortality. We investigated whether reduced pulmonary function is associated with increased mortality in persons with these conditions. METHODS: We examined 5,633 (projected, 62.4 million) US adults (age range, 18 to 79 years) in the Third National Health and Nutrition Examination Survey, who were never-smokers and were without known cardiovascular or obstructive lung disease. Cox regression (adjusted for age, sex, and ethnicity) was used to examine all-cause mortality risk across FVC categories (FVC: low, or= 95% predicted) among those with MetS, DM, or neither disease. RESULTS: The prevalence of DM and MetS significantly increased as predicted FVC decreased (p < 0.01). Age- and sex-adjusted mortality rates (per 1,000 person-years) increased in a stepwise manner as predicted FVC decreased in those patients with neither MetS nor DM (3.5 to 8.0), MetS (4.1 to 8.1), and DM (9.9 to 13.3). Compared to those with high FVC, those with low FVC had more than a fourfold increase in mortality among those with MetS (hazard ratio [HR], 4.27; 95% confidence interval [CI], 1.59 to 11.45; p < 0.01) and more than a twofold increase among those with neither disease (HR, 2.40; 95% CI, 1.06 to 5.43; p < 0.05). Also, every 10% reduction in FVC was associated with a 77% higher mortality (HR, 1.77; 95% CI, 1.33 to 2.37; p < 0.05) among persons with MetS. However, in those with DM, FVC did not contribute further to mortality risk. CONCLUSION: In persons with MetS, a reduced FVC is associated with further increases in mortality, suggesting that the evaluation of lung function may be useful for risk stratification in those with MetS.