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Start Preamble Centers for Medicare & viagra online no prescription. Medicaid Services (CMS), HHS. Extension of timeline for publication of final rule. This notice announces an extension of the viagra online no prescription timeline for publication of a Medicare final rule in accordance with the Social Security Act, which allows us to extend the timeline for publication of the final rule. As of August 26, 2020, the timeline for publication of the final rule to finalize the provisions of the October 17, 2019 proposed rule (84 FR 55766) is extended until August 31, 2021.

Start Further Info Lisa O. Wilson, (410) viagra online no prescription 786-8852. End Further Info End Preamble Start Supplemental Information In the October 17, 2019 Federal Register (84 FR 55766), we published a proposed rule that addressed undue regulatory impact and burden of the physician self-referral law. The proposed rule was issued in conjunction with the Centers for Medicare &. Medicaid Services' (CMS) Patients over Paperwork initiative and the Department of Health and Human Services' (the Department or HHS) Regulatory Sprint to Coordinated viagra online no prescription Care.

In the proposed rule, we proposed exceptions to the physician self-referral law for certain value-based compensation arrangements between or among physicians, providers, and suppliers. A new exception for certain arrangements under which a physician receives limited remuneration for items or services actually provided by the physician. A new exception for donations of cybersecurity technology and related viagra online no prescription services. And amendments to the existing exception for electronic health records (EHR) items and services. The proposed rule also provides critically necessary guidance for physicians and health care providers and suppliers whose financial relationships are governed by the physician self-referral statute and regulations.

This notice announces an extension of the timeline for publication of the viagra online no prescription final rule and the continuation of effectiveness of the proposed rule. Section 1871(a)(3)(A) of the Social Security Act (the Act) requires us to establish and publish a regular timeline for the publication of final regulations based on the previous publication of a proposed regulation. In accordance with section 1871(a)(3)(B) of the Act, the timeline may vary among different regulations based on differences in the complexity of the regulation, the number and scope of comments received, and other relevant factors, but may not be longer than 3 years except under exceptional circumstances. In addition, in accordance with section 1871(a)(3)(B) of the Act, the Secretary may extend the initial targeted publication date of the viagra online no prescription final regulation if the Secretary, no later than the regulation's previously established proposed publication date, publishes a notice with the new target date, and such notice includes a brief explanation of the justification for the variation. We announced in the Spring 2020 Unified Agenda (June 30, 2020, www.reginfo.gov) that we would issue the final rule in August 2020.

However, we are still working through the Start Printed Page 52941complexity of the issues raised by comments received on the proposed rule and therefore we are not able to meet the announced publication target date. This notice extends the timeline for publication of viagra online no prescription the final rule until August 31, 2021. Start Signature Dated. August 24, 2020. Wilma M.

Robinson, Deputy Executive Secretary to the Department, Department of Health viagra online no prescription and Human Services. End Signature End Supplemental Information [FR Doc. 2020-18867 Filed 8-26-20. 8:45 am]BILLING CODE 4120-01-PToday, viagra online no prescription the U.S. Department of Health and Human Services (HHS), through the Health Resources and Services Administration (HRSA), announced over $117 million in quality improvement awards to 1,318 health centers across all U.S.

States, territories and the District of Columbia. HRSA-funded health centers will use these funds to further strengthen quality improvement activities and expand quality primary health care service delivery.“These quality improvement awards support health centers across the country in delivering care to nearly 30 viagra online no prescription million people, providing a convenient source of quality care that has grown even more important during the COVID-19 pandemic,” said HHS Secretary Alex Azar. €œThese awards help ensure that all patients who visit a HRSA-funded health center continue to receive the highest quality of care, including access to COVID-19 testing and treatment.”Health centers deliver comprehensive care to people who are low-income, uninsured or face other obstacles to getting health care. On top of the safety-net that they provide, health centers have been on the front lines preventing and responding to the COVID-19 public health emergency, including providing over 3 million COVID-19 tests. Health centers continue to provide essential services for our nation’s most vulnerable and medically underserved populations, including those who often do not have access to care, before, during and after the COVID-19 pandemic.HRSA’s quality improvement awards recognize the highest performing health centers nationwide as well as those health centers that have made significant quality viagra online no prescription improvements from the previous year.Health centers are recognized for achievements in various areas.

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€‚For the podcast associated with this article, please visit https://academic.oup.com/eurheartj/pages/Podcasts.This Focus Issue on heart failure (HF) provides novel clinically relevant information on sodium–glucose co-transporter-2 (SGLT2) inhibitors which, initially proposed for the treatment of type 2 diabetes mellitus (T2D), have been found to improve the outcome of HF with reduced ejection fraction (HFrEF) when administered on the top of drugs known to improve the outcome of HF and are recommended in current European Guidelines.1,2Acording to modelling estimates, when compared with no neurohormonal blockade, the use of a broad-based combination of disease-modifying drugs at target doses in patients with HF viagra ebay may reduce the risk of death by Related Site as much as 75%. It is surprising that in spite of this powerful therapeutic armamentarium, <1% of patients with chronic HF are currently receiving recommended drugs at doses that have been shown to prolong life.3 The issue opens with a Current Opinion article entitled ‘Totality of evidence in trials of sodium–glucose co-transporter-2 inhibitors in the patients with heart failure with reduced ejection fraction. Implications for viagra ebay clinical practice’ by Milton Packer from the Baylor University Medical Center at Dallas in Texas, USA and colleagues.

The authors provide a perspective on the totality of evidence with SGLT2 inhibitors in patients with HFrEF.4 This paper is the first to issue a call for a major change in clinical practice based on the concordant results of DAPA-HF and EMPEROR-Reduced trials. The analyses and interpretations that are presented in this manuscript will undoubtedly generate considerable discussion and debate for a long time.Concern about hypotension often leads to withholding of beneficial therapy in patients with HFrEF. In a clinical research manuscript entitled ‘Effect of dapagliflozin according to baseline systolic blood pressure in the Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure viagra ebay trial (DAPA-HF)’ John McMurray from the Western Infirmary in Glasgow, UK and colleagues on behalf of the DAPA-HF Investigators and Committees evaluated the efficacy and safety of dapagliflozin according to baseline systolic blood pressure (SBP) in DAPA-HF trial.5 Key inclusion criteria were.

New York Heart Association (NYHA) class II–IV, left ventricular ejection fraction (LVEF) ≤40%, elevated N-terminal probrain natriuretic peptide (NT-proBNP) level, and SBP ≥95 mmHg. The primary outcome was a composite viagra ebay of worsening HF or cardiovascular death. The efficacy and safety of dapagliflozin was examined using SBP as both a categorical and a continuous variable.

The placebo-corrected reduction in SBP from baseline to 2 weeks with dapagliflozin was –2.54 mmHg. The benefit and safety of viagra ebay dapagliflozin were consistent across the range of SBP. Study drug discontinuation did not differ between dapagliflozin and placebo across the SBP categories examined.The authors conclude that dapagliflozin had a small effect on SBP in patients with HFrEF and was superior to placebo in improving outcomes, and well tolerated, across the range of SBP included in DAPA-HF.

The manuscript is accompanied by an Editorial by Francesco Cosentino from the University Hospital Solna in Stockholm, Sweden who comments viagra ebay that altogether, the results of the current post-hoc analysis demonstrating efficacy and safety of dapagliflozin regardless of SBP values might significantly contribute to foster the implementation of dapagliflozin use in HF clinical practice by dissipating any potential safety concern linked with its hypotensive effects.6In a clinical research article entitled ‘A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes. The DAPA-LVH trial’, Chim Lang from the University of Dundee in the UK and colleagues tested the hypothesis that dapagliflozin may regress left ventricular hypertrophy (LVH) in people with T2D.7 The authors randomly assigned 66 patients with T2D, LVH, and controlled blood pressure to receive dapagliflozin 10 mg once daily or placebo for 12 months. The primary endpoint was change in absolute left ventricular mass (LVM), assessed by cardiac magnetic resonance imaging (MRI).

In the intention-to-treat analysis, dapagliflozin significantly reduced LVM compared with placebo, with an viagra ebay absolute mean change of –2.82 g. Additional sensitivity analysis adjusting for baseline LVM, baseline blood pressure, weight, and SBP change showed the LVM change to remain statistically significant. Dapagliflozin significantly reduced pre-specified secondary endpoints including ambulatory 24-h SBP, nocturnal SBP, body weight, visceral adipose tissue, subcutaneous adipose tissue, insulin resistance, and high-sensitivity C-reactive protein viagra ebay.

Figure 1Column bar charts showing the mean regression of left ventricular mass following dapagliflozin treatment compared to placebo (from Brown AJM, Gandy S, McCrimmon R, Houston JG, Struthers AD, Lang CC. A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes. The DAPA-LVH viagra ebay trial.

See pages 3421–3432).Figure 1Column bar charts showing the mean regression of left ventricular mass following dapagliflozin treatment compared to placebo (from Brown AJM, Gandy S, McCrimmon R, Houston JG, Struthers AD, Lang CC. A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes. The DAPA-LVH viagra ebay trial.

See pages 3421–3432).Lang and colleagues conclude that dapagliflozin treatment significantly reduced LVM in patients with T2D and LVH. The regression of LVM suggests that dapagliflozin can initiate reverse remodelling and changes in left ventricular structure that may partly contribute to cardioprotective effects viagra ebay of dapagliflozin. This manuscript is accompanied by an Editorial by Francesco Paneni from the University of Zurich in Switzerland and colleagues.8 They note that the above-mentioned effects of SGLT2 inhibitors set the ground for a possible beneficial effect of these drugs in patients with HFpEF, where microvascular dysfunction, cardiomyocyte inflammation, and cardiometabolic alterations take centre stage.While several landmark studies have long established that implantable cardioverter-defibrillator (ICD) therapy improves survival for primary prevention of sudden cardiac death ,9 risk stratification parameters and methods for this purpose are clinically underused.

In a clinical research article entitled ‘Clinical effectiveness of primary prevention implantable cardioverter-defibrillators. Results of the EU-CERT-ICD controlled multicentre cohort study’ Markus Zabel from the Universitätsmedizin Göttingen in Germany and colleagues from the EU-CERT-ICD Study Investigators assessed the current clinical effectiveness of primary prevention by ICD therapy in a prospective investigator-initiated, controlled cohort study, conducted in 44 centres and viagra ebay 15 European countries. The study sought to assess current clinical effectiveness of primary prophylactic ICD implantation.10 The authors recruited 2327 patients with ischaemic or dilated cardiomyopathy and guideline indications for prophylactic ICD implantation.

The primary endpoint was all-cause viagra ebay mortality. Baseline and follow-up data from 2247 patients were analysable. 1516 patients with first ICD implantation (ICD group) and 731 patients without ICD serving as controls.

Multivariable models and propensity scoring for adjustment were viagra ebay used to compare the two groups for mortality. Adjusted mortality associated with ICD vs. Control was significantly lower (hazard ratio 0.731) viagra ebay.

Subgroup analyses indicated no ICD benefit in diabetics or in those aged ≥75 years. Figure 2Secondary efficacy endpoints comparing cardiosphere-derived cells and placebo at 6 months. Change in (A) viagra ebay left ventricular end-diastolic volume.

(B) left ventricular end-systolic volume. And (C) N-terminal pro b-type natriuretic peptide levels. At 6 viagra ebay months.

CDC, cardiosphere-derived cell. LVEDV, left ventricular end-diastolic viagra ebay volume. LVESV, left ventricular end-systolic volume.

NT-proBNP, N-terminal pro b-type natriuretic peptide (from Makkar RR, Kereiakes DJ, Aguirre F, Kowalchuk G, Chakravarty T, Malliaras K, Francis GS, Povsic TJ, Schatz R, Traverse JH, Pogoda JM, Smith RR, Marbán L, Ascheim DD, Ostovaneh MR, Lima JAC, DeMaria A, Marbán E, Henry TD. Intracoronary ALLogeneic heart STem cells to Achieve viagra ebay myocardial Regeneration (ALLSTAR). A randomized, placebo-controlled, double-blinded trial.

See pages viagra ebay 3451--3458).Figure 2Secondary efficacy endpoints comparing cardiosphere-derived cells and placebo at 6 months. Change in (A) left ventricular end-diastolic volume. (B) left ventricular end-systolic volume.

And (C) N-terminal viagra ebay pro b-type natriuretic peptide levels. At 6 months. CDC, cardiosphere-derived viagra ebay cell.

LVEDV, left ventricular end-diastolic volume. LVESV, left ventricular end-systolic volume. NT-proBNP, N-terminal pro b-type natriuretic peptide (from Makkar RR, Kereiakes DJ, Aguirre F, Kowalchuk G, Chakravarty T, Malliaras K, Francis GS, Povsic TJ, Schatz R, Traverse JH, Pogoda JM, Smith RR, Marbán L, viagra ebay Ascheim DD, Ostovaneh MR, Lima JAC, DeMaria A, Marbán E, Henry TD.

Intracoronary ALLogeneic heart STem cells to Achieve myocardial Regeneration (ALLSTAR). A randomized, placebo-controlled, double-blinded trial. See pages 3451--3458).The authors conclude that in contemporary ischaemic/dilated cardiomyopathy patients (LVEF ≤35%, narrow QRS), primary prophylactic ICD treatment was associated with a substantial reduction in mortality, although this improvement was not viagra ebay consistent across the whole population.

The manuscript is accompanied by an Editorial by N.A. Mark Estes III from the Heart and Vascular Institute UPMC in Pittsburgh, Pennsylvania, USA.11 The authors note that clinicians should be mindful of available risk stratification models and subgroup analyses from the EU-CERT-ICD and other studies viagra ebay. It follows that the process of shared decision-making should include careful consideration of the patient’s wishes and values, with an individualized assessment of potential benefit and risks of primary prevention of sudden death by ICD implantation.Cardiosphere-derived cells (CDCs) are cardiac progenitor cells which exhibit disease-modifying bioactivity in various models of cardiomyopathy and in previous clinical studies of acute myocardial infarction (MI), dilated cardiomyopathy, and Duchenne muscular dystrophy.12,13 In a clinical research article entitled ‘Intracoronary ALLogeneic heart STem cells to Achieve myocardial Regeneration (ALLSTAR).

A randomized, placebo-controlled, double-blinded trial’, Raj Makkar from the Cedars-Sinai Heart Institute in Los Angeles, California, USA and colleagues assessed the safety and efficacy of intracoronary administration of allogeneic CDCs in the multicentre, randomized, double-blind, placebo-controlled, intracoronary ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration (ALLSTAR) trial.14 The authors enrolled patients 4 weeks to 12 months after MI, with LVEF ≤45% and left ventricular LV scar size ≥15% of LVM by MRI. A pre-specified interim analysis was performed when 6-month MRI data were viagra ebay available. The trial was subsequently stopped due to the low probability of detecting a significant treatment effect of CDCs based on the primary endpoint.

Patients were randomly allocated in viagra ebay a 2:1 ratio to receive CDCs or placebo in the infarct-related artery by the stop–flow technique. The primary safety endpoint was the occurrence, during 1-month post-intracoronary infusion, of acute myocarditis attributable to allogeneic CDCs, ventricular tachycardia- or ventricular fibrillation-related death, sudden unexpected death, or a major adverse cardiac event (death or hospitalization for HF or non-fatal MI). The primary efficacy endpoint was the relative percentage change in infarct size at 12 months post-infusion as assessed by contrast-enhanced cardiac MRI.

Makkar and colleagues randomly allocated 90 patients to the CDC group and 44 viagra ebay to the placebo group. The mean baseline LVEF was 40% and the mean scar size was 22% of the LVM. No primary viagra ebay safety endpoint events occurred.

There was no difference in the percentage change from baseline in scar size between CDC and placebo groups at 6 months. Compared with placebo, there were significant reductions in LV end-diastolic volume, LV end-systolic volume, and NT-proBNP at 6 months in CDC-treated patients.The authors conclude that intracoronary infusion of allogeneic CDCs in patients with post-MI left ventricular dysfunction was safe but did not reduce scar size relative to placebo at 6 months. The manuscript is accompanied by an Editorial by Francisco Fernandez-Aviles viagra ebay from the Hospital General Universitario Gregorio Marañón in Madrid, Spain and colleagues.15 The authors feel that various points need to be better addressed before proceeding again to clinical trials, if we want to move the field of cardiovascular regenerative and reparative medicine forward, for the sake of the cardiovascular health of millions of patients.Treatment of pathological cardiac remodelling and subsequent HF represents an unmet clinical need.

Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes including that of heart diseases.16,17 In a Basic Science article entitled ‘Targeting muscle-enriched long non-coding RNA H19 reverses pathological cardiac hypertrophy’, Thomas Thum from the Hannover Medical School in Germany, and colleagues report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy.18 Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase, but a strong sustained repression upon reaching the decompensated phase of HF. The translational potential of H19 was highlighted by its repression in a large animal (pig) model of LVH, in diseased human heart samples, in human stem cell-derived cardiomyocytes, and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy viagra ebay in H19 knockout mice was aggravated compared with wild-type mice.

In contrast, vector-based, cardiomyocyte-directed gene therapy using murine but also human H19 strongly attenuated HF even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses, and chromatin immunoprecipitation-DNA sequencing, the authors identified a link viagra ebay between H19 and prohypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the antihypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity.Thum and colleagues conclude that H19 is highly conserved and down-regulated in failing hearts from mice, pigs, and humans.

H19 gene therapy prevents and reverses experimental pressure overload-induced HF. H19 acts viagra ebay as an antihypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling. The manuscript is accompanied by an Editorial by Gianluigi Condorelli from the Humanitas University in Rozzano, Italy and colleagues.

The authors note that dysregulation of epigenetic mechanisms leading to aberrant loss of cardiomyocyte homeostasis is a critical point to consider in understanding the onset of cardiovascular viagra ebay pathologies. Thus exploiting lncRNAs as therapeutic agents in myocardial disease could pave the way for efficaciously combatting one of the greatest healthcare burdens worldwide.19With the advent of omics, an innovative inductive method has provided researchers with possible ways new to monitor health and disease. This approach incorporates data from studies of the genome, transcriptome, proteome, and metabolome to focus on the assessment of a varied range of biomolecules.20 In a clinical review article entitled ‘Omics phenotyping in heart failure.

The next frontier’ Antoni Bayes-Genis from the Cardiology Service, Hospital Universitari Germans Trias i Pujol in Badalona, Spain and colleagues provide a state-of-the-art review aiming to provide an up-to-date look at breakthrough omic technologies that are helping to unravel HF disease mechanisms and heterogeneity.21 Genomics, transcriptomics, proteomics, and metabolomics in HF viagra ebay are reviewed in depth. In addition, there is a thorough, expert discussion regarding the value of omics in identifying novel disease pathways, advancing understanding of disease mechanisms, differentiating HF phenotypes, yielding biomarkers for diagnosis or prognosis, or identifying new therapeutic targets in HF. The combination of multiple omics technologies may create a more comprehensive picture of the factors viagra ebay and pathophysiology involved in HF than achieved by either one alone, and provides a rich resource for predictive phenotype modelling.

However, the successful translation of omics tools as solutions to clinical HF requires that the observations are robust and reproducible, and can be validated across multiple independent populations to ensure confidence in clinical decision-making.This issue is also complemented by a Discussion Forum contribution. In a contribution entitled ‘Heart failure development in obesity. Mechanistic pathways’ Kristjan Karason from the Sahlgrenska University Hospital in Gothenburg, Sweden and viagra ebay colleagues provide a reply to a recent comment entitled ‘Incident heart failure risk after bariatric surgery.

The role of epicardial fat’.22,23The editors hope that this issue of the European Heart Journal will be of interest to its readers.With thanks to Amelia Meier-Batschelet, Johanna Hugger, and Martin Meyer for help with compilation of this article. References1Docherty KF, Jhund PS, Inzucchi SE, Køber L, Kosiborod MN, Martinez FA, Ponikowski P, DeMets DL, Sabatine MS, Bengtsson O, Sjöstrand M, Langkilde AM, Desai AS, Diez M, Howlett JG, Katova T, Ljungman CEA, O’Meara E, Petrie MC, Schou M, Verma S, Vinh PN, Solomon SD, McMurray JJV. Effects of dapagliflozin in DAPA-HF according to background heart viagra ebay failure therapy.

Eur Heart J 2020;41:2379–2392.2Ponikowski P, Voors AA,, Anker SD, Bueno H, Cleland JGF, Coats AJS, Falk V, González-Juanatey JR, Harjola VP, Jankowska EA, Jessup M, Linde C, Nihoyannopoulos P, Parissis JT, Pieske B, Riley JP, Rosano GMC, Ruilope LM, Ruschitzka F, Rutten FH, van der Meer P. 2016 ESC Guidelines for the diagnosis and treatment of acute viagra ebay and chronic heart failure. The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC).

Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart viagra ebay J 2016;37:2129–2200.3Packer M. Are the benefits of SGLT2 inhibitors in heart failure and a reduced ejection fraction influenced by background therapy?.

Expectations viagra ebay and realities of a new standard of care. Eur Heart J 2020;41:2393–2396.4Butler J, Zannad F, Filippatos G, Anker SD, Packer M. Totality of evidence in trials of sodium–glucose co-transporter-2 inhibitors in the patients with heart failure with reduced ejection fraction.

Implications for clinical practice viagra ebay. Eur Heart J 2020;41:3398–3401.5Serenelli M, Böhm M, Inzucchi SE, Køber L, Kosiborod MN, Martinez FA, Ponikowski P,, Sabatine MS, Solomon SD, DeMets DL, Bengtsson O, Sjöstrand M, Langkilde AM, Anand IS, Chiang CE, Chopra VK, de Boer RA, Diez M, Dukát A, Ge J, Howlett JG, Katova T, Kitakaze M, Ljungman CEA, Verma S,, Docherty KF, Jhund PS, McMurray JJV. Effect of dapagliflozin according to baseline systolic blood pressure viagra ebay in the Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure trial (DAPA-HF).

Eur Heart J 2020;41:3402–3418.6Savarese G, Cosentino F. The interaction between dapagliflozin and blood pressure in heart failure. New evidence dissipating concerns viagra ebay.

Eur Heart J 2020;41:3419–3420.7Brown AJM, Gandy S, McCrimmon R, Houston JG, Struthers AD, Lang CC. A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people viagra ebay with type two diabetes. The DAPA-LVH trial.

Eur Heart J 2020;41:3421–3432.8Paneni F, Costantino S, Hamdani N. Regression of viagra ebay left ventricular hypertrophy with SGLT2 inhibitors. Eur Heart J 2020;41:3433–3436.9Priori SG, Blomström-Lundqvist C, Mazzanti A, Blom N, Borggrefe M, Camm J, Elliott PM, Fitzsimons D, Hatala R, Hindricks G, Kirchhof P, Kjeldsen K, Kuck KH, Hernandez-Madrid A, Nikolaou N, Norekvål TM, Spaulding C, Van Veldhuisen DJ.

2015 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of sudden cardiac death. The Task viagra ebay Force for the Management of Patients with Ventricular Arrhythmias and the Prevention of Sudden Cardiac Death of the European Society of Cardiology (ESC). Endorsed by.

Association for viagra ebay European Paediatric and Congenital Cardiology (AEPC). Eur Heart J 2015;36:2793–2867.10Zabel M, Willems R, Lubinski A, Bauer A, Brugada J, Conen D, Flevari P, Hasenfuß G, Svetlosak M, Huikuri HV, Malik M, Pavlović N, Schmidt G, Sritharan R, Schlögl S, Szavits-Nossan J, Traykov V, Tuinenburg AE, Willich SN, Harden M, Friede T, Svendsen JH, Sticherling C, Merkely B. Clinical effectiveness of primary prevention implantable cardioverter-defibrillators.

Results of viagra ebay the EU-CERT-ICD controlled multicentre cohort study. Eur Heart J 2020;41:3437–3447.11Estes MNA, Saba S. Primary prevention of sudden death viagra ebay with the implantable cardioverter defibrillator.

Bridging the evidence gap. Eur Heart J 2020;41:3448–3450.12Aminzadeh MA, Tseliou E, Sun B, Cheng K, Malliaras K, Makkar RR, Marbán E. Therapeutic efficacy of cardiosphere-derived cells in a transgenic mouse model of non-ischaemic dilated viagra ebay cardiomyopathy.

Eur Heart J 2015;36:751–762.13Fadini GP, Mehta A, Dhindsa DS, Bonora BM, Sreejit G, Nagareddy P, Quyyumi AA. Circulating stem viagra ebay cells and cardiovascular outcomes. From basic science to the clinic.

Eur Heart J 2020. Doi:10.1093/eurheartj/ehz923.14Makkar RR, Kereiakes DJ, Aguirre F, Kowalchuk G, Chakravarty T, Malliaras K, Francis GS, Povsic TJ, viagra ebay Schatz R, Traverse JH, Pogoda JM, Smith RR, Marbán L, Ascheim DD, Ostovaneh MR, Lima JAC, DeMaria A, Marbán E, Henry TD. Intracoronary ALLogeneic heart STem cells to Achieve myocardial Regeneration (ALLSTAR).

A randomized, placebo-controlled, double-blinded trial. Eur Heart J 2020;41:3451–3458.15Sanz-Ruiz R, viagra ebay Fernández-Avilés F. Cardiovascular regenerative and reparative medicine.

Is myocardial infarction the model? viagra ebay. Eur Heart J 2020;41:3459–3461.16Ounzain S, Micheletti R, Beckmann T, Schroen B, Alexanian M, Pezzuto I, Crippa S, Nemir M, Sarre A, Johnson R, Dauvillier J, Burdet F, Ibberson M, Guigó R, Xenarios I, Heymans S, Pedrazzini T. Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

Eur Heart viagra ebay J 2015;36:353–368.17Lüscher TF. Novel molecular mechanisms of vascular disease. Non-coding RNAs, inflammation, viagra ebay and radiation.

Eur Heart J. 2020;40:2467–2470.18Viereck J, Bührke A, Foinquinos A, Chatterjee S, Kleeberger JA, Xiao K, Janssen-Peters H, Batkai S, Ramanujam D, Kraft T, Cebotari S, Gueler F, Beyer AM, Schmitz J, Bräsen JH, Schmitto JD, Gyöngyösi M, Löser A, Hirt MN, Eschenhagen T, Engelhardt S, Bär C, Thum T. Targeting muscle-enriched viagra ebay long non-coding RNA H19 reverses pathological cardiac hypertrophy.

Eur Heart J 2020;41:3462–3474.19Pagiatakis C, Hall IF, Condorelli G. Long non-coding viagra ebay RNA H19. A new avenue for RNA therapeutics in cardiac hypertrophy?.

Eur Heart J 2020;41:3475–3476.20Hoogeveen RM, Pereira JPB, Nurmohamed NS, Zampoleri V, Bom MJ, Baragetti A, Boekholdt SM, Knaapen P, Khaw KT, Wareham NJ, Groen AK, Catapano AL, Koenig W, Levin E, Stroes ESG. Improved cardiovascular risk prediction using targeted plasma proteomics viagra ebay in primary prevention. Eur Heart J 2020;ehaa648.

21Bayes-Genis A, Liu PP, Lanfear DE, de Boer RA, González A, Thum T, Emdin M, Januzzi JL. Omics phenotyping viagra ebay in heart failure. The next frontier.

Eur Heart J viagra ebay 2020;41:3477–3484.22Karason K, Jamaly S. Heart failure development in obesity. Mechanistic pathways.

Eur Heart J viagra ebay 2020;41:3485.23van Woerden G, van Veldhuisen SL, Rienstra M. Incident heart failure risk after bariatric surgery. The role viagra ebay of epicardial fat.

Eur Heart J 2020;41:1775. Published on behalf of the European Society of Cardiology. All rights viagra ebay reserved.

© The Author(s) 2020. For permissions, viagra ebay please email. Journals.permissions@oup.com.Case presentationA 32-year-old cardiology resident was scheduled to round on the COVID-19 wards at a large, government teaching hospital in Bahrain.

To cover the increasing workload, the hospital required additional medical personnel to provide care for the numerous COVID-19 patients that were being seen. Prior to examining COVID-19-positive patients, she donned appropriate personal protective equipment (PPE)—a gown, gloves, N95 viagra ebay mask, and face shield. As part of her physical exam, she was obliged to auscultate her patients with a stethoscope, listening for cardiopulmonary abnormalities that can be comorbid with severe COVID-19 infection.

Thus, she was required to unzip her gown and keep her stethoscope either in her ears or around her neck. She used a standard-length Littman Cardiology™ stethoscope, requiring her to be in close viagra ebay proximity to the patient (i.e. Lean over to the patient’s level).One day after her rounds, she developed a sore throat.

She subsequently was tested positive for COVID-19 via polymerase chain reaction (PCR) viagra ebay. The resident cardiologist remembered one patient that she had examined where she suspected the transmission occurred. She recalls examining a patient who was COVID-19 positive.

Prior to the patient’s intubation she applied her viagra ebay own stethoscope directly to the patient’s chest to perform auscultation. The resident was perspiring and beginning to feel exhausted from her prior rounding and was breathing heavily as she unzipped her gown to place the stethoscope back within. The resident believes that COVID-19 viral particles which were transmitted to the stethoscope became aerosolized and inhaled as she brought the stethoscope close to her mouth while tucking it back into viagra ebay her gown.

The resident recovered, re-tested negative for COVID-19, and has now returned to her normal duties.The COVID-19 pandemic has called into question the triple-faceted role of the stethoscope. A diagnostic tool, symbol of patient–provider connection, and possible vector for infectious disease (Figure 1). A recent article in viagra ebay the American Journal of Medicine discusses developments in each arm of this triple role with reference to COVID-19, arguing that developments in stethoscope diagnostic technology, a need to bolster clinical skills, and developments in stethoscope hygiene methods will perpetuate both its relevance and safety.

This argument was made in light of those who believe the stethoscope will become obsolete with the development of more advanced technologies, as well as its potential to transmit disease.1 It is clear that a contaminated stethoscope might pose a danger to patients and providers, and can be a potential vector for the transmission of COVID-19, as illustrated in the case above. Thus, providers should seek to educate themselves on stethoscope contamination, viagra ebay assess the current methods of hygiene, and innovate accordingly rather than cast the stethoscope aside. Figure 1The three-faceted role of the stethoscope.

The stethoscope lies at the intersection of three roles in medicine. Diagnostic tool viagra ebay. Connection between provider and patients.

And a potential vector for infectious disease. As increased infection control vigilance has placed the stethoscope in a position of contention viagra ebay. Each facet of the stethoscope must be weighed in consideration of medicines’s cherished symbol.Figure 1The three-faceted role of the stethoscope.

The stethoscope viagra ebay lies at the intersection of three roles in medicine. Diagnostic tool. Connection between provider and patients.

And a potential viagra ebay vector for infectious disease. As increased infection control vigilance has placed the stethoscope in a position of contention. Each facet viagra ebay of the stethoscope must be weighed in consideration of medicines’s cherished symbol.Studies have demonstrated that stethoscopes can harbour similar levels and types of microbes to those on one’s hand.2 Thus, it is no surprise that the stethoscope has been christened as the physician’s ‘third hand’, with reference both to its potential for pathogen transmission and its integral role in patient–provider connection.

Despite this, no clear guidelines exist for performing stethoscope hygiene. The Centers for Disease Control (CDC) classifies the stethoscope as a ‘non-critical’ medical device (i.e. Only in contact with intact skin, not with bodily fluids), and recommends cleaning between as often as after contact with each viagra ebay patient to once weekly using an alcohol or bleach-based disinfectant.3 It has been demonstrated that viruses, including COVID-19,4 are capable of surviving on skin and other surfaces for an extended period of time.5 Thus, current guidelines may not adequately reflect the risk that stethoscope contamination poses.COVID-19 has fostered an era of increased infection control vigilance, and thus the benefits of the stethoscope must be rationally weighed against the risks.

In the vignette posed here, the cardiology resident felt the need to use her stethoscope to assess the COVID-19 patients on her round. Her likely rationale was the utility it provides in assessing viagra ebay the variety of cardiopulmonary abnormalities that can manifest during a COVID-19 infection. One of the most common manifestations of COVID-19 infection is multifocal pneumonia, often occurring prior to acute respiratory distress and need for mechanical ventilation.6 While pneumonia is diagnosed most definitively using imaging modalities (CT and X-ray) and laboratory testing, resource-limited scenarios might necessitate the usage of a stethoscope to listen for pulmonary indications (coarse breath sounds).

Furthermore, there is growing evidence that cardiovascular disease is highly comorbid with COVID-19 infection, leading to worse outcomes. The most common cardiovascular comorbidities among hospitalized COVID-19 patients are hypertension, coronary artery disease, and diabetes mellitus.7,8 In addition, recent viagra ebay reports have implicated COVID-19 in causing myocardial injury and left ventricular systolic dysfunction.9 Considering the sequelae of COVID-19 cardiopulmonary manifestations, auscultation using a stethoscope can be highly warranted. Therefore, emphasis must be placed on ensuring that the stethoscope can be used safely.Assessments of stethoscope hygiene practices have widely demonstrated deficits in adherence and method.

Direct observational studies have demonstrated stethoscope hygiene rates using recommended methods (wiping with alcohol, bleach, hydrogen peroxide, etc.) between 11.3% and 24%, with unconventional practices also being reported such as placing a glove over the stethoscope prior to auscultation or washing it with water/hand towel in a sink.10,11 Such findings imply that while stethoscope hygiene practices are deficient, providers who are cognizant of stethoscope contamination are struggling to find an effective form of hygiene that does not impede workflow—a proverbial ‘cry for help.’ With regard to current methods of stethoscope hygiene, providers cite lack of access to cleaning supplies, forgetfulness, or a lack of time as reasons for not performing stethoscope hygiene.12Healthcare guidelines advise against using personal stethoscopes in contact precaution settings in order to limit the potential for cross-contamination. Rather, single-patient viagra ebay disposable stethoscopes are often used for such patients. However, the audio quality of single-patient stethoscopes is quite poor,13 and it has been demonstrated that these stethoscopes can be contaminated with pathogens that can potentially be transmitted to providers, who must share this stethoscope.14 Proper cleaning of these stethoscopes between usage may not occur in high-workflow environments, such as the intensive care unit (ICU).

Thus, a more feasible and effective modality of stethoscope hygiene is viagra ebay warranted.A ray of hope for stethoscope hygiene is technological innovation. Among the solutions presented in recent years have been a UV-LED case for the stethoscope diaphragm,1, stethoscopes made from antimicrobial copper alloys,16 and disposable stethoscope diaphragm covers.17 The challenge imposed by the first two innovations is a lack of complete microbial disinfection. Given that it is unknown what viral dose threshold corresponds to COVID-19 pathogenesis, current infection control standards might necessitate a method that ensures zero transmission.

Stethoscope diaphragm covers alone can provide an aseptic contact surface during auscultation,17 but one is likely to encounter the same impediments stated for conventional stethoscope cleaning.12 A company based in San Diego, USA (AseptiScope Inc., San Diego, CA, USA) has attempted to overcome this issue by developing a touch-free diaphragm barrier dispenser.1 A recent article discussed the role of stethoscope contamination during COVID-19, stating that a specific barrier for the stethoscope is needed to prevent stethoscope contamination and subsequent transmission to patients and providers.18 A touch-free stethoscope diaphragm dispenser might be a feasible solution for this need.In the viagra ebay era of COVID-19, the stethoscope carries both profound utility as well as risk to patients if effective hygiene practices are not implemented. Thus, providers need to exercise caution when auscultating patients with COVID-19 given the risk for cross-contamination. However, rather than casting aside the stethoscope due to this risk, safety should be bolstered through education, hygiene practice, and viagra ebay consideration of innovative solutions.Conflict of interest.

A.S.M. Is a co-founder and the Chief Clinical Officer for AseptiScope Inc. (San Diego, viagra ebay CA, USA).

None of the other authors have conflicts to disclose. ReferencesReferences are available viagra ebay as supplementary material at European Heart Journal online. Published on behalf of the European Society of Cardiology.

All rights reserved. © The viagra ebay Author(s) 2020. For permissions, please email.

€‚For the podcast associated with this article, please visit https://academic.oup.com/eurheartj/pages/Podcasts.This Focus Issue on heart failure (HF) provides novel clinically relevant information on sodium–glucose co-transporter-2 (SGLT2) inhibitors which, initially proposed for the treatment of type 2 diabetes mellitus (T2D), have been found to improve the outcome of HF with reduced ejection fraction (HFrEF) when administered on the top of drugs known to improve the outcome of HF and are recommended in current European Guidelines.1,2Acording to modelling estimates, when compared with no neurohormonal blockade, the use of a broad-based combination of disease-modifying drugs viagra online no prescription at target doses in patients with HF may reduce the risk of death by as much as 75%. It is surprising that in spite of this powerful therapeutic armamentarium, <1% of patients with chronic HF are currently receiving recommended drugs at doses that have been shown to prolong life.3 The issue opens with a Current Opinion article entitled ‘Totality of evidence in trials of sodium–glucose co-transporter-2 inhibitors in the patients with heart failure with reduced ejection fraction. Implications for clinical viagra online no prescription practice’ by Milton Packer from the Baylor University Medical Center at Dallas in Texas, USA and colleagues.

The authors provide a perspective on the totality of evidence with SGLT2 inhibitors in patients with HFrEF.4 This paper is the first to issue a call for a major change in clinical practice based on the concordant results of DAPA-HF and EMPEROR-Reduced trials. The analyses and interpretations that are presented in this manuscript will undoubtedly generate considerable discussion and debate for a long time.Concern about hypotension often leads to withholding of beneficial therapy in patients with HFrEF. In a clinical research manuscript entitled ‘Effect of dapagliflozin according to baseline systolic blood pressure in the Dapagliflozin and Prevention of Adverse Outcomes in Heart Failure trial (DAPA-HF)’ John McMurray from the Western Infirmary in Glasgow, UK and colleagues on behalf of the DAPA-HF Investigators and Committees evaluated the efficacy and safety of dapagliflozin according to baseline systolic blood pressure (SBP) viagra online no prescription in DAPA-HF trial.5 Key inclusion criteria were.

New York Heart Association (NYHA) class II–IV, left ventricular ejection fraction (LVEF) ≤40%, elevated N-terminal probrain natriuretic peptide (NT-proBNP) level, and SBP ≥95 mmHg. The primary outcome was a composite of worsening HF viagra online no prescription or cardiovascular death. The efficacy and safety of dapagliflozin was examined using SBP as both a categorical and a continuous variable.

The placebo-corrected reduction in SBP from baseline to 2 weeks with dapagliflozin was –2.54 mmHg. The benefit and safety of dapagliflozin were consistent across the range viagra online no prescription of SBP. Study drug discontinuation did not differ between dapagliflozin and placebo across the SBP categories examined.The authors conclude that dapagliflozin had a small effect on SBP in patients with HFrEF and was superior to placebo in improving outcomes, and well tolerated, across the range of SBP included in DAPA-HF.

The manuscript is accompanied by an Editorial by Francesco Cosentino from the University Hospital Solna in Stockholm, Sweden viagra online no prescription who comments that altogether, the results of the current post-hoc analysis demonstrating efficacy and safety of dapagliflozin regardless of SBP values might significantly contribute to foster the implementation of dapagliflozin use in HF clinical practice by dissipating any potential safety concern linked with its hypotensive effects.6In a clinical research article entitled ‘A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes. The DAPA-LVH trial’, Chim Lang from the University of Dundee in the UK and colleagues tested the hypothesis that dapagliflozin may regress left ventricular hypertrophy (LVH) in people with T2D.7 The authors randomly assigned 66 patients with T2D, LVH, and controlled blood pressure to receive dapagliflozin 10 mg once daily or placebo for 12 months. The primary endpoint was change in absolute left ventricular mass (LVM), assessed by cardiac magnetic resonance imaging (MRI).

In the viagra online no prescription intention-to-treat analysis, dapagliflozin significantly reduced LVM compared with placebo, with an absolute mean change of –2.82 g. Additional sensitivity analysis adjusting for baseline LVM, baseline blood pressure, weight, and SBP change showed the LVM change to remain statistically significant. Dapagliflozin significantly reduced pre-specified secondary endpoints including ambulatory 24-h SBP, nocturnal SBP, body weight, visceral adipose viagra online no prescription tissue, subcutaneous adipose tissue, insulin resistance, and high-sensitivity C-reactive protein.

Figure 1Column bar charts showing the mean regression of left ventricular mass following dapagliflozin treatment compared to placebo (from Brown AJM, Gandy S, McCrimmon R, Houston JG, Struthers AD, Lang CC. A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes. The DAPA-LVH viagra online no prescription trial.

See pages 3421–3432).Figure 1Column bar charts showing the mean regression of left ventricular mass following dapagliflozin treatment compared to placebo (from Brown AJM, Gandy S, McCrimmon R, Houston JG, Struthers AD, Lang CC. A randomized controlled trial of dapagliflozin on left ventricular hypertrophy in people with type two diabetes. The DAPA-LVH viagra online no prescription trial.

See pages 3421–3432).Lang and colleagues conclude that dapagliflozin treatment significantly reduced LVM in patients with T2D and LVH. The regression of LVM suggests that dapagliflozin viagra online no prescription can initiate reverse remodelling and changes in left ventricular structure that may partly contribute to cardioprotective effects of dapagliflozin. This manuscript is accompanied by an Editorial by Francesco Paneni from the University of Zurich in Switzerland and colleagues.8 They note that the above-mentioned effects of SGLT2 inhibitors set the ground for a possible beneficial effect of these drugs in patients with HFpEF, where microvascular dysfunction, cardiomyocyte inflammation, and cardiometabolic alterations take centre stage.While several landmark studies have long established that implantable cardioverter-defibrillator (ICD) therapy improves survival for primary prevention of sudden cardiac death ,9 risk stratification parameters and methods for this purpose are clinically underused.

In a clinical research article entitled ‘Clinical effectiveness of primary prevention implantable cardioverter-defibrillators. Results of the EU-CERT-ICD controlled viagra online no prescription multicentre cohort study’ Markus Zabel from the Universitätsmedizin Göttingen in Germany and colleagues from the EU-CERT-ICD Study Investigators assessed the current clinical effectiveness of primary prevention by ICD therapy in a prospective investigator-initiated, controlled cohort study, conducted in 44 centres and 15 European countries. The study sought to assess current clinical effectiveness of primary prophylactic ICD implantation.10 The authors recruited 2327 patients with ischaemic or dilated cardiomyopathy and guideline indications for prophylactic ICD implantation.

The primary viagra online no prescription endpoint was all-cause mortality. Baseline and follow-up data from 2247 patients were analysable. 1516 patients with first ICD implantation (ICD group) and 731 patients without ICD serving as controls.

Multivariable models and viagra online no prescription propensity scoring for adjustment were used to compare the two groups for mortality. Adjusted mortality associated with ICD vs. Control was significantly viagra online no prescription lower (hazard ratio 0.731).

Subgroup analyses indicated no ICD benefit in diabetics or in those aged ≥75 years. Figure 2Secondary efficacy endpoints comparing cardiosphere-derived cells and placebo at 6 months. Change in (A) left ventricular end-diastolic volume viagra online no prescription.

(B) left ventricular end-systolic volume. And (C) N-terminal pro b-type natriuretic peptide levels. At 6 viagra online no prescription months.

CDC, cardiosphere-derived cell. LVEDV, left viagra online no prescription ventricular end-diastolic volume. LVESV, left ventricular end-systolic volume.

NT-proBNP, N-terminal pro b-type natriuretic peptide (from Makkar RR, Kereiakes DJ, Aguirre F, Kowalchuk G, Chakravarty T, Malliaras K, Francis GS, Povsic TJ, Schatz R, Traverse JH, Pogoda JM, Smith RR, Marbán L, Ascheim DD, Ostovaneh MR, Lima JAC, DeMaria A, Marbán E, Henry TD. Intracoronary ALLogeneic viagra online no prescription heart STem cells to Achieve myocardial Regeneration (ALLSTAR). A randomized, placebo-controlled, double-blinded trial.

See pages 3451--3458).Figure 2Secondary efficacy endpoints viagra online no prescription comparing cardiosphere-derived cells and placebo at 6 months. Change in (A) left ventricular end-diastolic volume. (B) left ventricular end-systolic volume.

And (C) N-terminal pro b-type natriuretic peptide viagra online no prescription levels. At 6 months. CDC, cardiosphere-derived cell viagra online no prescription.

LVEDV, left ventricular end-diastolic volume. LVESV, left ventricular end-systolic volume. NT-proBNP, N-terminal pro b-type natriuretic viagra online no prescription peptide (from Makkar RR, Kereiakes DJ, Aguirre F, Kowalchuk G, Chakravarty T, Malliaras K, Francis GS, Povsic TJ, Schatz R, Traverse JH, Pogoda JM, Smith RR, Marbán L, Ascheim DD, Ostovaneh MR, Lima JAC, DeMaria A, Marbán E, Henry TD.

Intracoronary ALLogeneic heart STem cells to Achieve myocardial Regeneration (ALLSTAR). A randomized, placebo-controlled, double-blinded trial. See pages 3451--3458).The authors conclude that in contemporary ischaemic/dilated cardiomyopathy patients (LVEF ≤35%, narrow QRS), primary prophylactic ICD treatment was associated viagra online no prescription with a substantial reduction in mortality, although this improvement was not consistent across the whole population.

The manuscript is accompanied by an Editorial by N.A. Mark Estes III from the Heart and Vascular Institute UPMC in Pittsburgh, Pennsylvania, viagra online no prescription USA.11 The authors note that clinicians should be mindful of available risk stratification models and subgroup analyses from the EU-CERT-ICD and other studies. It follows that the process of shared decision-making should include careful consideration of the patient’s wishes and values, with an individualized assessment of potential benefit and risks of primary prevention of sudden death by ICD implantation.Cardiosphere-derived cells (CDCs) are cardiac progenitor cells which exhibit disease-modifying bioactivity in various models of cardiomyopathy and in previous clinical studies of acute myocardial infarction (MI), dilated cardiomyopathy, and Duchenne muscular dystrophy.12,13 In a clinical research article entitled ‘Intracoronary ALLogeneic heart STem cells to Achieve myocardial Regeneration (ALLSTAR).

A randomized, placebo-controlled, double-blinded trial’, Raj Makkar from the Cedars-Sinai Heart Institute in Los Angeles, California, USA and colleagues assessed the safety and efficacy of intracoronary administration of allogeneic CDCs in the multicentre, randomized, double-blind, placebo-controlled, intracoronary ALLogeneic Heart STem Cells to Achieve Myocardial Regeneration (ALLSTAR) trial.14 The authors enrolled patients 4 weeks to 12 months after MI, with LVEF ≤45% and left ventricular LV scar size ≥15% of LVM by MRI. A pre-specified viagra online no prescription interim analysis was performed when 6-month MRI data were available. The trial was subsequently stopped due to the low probability of detecting a significant treatment effect of CDCs based on the primary endpoint.

Patients were randomly allocated in a 2:1 ratio to receive CDCs or placebo in the infarct-related viagra online no prescription artery by the stop–flow technique. The primary safety endpoint was the occurrence, during 1-month post-intracoronary infusion, of acute myocarditis attributable to allogeneic CDCs, ventricular tachycardia- or ventricular fibrillation-related death, sudden unexpected death, or a major adverse cardiac event (death or hospitalization for HF or non-fatal MI). The primary efficacy endpoint was the relative percentage change in infarct size at 12 months post-infusion as assessed by contrast-enhanced cardiac MRI.

Makkar and colleagues randomly allocated 90 patients to the CDC group and 44 to the viagra online no prescription placebo group. The mean baseline LVEF was 40% and the mean scar size was 22% of the LVM. No primary safety endpoint viagra online no prescription events occurred.

There was no difference in the percentage change from baseline in scar size between CDC and placebo groups at 6 months. Compared with placebo, there were significant reductions in LV end-diastolic volume, LV end-systolic volume, and NT-proBNP at 6 months in CDC-treated patients.The authors conclude that intracoronary infusion of allogeneic CDCs in patients with post-MI left ventricular dysfunction was safe but did not reduce scar size relative to placebo at 6 months. The manuscript is accompanied by an Editorial by Francisco Fernandez-Aviles from the Hospital General Universitario Gregorio Marañón in Madrid, Spain and colleagues.15 The authors feel that various points need to be better addressed before proceeding again to clinical trials, if we want to move the field of cardiovascular regenerative and reparative medicine forward, for the sake of the cardiovascular viagra online no prescription health of millions of patients.Treatment of pathological cardiac remodelling and subsequent HF represents an unmet clinical need.

Long non-coding RNAs (lncRNAs) are emerging as crucial molecular orchestrators of disease processes including that of heart diseases.16,17 In a Basic Science article entitled ‘Targeting muscle-enriched long non-coding RNA H19 reverses pathological cardiac hypertrophy’, Thomas Thum from the Hannover Medical School in Germany, and colleagues report on the powerful therapeutic potential of the conserved lncRNA H19 in the treatment of pathological cardiac hypertrophy.18 Pressure overload-induced left ventricular cardiac remodelling revealed an up-regulation of H19 in the early phase, but a strong sustained repression upon reaching the decompensated phase of HF. The translational potential of H19 was highlighted by its repression in a large animal (pig) model of LVH, in diseased human heart samples, in human stem cell-derived cardiomyocytes, and in human engineered heart tissue in response to afterload enhancement. Pressure overload-induced cardiac hypertrophy in H19 viagra online no prescription knockout mice was aggravated compared with wild-type mice.

In contrast, vector-based, cardiomyocyte-directed gene therapy using murine but also human H19 strongly attenuated HF even when cardiac hypertrophy was already established. Mechanistically, using microarray, gene set enrichment analyses, and chromatin viagra online no prescription immunoprecipitation-DNA sequencing, the authors identified a link between H19 and prohypertrophic nuclear factor of activated T cells (NFAT) signalling. H19 physically interacts with the polycomb repressive complex 2 to suppress H3K27 tri-methylation of the antihypertrophic Tescalcin locus which in turn leads to reduced NFAT expression and activity.Thum and colleagues conclude that H19 is highly conserved and down-regulated in failing hearts from mice, pigs, and humans.

H19 gene therapy prevents and reverses experimental pressure overload-induced HF. H19 acts as an antihypertrophic lncRNA and viagra online no prescription represents a promising therapeutic target to combat pathological cardiac remodelling. The manuscript is accompanied by an Editorial by Gianluigi Condorelli from the Humanitas University in Rozzano, Italy and colleagues.

The authors note that dysregulation of epigenetic mechanisms leading to aberrant loss of cardiomyocyte homeostasis is a critical point to consider in understanding the viagra online no prescription onset of cardiovascular pathologies. Thus exploiting lncRNAs as therapeutic agents in myocardial disease could pave the way for efficaciously combatting one of the greatest healthcare burdens worldwide.19With the advent of omics, an innovative inductive method has provided researchers with possible ways new to monitor health and disease. This approach incorporates data from studies of the genome, transcriptome, proteome, and metabolome to focus on the assessment of a varied range of biomolecules.20 In a clinical review article entitled ‘Omics phenotyping in heart failure.

The next frontier’ Antoni Bayes-Genis from the Cardiology Service, Hospital Universitari Germans Trias i Pujol in Badalona, Spain and colleagues provide a state-of-the-art review aiming to provide viagra online no prescription an up-to-date look at breakthrough omic technologies that are helping to unravel HF disease mechanisms and heterogeneity.21 Genomics, transcriptomics, proteomics, and metabolomics in HF are reviewed in depth. In addition, there is a thorough, expert discussion regarding the value of omics in identifying novel disease pathways, advancing understanding of disease mechanisms, differentiating HF phenotypes, yielding biomarkers for diagnosis or prognosis, or identifying new therapeutic targets in HF. The combination of multiple omics technologies may create a more comprehensive picture of the factors and pathophysiology involved in HF than achieved by either one alone, and provides a rich viagra online no prescription resource for predictive phenotype modelling.

However, the successful translation of omics tools as solutions to clinical HF requires that the observations are robust and reproducible, and can be validated across multiple independent populations to ensure confidence in clinical decision-making.This issue is also complemented by a Discussion Forum contribution. In a contribution entitled ‘Heart failure development in obesity. Mechanistic pathways’ Kristjan Karason from the Sahlgrenska University Hospital in Gothenburg, viagra online no prescription Sweden and colleagues provide a reply to a recent comment entitled ‘Incident heart failure risk after bariatric surgery.

The role of epicardial fat’.22,23The editors hope that this issue of the European Heart Journal will be of interest to its readers.With thanks to Amelia Meier-Batschelet, Johanna Hugger, and Martin Meyer for help with compilation of this article. References1Docherty KF, Jhund PS, Inzucchi SE, Køber L, Kosiborod MN, Martinez FA, Ponikowski P, DeMets DL, Sabatine MS, Bengtsson O, Sjöstrand M, Langkilde AM, Desai AS, Diez M, Howlett JG, Katova T, Ljungman CEA, O’Meara E, Petrie MC, Schou M, Verma S, Vinh PN, Solomon SD, McMurray JJV. Effects of dapagliflozin in DAPA-HF according to background heart viagra online no prescription failure therapy.

Eur Heart J 2020;41:2379–2392.2Ponikowski P, Voors AA,, Anker SD, Bueno H, Cleland JGF, Coats AJS, Falk V, González-Juanatey JR, Harjola VP, Jankowska EA, Jessup M, Linde C, Nihoyannopoulos P, Parissis JT, Pieske B, Riley JP, Rosano GMC, Ruilope LM, Ruschitzka F, Rutten FH, van der Meer P. 2016 ESC Guidelines for the diagnosis and viagra online no prescription treatment of acute and chronic heart failure. The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC).

Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur Heart J 2016;37:2129–2200.3Packer M viagra online no prescription. Are the benefits of SGLT2 inhibitors in heart failure and a reduced ejection fraction influenced by background therapy?.

Expectations and realities of a new standard of viagra online no prescription care. Eur Heart J 2020;41:2393–2396.4Butler J, Zannad F, Filippatos G, Anker SD, Packer M. Totality of evidence in trials of sodium–glucose co-transporter-2 inhibitors in the patients with heart failure with reduced ejection fraction.

Implications for clinical practice viagra online no prescription. Eur Heart J 2020;41:3398–3401.5Serenelli M, Böhm M, Inzucchi SE, Køber L, Kosiborod MN, Martinez FA, Ponikowski P,, Sabatine MS, Solomon SD, DeMets DL, Bengtsson O, Sjöstrand M, Langkilde AM, Anand IS, Chiang CE, Chopra VK, de Boer RA, Diez M, Dukát A, Ge J, Howlett JG, Katova T, Kitakaze M, Ljungman CEA, Verma S,, Docherty KF, Jhund PS, McMurray JJV. Effect of dapagliflozin according to baseline systolic blood pressure in the Dapagliflozin and Prevention of viagra online no prescription Adverse Outcomes in Heart Failure trial (DAPA-HF).

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Association for European Paediatric viagra online no prescription and Congenital Cardiology (AEPC). Eur Heart J 2015;36:2793–2867.10Zabel M, Willems R, Lubinski A, Bauer A, Brugada J, Conen D, Flevari P, Hasenfuß G, Svetlosak M, Huikuri HV, Malik M, Pavlović N, Schmidt G, Sritharan R, Schlögl S, Szavits-Nossan J, Traykov V, Tuinenburg AE, Willich SN, Harden M, Friede T, Svendsen JH, Sticherling C, Merkely B. Clinical effectiveness of primary prevention implantable cardioverter-defibrillators.

Results of the EU-CERT-ICD viagra online no prescription controlled multicentre cohort study. Eur Heart J 2020;41:3437–3447.11Estes MNA, Saba S. Primary prevention of sudden death with the implantable viagra online no prescription cardioverter defibrillator.

Bridging the evidence gap. Eur Heart J 2020;41:3448–3450.12Aminzadeh MA, Tseliou E, Sun B, Cheng K, Malliaras K, Makkar RR, Marbán E. Therapeutic efficacy of cardiosphere-derived cells in a transgenic mouse model of non-ischaemic viagra online no prescription dilated cardiomyopathy.

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A randomized, placebo-controlled, double-blinded trial. Eur Heart J 2020;41:3451–3458.15Sanz-Ruiz R, Fernández-Avilés F viagra online no prescription. Cardiovascular regenerative and reparative medicine.

Is myocardial infarction the viagra online no prescription model?. Eur Heart J 2020;41:3459–3461.16Ounzain S, Micheletti R, Beckmann T, Schroen B, Alexanian M, Pezzuto I, Crippa S, Nemir M, Sarre A, Johnson R, Dauvillier J, Burdet F, Ibberson M, Guigó R, Xenarios I, Heymans S, Pedrazzini T. Genome-wide profiling of the cardiac transcriptome after myocardial infarction identifies novel heart-specific long non-coding RNAs.

Eur Heart J 2015;36:353–368.17Lüscher TF viagra online no prescription. Novel molecular mechanisms of vascular disease. Non-coding RNAs, inflammation, and radiation viagra online no prescription.

Eur Heart J. 2020;40:2467–2470.18Viereck J, Bührke A, Foinquinos A, Chatterjee S, Kleeberger JA, Xiao K, Janssen-Peters H, Batkai S, Ramanujam D, Kraft T, Cebotari S, Gueler F, Beyer AM, Schmitz J, Bräsen JH, Schmitto JD, Gyöngyösi M, Löser A, Hirt MN, Eschenhagen T, Engelhardt S, Bär C, Thum T. Targeting muscle-enriched long non-coding RNA viagra online no prescription H19 reverses pathological cardiac hypertrophy.

Eur Heart J 2020;41:3462–3474.19Pagiatakis C, Hall IF, Condorelli G. Long non-coding RNA H19 viagra online no prescription. A new avenue for RNA therapeutics in cardiac hypertrophy?.

Eur Heart J 2020;41:3475–3476.20Hoogeveen RM, Pereira JPB, Nurmohamed NS, Zampoleri V, Bom MJ, Baragetti A, Boekholdt SM, Knaapen P, Khaw KT, Wareham NJ, Groen AK, Catapano AL, Koenig W, Levin E, Stroes ESG. Improved cardiovascular risk prediction using targeted plasma viagra online no prescription proteomics in primary prevention. Eur Heart J 2020;ehaa648.

21Bayes-Genis A, Liu PP, Lanfear DE, de Boer RA, González A, Thum T, Emdin M, Januzzi JL. Omics phenotyping viagra online no prescription in heart failure. The next frontier.

Eur Heart viagra online no prescription J 2020;41:3477–3484.22Karason K, Jamaly S. Heart failure development in obesity. Mechanistic pathways.

Eur Heart J 2020;41:3485.23van Woerden G, van Veldhuisen SL, Rienstra viagra online no prescription M. Incident heart failure risk after bariatric surgery. The role of epicardial viagra online no prescription fat.

Eur Heart J 2020;41:1775. Published on behalf of the European Society of Cardiology. All rights viagra online no prescription reserved.

© The Author(s) 2020. For permissions, viagra online no prescription please email. Journals.permissions@oup.com.Case presentationA 32-year-old cardiology resident was scheduled to round on the COVID-19 wards at a large, government teaching hospital in Bahrain.

To cover the increasing workload, the hospital required additional medical personnel to provide care for the numerous COVID-19 patients that were being seen. Prior to examining COVID-19-positive patients, she donned appropriate personal viagra online no prescription protective equipment (PPE)—a gown, gloves, N95 mask, and face shield. As part of her physical exam, she was obliged to auscultate her patients with a stethoscope, listening for cardiopulmonary abnormalities that can be comorbid with severe COVID-19 infection.

Thus, she was required to unzip her gown and keep her stethoscope either in her ears or around her neck. She used a standard-length Littman Cardiology™ stethoscope, viagra online no prescription requiring her to be in close proximity to the patient (i.e. Lean over to the patient’s level).One day after her rounds, she developed a sore throat.

She subsequently viagra online no prescription was tested positive for COVID-19 via polymerase chain reaction (PCR). The resident cardiologist remembered one patient that she had examined where she suspected the transmission occurred. She recalls examining a patient who was COVID-19 positive.

Prior to the patient’s intubation she applied her own stethoscope directly to the patient’s chest to viagra online no prescription perform auscultation. The resident was perspiring and beginning to feel exhausted from her prior rounding and was breathing heavily as she unzipped her gown to place the stethoscope back within. The resident believes that COVID-19 viral particles which were viagra online no prescription transmitted to the stethoscope became aerosolized and inhaled as she brought the stethoscope close to her mouth while tucking it back into her gown.

The resident recovered, re-tested negative for COVID-19, and has now returned to her normal duties.The COVID-19 pandemic has called into question the triple-faceted role of the stethoscope. A diagnostic tool, symbol of patient–provider connection, and possible vector for infectious disease (Figure 1). A recent article in the American Journal of Medicine discusses developments in each arm of this triple role with reference to COVID-19, arguing that developments in stethoscope diagnostic technology, a need to bolster clinical skills, and developments in stethoscope hygiene methods will perpetuate both its viagra online no prescription relevance and safety.

This argument was made in light of those who believe the stethoscope will become obsolete with the development of more advanced technologies, as well as its potential to transmit disease.1 It is clear that a contaminated stethoscope might pose a danger to patients and providers, and can be a potential vector for the transmission of COVID-19, as illustrated in the case above. Thus, providers should seek to educate themselves on stethoscope contamination, assess the current methods of hygiene, and innovate viagra online no prescription accordingly rather than cast the stethoscope aside. Figure 1The three-faceted role of the stethoscope.

The stethoscope lies at the intersection of three roles in medicine. Diagnostic tool viagra online no prescription. Connection between provider and patients.

And a potential vector for infectious disease. As increased infection control vigilance has placed the stethoscope in a position viagra online no prescription of contention. Each facet of the stethoscope must be weighed in consideration of medicines’s cherished symbol.Figure 1The three-faceted role of the stethoscope.

The stethoscope lies at the intersection of three viagra online no prescription roles in medicine. Diagnostic tool. Connection between provider and patients.

And a potential vector for infectious disease viagra online no prescription. As increased infection control vigilance has placed the stethoscope in a position of contention. Each facet of the stethoscope must be weighed in consideration of medicines’s cherished symbol.Studies have demonstrated that stethoscopes can harbour similar levels and types of microbes to those on one’s hand.2 Thus, it is no surprise that viagra online no prescription the stethoscope has been christened as the physician’s ‘third hand’, with reference both to its potential for pathogen transmission and its integral role in patient–provider connection.

Despite this, no clear guidelines exist for performing stethoscope hygiene. The Centers for Disease Control (CDC) classifies the stethoscope as a ‘non-critical’ medical device (i.e. Only in contact with intact skin, not with bodily fluids), and recommends cleaning viagra online no prescription between as often as after contact with each patient to once weekly using an alcohol or bleach-based disinfectant.3 It has been demonstrated that viruses, including COVID-19,4 are capable of surviving on skin and other surfaces for an extended period of time.5 Thus, current guidelines may not adequately reflect the risk that stethoscope contamination poses.COVID-19 has fostered an era of increased infection control vigilance, and thus the benefits of the stethoscope must be rationally weighed against the risks.

In the vignette posed here, the cardiology resident felt the need to use her stethoscope to assess the COVID-19 patients on her round. Her likely rationale was the utility it provides viagra online no prescription in assessing the variety of cardiopulmonary abnormalities that can manifest during a COVID-19 infection. One of the most common manifestations of COVID-19 infection is multifocal pneumonia, often occurring prior to acute respiratory distress and need for mechanical ventilation.6 While pneumonia is diagnosed most definitively using imaging modalities (CT and X-ray) and laboratory testing, resource-limited scenarios might necessitate the usage of a stethoscope to listen for pulmonary indications (coarse breath sounds).

Furthermore, there is growing evidence that cardiovascular disease is highly comorbid with COVID-19 infection, leading to worse outcomes. The most common cardiovascular comorbidities among hospitalized COVID-19 patients are hypertension, coronary artery disease, and diabetes mellitus.7,8 In addition, viagra online no prescription recent reports have implicated COVID-19 in causing myocardial injury and left ventricular systolic dysfunction.9 Considering the sequelae of COVID-19 cardiopulmonary manifestations, auscultation using a stethoscope can be highly warranted. Therefore, emphasis must be placed on ensuring that the stethoscope can be used safely.Assessments of stethoscope hygiene practices have widely demonstrated deficits in adherence and method.

Direct observational studies have demonstrated stethoscope hygiene rates using recommended methods (wiping with alcohol, bleach, hydrogen peroxide, etc.) between 11.3% and 24%, with unconventional practices also being reported such as placing a glove over the stethoscope prior to auscultation or washing it with water/hand towel in a sink.10,11 Such findings imply that while stethoscope hygiene practices are deficient, providers who are cognizant of stethoscope contamination are struggling to find an effective form of hygiene that does not impede workflow—a proverbial ‘cry for help.’ With regard to current methods of stethoscope hygiene, providers cite lack of access to cleaning supplies, forgetfulness, or a lack of time as reasons for not performing stethoscope hygiene.12Healthcare guidelines advise against using personal stethoscopes in contact precaution settings in order to limit the potential for cross-contamination. Rather, single-patient disposable stethoscopes are often used for such patients viagra online no prescription. However, the audio quality of single-patient stethoscopes is quite poor,13 and it has been demonstrated that these stethoscopes can be contaminated with pathogens that can potentially be transmitted to providers, who must share this stethoscope.14 Proper cleaning of these stethoscopes between usage may not occur in high-workflow environments, such as the intensive care unit (ICU).

Thus, a more feasible and effective modality of stethoscope hygiene is viagra online no prescription warranted.A ray of hope for stethoscope hygiene is technological innovation. Among the solutions presented in recent years have been a UV-LED case for the stethoscope diaphragm,1, stethoscopes made from antimicrobial copper alloys,16 and disposable stethoscope diaphragm covers.17 The challenge imposed by the first two innovations is a lack of complete microbial disinfection. Given that it is unknown what viral dose threshold corresponds to COVID-19 pathogenesis, current infection control standards might necessitate a method that ensures zero transmission.

Stethoscope diaphragm covers alone can provide an aseptic contact surface during auscultation,17 but one is likely to encounter the same impediments stated for conventional stethoscope cleaning.12 A company based in San Diego, USA (AseptiScope Inc., San Diego, CA, USA) has attempted to overcome this issue by developing a touch-free diaphragm barrier dispenser.1 A recent article discussed the role of stethoscope contamination during COVID-19, stating that a specific barrier for the stethoscope is needed to prevent stethoscope contamination and subsequent transmission to patients and providers.18 A touch-free stethoscope diaphragm dispenser might be a feasible solution for this need.In the era of COVID-19, the stethoscope carries both profound utility as well as risk to patients if effective hygiene practices viagra online no prescription are not implemented. Thus, providers need to exercise caution when auscultating patients with COVID-19 given the risk for cross-contamination. However, rather than casting aside the stethoscope due to this risk, safety should be bolstered through education, hygiene practice, and consideration of innovative solutions.Conflict viagra online no prescription of interest.

A.S.M. Is a co-founder and the Chief Clinical Officer for AseptiScope Inc. (San Diego, viagra online no prescription CA, USA).

None of the other authors have conflicts to disclose. ReferencesReferences are available as supplementary material at European Heart Journal viagra online no prescription online. Published on behalf of the European Society of Cardiology.

All rights reserved. © The Author(s) 2020 viagra online no prescription. For permissions, please email.

What should I tell my health care provider before I take Viagra?

They need to know if you have any of these conditions:

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In just six months, the world’s largest randomized control trial on COVID-19 therapeutics has generated conclusive evidence on the effectiveness of repurposed drugs for the treatment of COVID-19.Interim results from the Solidarity Therapeutics Trial, coordinated by the World Health Organization, indicate that remdesivir, hydroxychloroquine, lopinavir/ritonavir and interferon regimens appeared to have little or no effect on 28-day mortality or the in-hospital course of COVID-19 among hospitalized patients.The study, which spans more than 30 countries, looked at the effects of these treatments on how long does it take for viagra to start working overall mortality, initiation of ventilation, and duration of hospital stay in hospitalized patients. Other uses of the drugs, for example in treatment of patients in the community or for prevention, would have to be how long does it take for viagra to start working examined using different trials.The progress achieved by the Solidarity Therapeutics Trial shows that large international trials are possible, even during a pandemic, and offer the promise of quickly and reliably answering critical public health questions concerning therapeutics.The results of the trial are under review for publication in a medical journal and have been uploaded as preprint at medRxiv available at this link. Https://www.medrxiv.org/content/10.1101/2020.10.15.20209817v1The global platform of the Solidarity Trial is ready to rapidly evaluate promising new treatment options, with nearly 500 hospitals open as trial sites.Newer antiviral drugs, immunomodulators and anti-SARS COV-2 monoclonal antibodies are now being considered for evaluation.The World Health Organization has appointed two distinguished leaders to co-chair an Independent Commission on sexual abuse and exploitation during the response to the tenth Ebola Virus Disease epidemic in the provinces of North Kivu and Ituri, the Democratic Republic of the Congo.

The commission will be co-chaired by Her Excellency Aïchatou Mindaoudou, former minister of foreign affairs and of social development of how long does it take for viagra to start working Niger, who has held senior United Nations posts in Côte d’Ivoire and in Darfur. She will be joined by co-chair Julienne Lusenge of the Democratic Republic of the Congo, an internationally recognized human rights how long does it take for viagra to start working activist and advocate for survivors of sexual violence in conflict. The role of the Independent Commission will be to swiftly establish the facts, identify and support survivors, ensure that any ongoing abuse has stopped, and hold perpetrators to account.

It will comprise up to seven members, including the how long does it take for viagra to start working co-chairs, with expertise in sexual exploitation and abuse, emergency response, and investigations. The co-chairs will choose how long does it take for viagra to start working the other members of the Commission, which will be supported by a Secretariat based at WHO. To support the Independent Commission’s work, the Director-General has decided to use an open process to hire an independent and external organization with experience in conducting similar inquiries.

The tenth epidemic of Ebola Virus Disease in the provinces of North Kivu and Ituri – the world’s second largest Ebola outbreak on record – was declared over on 25 June 2020, after persisting for nearly two years how long does it take for viagra to start working in an active conflict zone, and causing 2,300 deaths. WHO has how long does it take for viagra to start working a zero tolerance policy with regard to sexual exploitation and abuse. We reiterate our strong commitment to preventing and protecting against sexual exploitation and abuse in all our operations around the world..

In just six months, the world’s largest randomized control trial on COVID-19 therapeutics has generated conclusive evidence on the effectiveness of repurposed drugs for the treatment of COVID-19.Interim results from the Solidarity Therapeutics Trial, coordinated by the World Health Organization, indicate that remdesivir, hydroxychloroquine, lopinavir/ritonavir and interferon regimens appeared to have little or no effect on viagra online no prescription 28-day mortality or the in-hospital course of COVID-19 among hospitalized patients.The study, which spans more than 30 countries, looked at the effects of these treatments on overall mortality, initiation of ventilation, and duration of hospital stay in hospitalized patients. Other uses of the drugs, for example in treatment of patients in the community or for prevention, would have to be examined using different trials.The progress achieved by the Solidarity Therapeutics Trial shows that large international trials are possible, even during a pandemic, and offer the promise viagra online no prescription of quickly and reliably answering critical public health questions concerning therapeutics.The results of the trial are under review for publication in a medical journal and have been uploaded as preprint at medRxiv available at this link. Https://www.medrxiv.org/content/10.1101/2020.10.15.20209817v1The global platform of the Solidarity Trial is ready to rapidly evaluate promising new treatment options, with nearly 500 hospitals open as trial sites.Newer antiviral drugs, immunomodulators and anti-SARS COV-2 monoclonal antibodies are now being considered for evaluation.The World Health Organization has appointed two distinguished leaders to co-chair an Independent Commission on sexual abuse and exploitation during the response to the tenth Ebola Virus Disease epidemic in the provinces of North Kivu and Ituri, the Democratic Republic of the Congo. The commission will be viagra online no prescription co-chaired by Her Excellency Aïchatou Mindaoudou, former minister of foreign affairs and of social development of Niger, who has held senior United Nations posts in Côte d’Ivoire and in Darfur. She will be joined by co-chair Julienne Lusenge of the Democratic Republic of the viagra online no prescription Congo, an internationally recognized human rights activist and advocate for survivors of sexual violence in conflict.

The role of the Independent Commission will be to swiftly establish the facts, identify and support survivors, ensure that any ongoing abuse has stopped, and hold perpetrators to account. It will comprise up to seven members, including the co-chairs, with expertise in sexual exploitation and abuse, viagra online no prescription emergency response, and investigations. The co-chairs will choose viagra online no prescription the other members of the Commission, which will be supported by a Secretariat based at WHO. To support the Independent Commission’s work, the Director-General has decided to use an open process to hire an independent and external organization with experience in conducting similar inquiries. The tenth epidemic of Ebola Virus Disease in the provinces of North Kivu and Ituri – the world’s second largest Ebola outbreak on record – was declared over on 25 June 2020, after persisting viagra online no prescription for nearly two years in an active conflict zone, and causing 2,300 deaths.

WHO has a zero tolerance policy viagra online no prescription with regard to sexual exploitation and abuse. We reiterate our strong commitment to preventing and protecting against sexual exploitation and abuse in all our operations around the world..

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August 28, main ingredient in viagra 2020Contact. Office of CommunicationsPhone. 202-693-1999U.S.

Department of Labor Issues Revised Final Beryllium StandardsFor Construction and Shipyards WASHINGTON, DC - The U.S. Department of Labor's Occupational Safety and Health Administration (OSHA) today published a final rule revising the beryllium standards for construction and shipyards. The final rule includes changes designed to clarify the standards and simplify or improve compliance.

These changes maintain protection for workers while ensuring that the standard is well understood and compliance is simple and straightforward. The final rule amends the following paragraphs in the beryllium standards for construction and shipyards. Definitions, Methods of Compliance, Respiratory Protection, Personal Protective Clothing and Equipment, Housekeeping, Hazard Communication, Medical Surveillance, and Recordkeeping.

OSHA has removed the Hygiene Areas and Practices paragraph from the final standards because the necessary protections are provided by existing OSHA standards for sanitation. The effective date of the revisions in this final rule is September 30, 2020. OSHA began enforcing the new permissible exposure limits in the 2017 beryllium standards for construction and shipyards in May 2018.

OSHA will begin enforcing the remaining provisions of the standards on September 30, 2020. The final standard will affect approximately 12,000 workers employed in nearly 2,800 establishments in the construction and shipyard industries. The final standards are estimated to yield $2.5 million in total annualized cost savings to employers.

Under the Occupational Safety and Health Act of 1970, employers are responsible for providing safe and healthful workplaces for their employees. OSHA's role is to help ensure these conditions for America's working men and women by setting and enforcing standards, and providing training, education, and assistance. For more information, visit www.osha.gov.

The mission of the Department of Labor is to foster, promote, and develop the welfare of the wage earners, job seekers, and retirees of the United States. Improve working conditions. Advance opportunities for profitable employment.

And assure work-related benefits and rights. # # # U.S. Department of Labor news materials are accessible at http://www.dol.gov.

The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay).August 27, 2020U.S. Department of Labor Announces ActionsTo Assist Americans Impacted By Hurricane Laura WASHINGTON, DC – The U.S.

Department of Labor today announced actions it is taking to assist Americans in states affected by Hurricane Laura. In response to the anticipated needs of those living in states in the path of Hurricane Laura, the Department and its agencies are taking the following actions. The Occupational Safety and Health Administration (OSHA) has actively engaged with the U.S.

Department of Homeland Security, the Federal Emergency Management Administration, the Environmental Protection Agency, and other federal agencies and is prepared to provide assistance. The Wage and Hour Division (WHD) will be prioritizing all calls in the affected areas to continue to provide uninterrupted service to workers and employers. The Employment and Training Administration (ETA) is prepared to provide Disaster Dislocated Worker Grants to help affected states address workforce needs.

The disbursement of funds will be determined as needs are assessed by state and local partners. ETA is also prepared to assist in administering Disaster Unemployment Assistance. The Employee Benefits Security Administration (EBSA) will coordinate with other federal agencies, including the U.S.

Department of Treasury, the IRS and the Pension Benefit Guaranty Corp. On the release of compliance guidance for employee benefit plans, and plan participants and beneficiaries in response to Hurricane Laura. General information on disaster relief under the Employee Retirement Income Security Act (ERISA) is available on EBSA's website at Disaster Relief Information for Employers and Advisers and Disaster Relief Information for Workers and Families, or by contacting EBSA online or by calling 1-866-444-3272.

The Office of Federal Contract Compliance Programs (OFCCP) issued a Temporary Exemption from certain federal contracting requirements. For a period of three months, from August 27, 2020, to November 27, 2020, new federal contracts to provide relief, clean-up or rebuilding efforts will be exempt from having to develop written affirmative action programs as required by Executive Order 11246. The Mine Safety and Health Administration (MSHA) is responding to Hurricane Laura's impact on mines, and stands ready to respond more generally with specialized equipment and personnel.

And The Veterans' Employment and Training Service (VETS) is working with its grantees to identify further flexibilities and additional funding needs for its programs. VETS staff is prepared to assist employers, members of the National Guard and Reserves and members of the National Disaster Medical System and Urban Search and Rescue who deploy in support of rescue and recovery operations. The Department will continue to monitor developments regarding Hurricane Laura and take additional actions as necessary.

For additional information, please visit the Department's Severe Storm and Flood Recovery Assistance webpage. The mission of the Department of Labor is to foster, promote and develop the welfare of the wage earners, job seekers and retirees of the United States. Improve working conditions.

Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # Media Contact.

Eric Holland, 202-693-4676, holland.eric.w@dol.gov Release Number. 20-1654-NAT U.S. Department of Labor news materials are accessible at http://www.dol.gov.

The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay)..

August 28, viagra online no prescription 2020Contact this link. Office of CommunicationsPhone. 202-693-1999U.S. Department of Labor Issues Revised Final Beryllium StandardsFor Construction and Shipyards WASHINGTON, DC - The U.S.

Department of Labor's Occupational Safety and Health Administration (OSHA) today published a final rule revising the beryllium standards for construction and shipyards. The final rule includes changes designed to clarify the standards and simplify or improve compliance. These changes maintain protection for workers while ensuring that the standard is well understood and compliance is simple and straightforward. The final rule amends the following paragraphs in the beryllium standards for construction and shipyards.

Definitions, Methods of Compliance, Respiratory Protection, Personal Protective Clothing and Equipment, Housekeeping, Hazard Communication, Medical Surveillance, and Recordkeeping. OSHA has removed the Hygiene Areas and Practices paragraph from the final standards because the necessary protections are provided by existing OSHA standards for sanitation. The effective date of the revisions in this final rule is September 30, 2020. OSHA began enforcing the new permissible exposure limits in the 2017 beryllium standards for construction and shipyards in May 2018.

OSHA will begin enforcing the remaining provisions of the standards on September 30, 2020. The final standard will affect approximately 12,000 workers employed in nearly 2,800 establishments in the construction and shipyard industries. The final standards are estimated to yield $2.5 million in total annualized cost savings to employers. Under the Occupational Safety and Health Act of 1970, employers are responsible for providing safe and healthful workplaces for their employees.

OSHA's role is to help ensure these conditions for America's working men and women by setting and enforcing standards, and providing training, education, and assistance. For more information, visit www.osha.gov. The mission of the Department of Labor is to foster, promote, and develop the welfare of the wage earners, job seekers, and retirees of the United States. Improve working conditions.

Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # U.S. Department of Labor news materials are accessible at http://www.dol.gov.

The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay).August 27, 2020U.S. Department of Labor Announces ActionsTo Assist Americans Impacted By Hurricane Laura WASHINGTON, DC – The U.S. Department of Labor today announced actions it is taking to assist Americans in states affected by Hurricane Laura.

In response to the anticipated needs of those living in states in my review here the path of Hurricane Laura, the Department and its agencies are taking the following actions. The Occupational Safety and Health Administration (OSHA) has actively engaged with the U.S. Department of Homeland Security, the Federal Emergency Management Administration, the Environmental Protection Agency, and other federal agencies and is prepared to provide assistance. The Wage and Hour Division (WHD) will be prioritizing all calls in the affected areas to continue to provide uninterrupted service to workers and employers.

The Employment and Training Administration (ETA) is prepared to provide Disaster Dislocated Worker Grants to help affected states address workforce needs. The disbursement of funds will be determined as needs are assessed by state and local partners. ETA is also prepared to assist in administering Disaster Unemployment Assistance. The Employee Benefits Security Administration (EBSA) will coordinate with other federal agencies, including the U.S.

Department of Treasury, the IRS and the Pension Benefit Guaranty Corp. On the release of compliance guidance for employee benefit plans, and plan participants and beneficiaries in response to Hurricane Laura. General information on disaster relief under the Employee Retirement Income Security Act (ERISA) is available on EBSA's website at Disaster Relief Information for Employers and Advisers and Disaster Relief Information for Workers and Families, or by contacting EBSA online or by calling 1-866-444-3272. The Office of Federal Contract Compliance Programs (OFCCP) issued a Temporary Exemption from certain federal contracting requirements.

For a period of three months, from August 27, 2020, to November 27, 2020, new federal contracts to provide relief, clean-up or rebuilding efforts will be exempt from having to develop written affirmative action programs as required by Executive Order 11246. The Mine Safety and Health Administration (MSHA) is responding to Hurricane Laura's impact on mines, and stands ready to respond more generally with specialized equipment and personnel. And The Veterans' Employment and Training Service (VETS) is working with its grantees to identify further flexibilities and additional funding needs for its programs. VETS staff is prepared to assist employers, members of the National Guard and Reserves and members of the National Disaster Medical System and Urban Search and Rescue who deploy in support of rescue and recovery operations.

The Department will continue to monitor developments regarding Hurricane Laura and take additional actions as necessary. For additional information, please visit the Department's Severe Storm and Flood Recovery Assistance webpage. The mission of the Department of Labor is to foster, promote and develop the welfare of the wage earners, job seekers and retirees of the United States. Improve working conditions.

Advance opportunities for profitable employment. And assure work-related benefits and rights. # # # Media Contact. Eric Holland, 202-693-4676, holland.eric.w@dol.gov Release Number.

20-1654-NAT U.S. Department of Labor news materials are accessible at http://www.dol.gov. The Department's Reasonable Accommodation Resource Center converts departmental information and documents into alternative formats, which include Braille and large print. For alternative format requests, please contact the Department at (202) 693-7828 (voice) or (800) 877-8339 (federal relay)..

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Latest Prevention & recreational viagra reddit. Wellness News THURSDAY, recreational viagra reddit Sept. 10, 2020 (American Heart Association News)Like ordering a ride or food delivery on your smartphone, keeping track of your heart rate, blood pressure or weight is just a few taps away thanks to thousands of free or inexpensive health apps.But with each click, you may be unwittingly handing over your health data to a third party.As health apps skyrocket in popularity, experts and medical organizations have begun warning consumers of the hidden dangers. In May, the American Medical Association called on lawmakers and the health care industry to install "regulatory guardrails" to protect all recreational viagra reddit types of patient privacy in the digital age.Until that happens, health app users are largely unprotected from having their data passed along to tech giants and marketing companies that might target them with ads, said Mohammed Abdullah, senior author of a new study about privacy issues and apps.The study, being presented at the American Heart Association's virtual Hypertension Scientific Sessions that begins Thursday, examined 35 diabetes mobile apps and found that all of them gave data to a third party, even in cases where the app's privacy policy said it wouldn't.

The research is considered preliminary until published in a peer-reviewed journal."Right now, there are no limitations on what companies can do with this data," said Abdullah, a medical student at the University of Texas Medical Branch in Galveston. "As technology and health care become further intertwined and companies spend billions of dollars on health care-related apps, it's becoming more and more important to make sure we have checks and balances in place."That's because the data on health apps, he said, is not safeguarded by HIPAA, the 1996 law that protects recreational viagra reddit health information gathered by doctors and health systems."Right now, it's like the Wild West, with zero protection," said Dr. David Grande, author of a study about health privacy in the digital age published in July in recreational viagra reddit JAMA Network Open. "Health privacy concerns are growing at an astronomical pace, but we still have a very antiquated view of them."For example, Grande said many Americans are unaware that once their health data is collected, it's available online forever.

In Europe, "right recreational viagra reddit to be forgotten" online privacy laws offer consumers some protection. But in the U.S., digital health info is "immortal," he said."People don't understand all the digital footprints they're leaving behind each time they interact with heath apps, and frankly, it's very hard to understand. Who on earth would want to read a long, complicated privacy recreational viagra reddit agreement?. " said Grande, policy director at the University of Pennsylvania's Leonard Davis Institute of Health Economics in Philadelphia.As arduous as that task might seem, Abdullah urges people to take five minutes to read the agreements and find out what might happen to their data once they click "agree.""You have to weigh the risks and benefits," he said.

"The app might help patients track their blood recreational viagra reddit sugar, but is it worth using if you know your data might possibly be shared?. "For consumers concerned with privacy, one red flag is the presence of ads on the health app."If you open the app and find ad services, you can recreational viagra reddit be sure your data is being sent off to a third party in some way, shape or form," Abdullah said.Another tip is to check the app's automatic settings and make changes that will protect privacy, like turning off your location. But that, too, has a drawback, Grande said. "In some cases, turning off privacy settings makes an app harder to use."Like many internet-based services, health apps are usually free to download, with app-makers earning money through advertising or selling data to third parties, he said.However, that business model could change if lawmakers start enacting stricter guidelines and consumers become more willing to pay for health apps."Consumers put health very high on their list in terms recreational viagra reddit of where they want privacy protection," Grande said.

"As they grow more uncomfortable with every aspect of their life being tracked, I think the thirst for regulation and privacy control will grow, too."American Heart Association News covers heart and brain health. Not all recreational viagra reddit views expressed in this story reflect the official position of the American Heart Association. Copyright is owned or held by the American Heart Association, Inc., and all rights are reserved. If you have questions or comments about this story, please email [email protected]Copyright © 2020 HealthDay recreational viagra reddit.

All rights recreational viagra reddit reserved. QUESTION In the U.S., 1 in every 4 deaths is caused by heart disease. See AnswerLatest Mental recreational viagra reddit Health News THURSDAY, Sept. 10, 2020 (American Heart Association News)With unemployment rates hovering at or near double digits, millions of people are at risk for eviction or foreclosure.

And a growing recreational viagra reddit body of research suggests the effects go beyond financial, taking a toll on both physical and mental health.The CARES Act passed in late March included a moratorium on some evictions and an additional $600 per week in unemployment benefits. But those federal protections expired. A patchwork of temporary local, state and federal eviction moratoriums are in place, but the long-term picture is still uncertain.In fact, an analysis recreational viagra reddit by Stout Risius Ross, a global consulting company, estimates more than 17 million U.S. Households – or more than 43% of rental households – are at risk for eviction over the coming months."The health impact is substantial, and it spans multiple realms," said Shakira Suglia, recreational viagra reddit an associate professor and director of graduate studies in the department of epidemiology at Rollins School of Public Health at Emory University in Atlanta.For example, a 2015 study in the journal Social Forces showed mothers who were evicted were more likely to experience depression and higher parental stress than those in stable homes, and they also reported worse health.

A nationwide survey conducted by the Centers for Disease Control and Prevention in 2015 found that people with self-reported cardiovascular disease were more likely to face housing insecurity than those who didn't have heart problems. Research also shows people who recreational viagra reddit face the threat of eviction are at greater risk for high blood pressure.Black and Latino communities are at even greater risk. Studies from cities throughout the country show that people of color, particularly Black and Latino people, make up about 80% of those facing eviction, according to a report last month from a group of nine academic groups and housing advocates.Matthew Desmond is a sociologist whose Eviction Lab at Princeton University was part of that report. He has conducted research showing that while Black women in Milwaukee neighborhoods made up less than 10% of the population, they accounted for 30% of recreational viagra reddit evictions.

Desmond won a 2017 Pulitzer Prize for his book "Evicted. Poverty and Profit in recreational viagra reddit the American City."Dr. Megan Sandel, an associate professor of pediatrics at Boston University School of Medicine, said recreational viagra reddit the pattern of evictions often follow the historic trends of disinvestment in communities from redlining, the unequal treatment in lending faced by many communities of color. "You see this perpetuation of housing discrimination even to this day."Black and Hispanic households are almost twice as likely as white households to lack housing security, according to a 2014 report from the Joint Center for Housing Studies at Harvard University.Sandel, who also is an associate professor of environmental health at Boston University, said federal rental assistance and extending unemployment insurance could help families, but long-term solutions are needed."When families are able to move to areas with less concentrated poverty, their kids have higher lifetime earnings and are able to move up the economic ladder," she said.

"We talk about health so much in terms of pills or interventions, but a stable, recreational viagra reddit decent, affordable home is the best intervention I can provide to my families. Right now, that's under threat for millions of Americans."Suglia, who co-authored an AHA scientific statement about housing and health, said an array of factors, such as stress, can impact health when a family is worried about paying their rent or being able to stay in their home.Under chronic stress, physiological systems may become dysregulated. Additionally, being in a constant state of worry may increase recreational viagra reddit the likelihood that people turn to smoking, alcohol, and fat and sugar-laden foods, she said. That all can have physical effects."When your housing becomes unaffordable, you may neglect medication, health care, food and heat," she said.

"All these things only exacerbate or create recreational viagra reddit additional health problems."While local and federal governments grapple with the issue, many nonprofit organizations are stepping in to help.For example, the American Heart Association and Enterprise Community Partners, a national affordable housing nonprofit, recently held a free webinar to help faith organizations learn strategies to convert unused property into affordable homes. Funded in part by the Kresge Foundation, the groups will hold additional workshops in late 2020 and recreational viagra reddit early 2021. SLIDESHOW 17 Everyday Ways to Ease Depression See Slideshow In Chicago's Washington Heights, the Endeleo Institute, named for a Swahili term for growth and progress, is doing similar work to repurpose assets in that predominantly Black neighborhood's 95th Street corridor. The group also is working recreational viagra reddit with the AHA, Northwestern University and other institutions to organize farmers markets and launch health education efforts.American Heart Association News covers heart and brain health.

Not all views expressed in this story reflect the official position of the American Heart Association. Copyright is owned or held by the American Heart Association, Inc., and all recreational viagra reddit rights are reserved. If you have questions or comments about this story, please email [email protected]Copyright © 2020 HealthDay. All rights reserved recreational viagra reddit.

From Mental Health Resources Featured Centers Health Solutions From Our recreational viagra reddit SponsorsLatest High Blood Pressure News THURSDAY, Sept. 10, 2020 (HealthDay News)Uncontrolled high blood pressure is becoming more common among Americans, putting them at increased risk for heart attack and stroke, a new study shows.Previous research showed that in 1999-2000, 32.2% of Americans maintained blood pressure less than 140/90 mm Hg, but the rate rose to 54.5% in 2013-2014. However, the rate fell to 48% in 2015-2016.Unfortunately, this new study found the proportion recreational viagra reddit of adults aged 40-59 with successfully managed blood pressure fell nearly 10 percentage points from 2009 to 2018 (56.3% vs. 46.6%, respectively).

Successful blood pressure management also fell recreational viagra reddit among adults 60 and older by almost 6 percentage points from 2009 to 2018 (53.6% vs. 47.9%, respectively).The study will be presented at a virtual American Heart Association meeting, being held Sept. 10-13. Such research is considered preliminary until published in a peer-reviewed journal."We cannot assume improvement in blood pressure management will continue, even after 35 years of success.

High blood pressure is a serious health risk and deserves constant attention to prevent as many heart attacks and strokes as possible," said lead author Dr. Brent Egan, a professor at the University of South Carolina School of Medicine.The reasons why fewer Americans have successfully managed blood pressure varies by age and requires further study, according to the researchers."A closer look at our findings revealed the fall in blood pressure control in older adults was mainly due to less effective use of blood pressure medication and management, so we need to focus on making sure the level of treatment is adequate for this age group," Egan said in a meeting news release.The American Heart Association and American Medical Association have launched a national program called "Target. BP" in response to the high rate of uncontrolled blood pressure.-- Robert PreidtCopyright © 2020 HealthDay. All rights reserved.

QUESTION Salt and sodium are the same. See Answer References SOURCE. American Heart Association, news release, Sept. 10, 2020Latest High Blood Pressure News By Serena GordonHealthDay ReporterFRIDAY, Sept.

11, 2020People with fatter legs appear less likely to have high blood pressure, new research suggests.The researchers suspect that measuring leg fat could help guide blood pressure prevention efforts. Those with bigger legs may not need to worry as much about high blood pressure -- a contributor to heart attack and stroke."Distribution of fat matters. Even though we think that fat is bad in all cases, it might be that leg fat is not as bad as we think," said the study's lead author, Aayush Visaria. He's a fourth year medical student at Rutgers New Jersey Medical School.Previous research has found that excess weight around the middle can increase the risk of heart disease and diabetes.

"Middle fat is really important because that's where all the organs are -- liver, pancreas, intestines -- and they're all affected by fat. Too much fat messes up the function of those organs," Visaria said.But that doesn't mean folks who gain more weight in their lower limbs don't need to maintain a healthy weight, too. "Regardless of where the fat is, a lot of fat isn't good. Having muscle is better than having fat.

Our study says, if you have fat, more fat in legs is better than having it in the abdomen," Visaria said.Dr. Vivek Bhalla is director of the Hypertension Center at Stanford University in California. "This study should not be interpreted to say that if a patient has higher leg fat that they will not develop high blood pressure. It is an important study to motivate further research into the factors as to why blood pressure would be lower," he explained.Bhalla said it's not yet clear how fat distribution might affect your high blood pressure risk.

He said it's possible that fat stored in different areas of the body may act in different ways.Visaria suggested that the difference may have something to do with triglyceride (a type of blood fat) levels. People in the study with more leg fat had decreased triglyceride levels, he noted.The new study included almost 6,000 adults participating in national health surveys between 2011 and 2016. Their average age was 37. About half were female.

Nearly one-quarter of the group had high blood pressure (defined in this study as blood pressure above 130/80 mm Hg).The researchers used special X-ray imaging to measure fat in the legs. These measurements were compared to overall body fat measurements. Men who had 34% fat in their legs were defined as having high leg fat. For women, the cutoff was 39%.Those with higher leg fat were 61% less likely to have high blood pressure than their slim-legged counterparts.

The findings held even after the researchers adjusted for factors such as age, sex, race/ethnicity, smoking, alcohol use, cholesterol levels and levels of waist fat.The researchers noted that this study wasn't designed to prove a cause-and-effect relationship. It could only show an association between higher leg fat and lower blood pressure. Visaria said more research is necessary, particularly in older people. He said the people in this study were between 20 and 59, so these findings may not be generalizable to people over 60.Bhalla pointed out that "obesity is a major epidemic in the United States and around the world, and like many things in medicine, it's not black or white, but rather there are shades of gray."In addition, Bhalla advised, "As we learn more about different types of fat, the distribution of fat, what factors are secreted by different types of fat and how that affects risk of common conditions -- [including] high blood pressure, heart disease and diabetes -- we need to keep in mind that we have to treat patients as individuals, measure their individual risk, and counsel patients appropriately." SLIDESHOW Low Blood Pressure (Hypotension).

Symptoms, Signs, Causes See Slideshow He also said it's important to remember the things people can do right now to lower their blood pressure, including:The findings were scheduled for presentation Thursday at a virtual meeting of the American Heart Association. Findings presented at meetings should be viewed as preliminary until they've been published in a peer-reviewed journal.Copyright © 2020 HealthDay. All rights reserved. From Heart Health Resources Featured Centers Health Solutions From Our Sponsors References SOURCES.

Aayush Visaria, MPH, 4th year medical student, Rutgers New Jersey Medical School, Newark, N.J.. Vivek Bhalla, MD, associate professor, medicine and nephrology, and director, Stanford Hypertension Center, AHA-Certified Comprehensive Hypertension Center, Stanford University School of Medicine, Calif.. Sept. 10, 2020, presentation, American Heart Association virtual hypertension meetingLatest HIV News By Alan MozesHealthDay ReporterTHURSDAY, Sept.

10, 2020 (HealthDay News)The daily drug regimen known as PrEP is a nearly foolproof way to prevent HIV infection. But a new study suggests that many high-risk Americans may be giving the medication a pass because of cost.The warning stems from a pricing analysis that tracked about 2.6 million PrEP prescriptions filled between 2014 and 2018.The researchers found that during that time frame, PrEP prices -- which were already high -- shot up an average of 5% per year."Pre-exposure prophylaxis, or PrEP, is when people at risk for HIV take a medication to prevent HIV," explained study author Dr. Nathan Furukawa. He is a medical officer in the division of HIV/AIDS prevention at the U.S.

Centers for Disease Control and Prevention.First introduced back in 2012, the regimen entails taking a single pill once a day (brand name Truvada), though the pill actually combines two antiretroviral drugs. According to the CDC, a second option -- Descovy -- is also approved for PrEP, though it is not yet clear whether it specifically protects women during vaginal intercourse.But the bottom line is that "PrEP is highly effective at preventing HIV from sex or injection drug use when taken consistently," stressed Furukawa. "If enough people at risk of HIV take PrEP, we can stop new HIV infections and end the HIV epidemic."Furukawa and his colleagues describe the widespread adoption of PrEP as "a foundational pillar" of the effort to rein in HIV among those most vulnerable, including gay and bisexual men, and drug users.But only about 18% of Americans deemed at high-risk for HIV had embraced PrEP as of 2018. And the latest findings, said Furukawa, suggest that its prohibitive cost may be to blame.The conclusion follows an examination of PrEP orders and prices compiled by the IQVIA prescription database.

The database tracks more than nine in 10 medications dispensed by retail pharmacies in the United States, and between 60% and 86% of medications obtained online.The investigators found that between 2014 and 2018, the number of Americans who began taking PrEP increased significantly, rising from about 20,000 to nearly 205,000.Yet, at the same time, the cost of a month's supply of 30 tablets rose from $1,350 to $1,638, the study authors noted.But isn't PrEP covered by insurance?. Yes and no, Furukawa said."PrEP is covered by nearly all insurance plans, most state Medicaid plans and Medicare," he noted. "However, out-of-pocket costs for patients who have not met their deductible may still be prohibitive."Out-of-pocket costs shot up by nearly 15% a year during the study period. That translates to an average out-of-pocket rise from $54 a month in 2014 to $94 a month by 2018, the findings showed."Even though insurers cover about 94% of PrEP medication costs, patients may still have difficulties paying the remaining out-of-pocket costs, since the cost of the medication is large," Furukawa explained.As for those who are underinsured or lack insurance altogether, state medication assistance programs and the manufacturer of Truvada (Gilead) may offer subsidies.

(Furukawa directed those interested to the nonprofit PrEPcost.org website.)Furukawa also acknowledged that there are other impediments to PrEP acceptance beyond cost, including lack of awareness and insufficient access to health care.Still, he suggested that lowering the cost of the medication could be an important factor in getting more people on board.That thought was seconded by Dr. Michael Horberg, associate medical director and director of HIV/AIDS and STD at the Kaiser Permanente Care Management Institute in Rockville, Md.While drug assistance programs are available, "pharmacy costs are always a big concern in preventive medicine," said Horberg.But PrEP cost is likely to fall in the coming years, he said, as a cheaper generic version of Truvada comes to market. And PrEP is also now classified as a level "A" recommended drug by the U.S. Preventive Services Task Force.

That designation should drive out-of-pocket costs down to as low as zero, Horberg added."Nonetheless, the pharmaceutical industry should aim to lower costs, and make this readily available to all U.S. Citizens who would benefit from PrEP," Horberg said.The findings were published Sept. 8 in the Annals of Internal Medicine.Copyright © 2020 HealthDay. All rights reserved.

SLIDESHOW A Timeline of the HIV/AIDS Pandemic See Slideshow References SOURCES. Nathan Furukawa, MD, MPH, medical officer, division of HIV/AIDS prevention, U.S. Centers for Disease Control and Prevention, Atlanta. Michael Horberg, MD, MAS, associate medical director and director, HIV/AIDS and STD, Kaiser Permanente Care Management Institute, Rockville, Md..

Latest Prevention viagra online no prescription &. Wellness News THURSDAY, Sept viagra online no prescription. 10, 2020 (American Heart Association News)Like ordering a ride or food delivery on your smartphone, keeping track of your heart rate, blood pressure or weight is just a few taps away thanks to thousands of free or inexpensive health apps.But with each click, you may be unwittingly handing over your health data to a third party.As health apps skyrocket in popularity, experts and medical organizations have begun warning consumers of the hidden dangers.

In May, viagra online no prescription the American Medical Association called on lawmakers and the health care industry to install "regulatory guardrails" to protect all types of patient privacy in the digital age.Until that happens, health app users are largely unprotected from having their data passed along to tech giants and marketing companies that might target them with ads, said Mohammed Abdullah, senior author of a new study about privacy issues and apps.The study, being presented at the American Heart Association's virtual Hypertension Scientific Sessions that begins Thursday, examined 35 diabetes mobile apps and found that all of them gave data to a third party, even in cases where the app's privacy policy said it wouldn't. The research is considered preliminary until published in a peer-reviewed journal."Right now, there are no limitations on what companies can do with this data," said Abdullah, a medical student at the University of Texas Medical Branch in Galveston. "As technology and health care become further intertwined and companies spend billions of dollars on health care-related apps, it's becoming more and more important to make sure we have checks and balances in place."That's because the data on health apps, he said, is not safeguarded by HIPAA, the 1996 law that protects health information gathered by doctors and health systems."Right now, it's like the Wild West, with viagra online no prescription zero protection," said Dr.

David Grande, author of a study about health privacy in the digital viagra online no prescription age published in July in JAMA Network Open. "Health privacy concerns are growing at an astronomical pace, but we still have a very antiquated view of them."For example, Grande said many Americans are unaware that once their health data is collected, it's available online forever. In Europe, "right to be forgotten" online privacy laws viagra online no prescription offer consumers some protection.

But in the U.S., digital health info is "immortal," he said."People don't understand all the digital footprints they're leaving behind each time they interact with heath apps, and frankly, it's very hard to understand. Who on earth would want to read a viagra online no prescription long, complicated privacy agreement?. " said Grande, policy director at the University of Pennsylvania's Leonard Davis Institute of Health Economics in Philadelphia.As arduous as that task might seem, Abdullah urges people to take five minutes to read the agreements and find out what might happen to their data once they click "agree.""You have to weigh the risks and benefits," he said.

"The app might help patients track their blood sugar, but is it worth using if you know your data might viagra online no prescription possibly be shared?. "For consumers concerned with privacy, one red flag is the presence of ads on the health app."If you open the app and find ad services, you can viagra online no prescription be sure your data is being sent off to a third party in some way, shape or form," Abdullah said.Another tip is to check the app's automatic settings and make changes that will protect privacy, like turning off your location. But that, too, has a drawback, Grande said.

"In some cases, turning off privacy settings makes an app harder to use."Like many internet-based services, viagra online no prescription health apps are usually free to download, with app-makers earning money through advertising or selling data to third parties, he said.However, that business model could change if lawmakers start enacting stricter guidelines and consumers become more willing to pay for health apps."Consumers put health very high on their list in terms of where they want privacy protection," Grande said. "As they grow more uncomfortable with every aspect of their life being tracked, I think the thirst for regulation and privacy control will grow, too."American Heart Association News covers heart and brain health. Not all views expressed in this story reflect the official position of the American Heart viagra online no prescription Association.

Copyright is owned or held by the American Heart Association, Inc., and all rights are reserved. If you have questions or comments about this story, please email [email protected]Copyright viagra online no prescription © 2020 HealthDay. All rights viagra online no prescription reserved.

QUESTION In the U.S., 1 in every 4 deaths is caused by heart disease. See AnswerLatest Mental Health News THURSDAY, viagra online no prescription Sept. 10, 2020 (American Heart Association News)With unemployment rates hovering at or near double digits, millions of people are at risk for eviction or foreclosure.

And a growing body of research suggests the effects go beyond financial, taking a toll on both physical and mental health.The CARES Act passed in late March included a moratorium on viagra online no prescription some evictions and an additional $600 per week in unemployment benefits. But those federal protections expired. A patchwork of temporary viagra online no prescription local, state and federal eviction moratoriums are in place, but the long-term picture is still uncertain.In fact, an analysis by Stout Risius Ross, a global consulting company, estimates more than 17 million U.S.

Households – or more than 43% of rental households – are at risk for eviction over viagra online no prescription the coming months."The health impact is substantial, and it spans multiple realms," said Shakira Suglia, an associate professor and director of graduate studies in the department of epidemiology at Rollins School of Public Health at Emory University in Atlanta.For example, a 2015 study in the journal Social Forces showed mothers who were evicted were more likely to experience depression and higher parental stress than those in stable homes, and they also reported worse health. A nationwide survey conducted by the Centers for Disease Control and Prevention in 2015 found that people with self-reported cardiovascular disease were more likely to face housing insecurity than those who didn't have heart problems. Research also shows people who face the threat of eviction are at greater risk for high blood pressure.Black and Latino communities are at even viagra online no prescription greater risk.

Studies from cities throughout the country show that people of color, particularly Black and Latino people, make up about 80% of those facing eviction, according to a report last month from a group of nine academic groups and housing advocates.Matthew Desmond is a sociologist whose Eviction Lab at Princeton University was part of that report. He has conducted research showing that while Black women in Milwaukee neighborhoods made up less viagra online no prescription than 10% of the population, they accounted for 30% of evictions. Desmond won a 2017 Pulitzer Prize for his book "Evicted.

Poverty and viagra online no prescription Profit in the American City."Dr. Megan Sandel, an associate professor of pediatrics at Boston University School of viagra online no prescription Medicine, said the pattern of evictions often follow the historic trends of disinvestment in communities from redlining, the unequal treatment in lending faced by many communities of color. "You see this perpetuation of housing discrimination even to this day."Black and Hispanic households are almost twice as likely as white households to lack housing security, according to a 2014 report from the Joint Center for Housing Studies at Harvard University.Sandel, who also is an associate professor of environmental health at Boston University, said federal rental assistance and extending unemployment insurance could help families, but long-term solutions are needed."When families are able to move to areas with less concentrated poverty, their kids have higher lifetime earnings and are able to move up the economic ladder," she said.

"We talk about health so much in terms of pills or interventions, but a stable, decent, affordable home is the best intervention viagra online no prescription I can provide to my families. Right now, that's under threat for millions of Americans."Suglia, who co-authored an AHA scientific statement about housing and health, said an array of factors, such as stress, can impact health when a family is worried about paying their rent or being able to stay in their home.Under chronic stress, physiological systems may become dysregulated. Additionally, being in a constant state of worry may increase the likelihood that people turn to smoking, viagra online no prescription alcohol, and fat and sugar-laden foods, she said.

That all can have physical effects."When your housing becomes unaffordable, you may neglect medication, health care, food and heat," she said. "All these things only exacerbate or create additional health problems."While local and federal governments grapple with the issue, many nonprofit organizations are stepping in to help.For example, the viagra online no prescription American Heart Association and Enterprise Community Partners, a national affordable housing nonprofit, recently held a free webinar to help faith organizations learn strategies to convert unused property into affordable homes. Funded in part by the Kresge Foundation, the groups will hold additional workshops in late 2020 and early 2021 viagra online no prescription.

SLIDESHOW 17 Everyday Ways to Ease Depression See Slideshow In Chicago's Washington Heights, the Endeleo Institute, named for a Swahili term for growth and progress, is doing similar work to repurpose assets in that predominantly Black neighborhood's 95th Street corridor. The group also is working with the AHA, Northwestern University and other institutions to organize farmers markets and launch health education viagra online no prescription efforts.American Heart Association News covers heart and brain health. Not all views expressed in this story reflect the official position of the American Heart Association.

Copyright is owned or held by the viagra online no prescription American Heart Association, Inc., and all rights are reserved. If you have questions or comments about this story, please email [email protected]Copyright © 2020 HealthDay. All rights viagra online no prescription reserved.

From Mental Health Resources Featured Centers Health Solutions From Our SponsorsLatest High Blood Pressure viagra online no prescription News THURSDAY, Sept. 10, 2020 (HealthDay News)Uncontrolled high blood pressure is becoming more common among Americans, putting them at increased risk for heart attack and stroke, a new study shows.Previous research showed that in 1999-2000, 32.2% of Americans maintained blood pressure less than 140/90 mm Hg, but the rate rose to 54.5% in 2013-2014. However, the rate fell to 48% in 2015-2016.Unfortunately, this new study found the proportion of adults aged 40-59 with successfully managed blood pressure fell nearly 10 viagra online no prescription percentage points from 2009 to 2018 (56.3% vs.

46.6%, respectively). Successful blood pressure management also viagra online no prescription fell among adults 60 and older by almost 6 percentage points from 2009 to 2018 (53.6% vs. 47.9%, respectively).The study will be presented at a virtual American Heart Association meeting, being held Sept.

10-13. Such research is considered preliminary until published in a peer-reviewed journal."We cannot assume improvement in blood pressure management will continue, even after 35 years of success. High blood pressure is a serious health risk and deserves constant attention to prevent as many heart attacks and strokes as possible," said lead author Dr.

Brent Egan, a professor at the University of South Carolina School of Medicine.The reasons why fewer Americans have successfully managed blood pressure varies by age and requires further study, according to the researchers."A closer look at our findings revealed the fall in blood pressure control in older adults was mainly due to less effective use of blood pressure medication and management, so we need to focus on making sure the level of treatment is adequate for this age group," Egan said in a meeting news release.The American Heart Association and American Medical Association have launched a national program called "Target. BP" in response to the high rate of uncontrolled blood pressure.-- Robert PreidtCopyright © 2020 HealthDay. All rights reserved.

QUESTION Salt and sodium are the same. See Answer References SOURCE. American Heart Association, news release, Sept.

10, 2020Latest High Blood Pressure News By Serena GordonHealthDay ReporterFRIDAY, Sept. 11, 2020People with fatter legs appear less likely to have high blood pressure, new research suggests.The researchers suspect that measuring leg fat could help guide blood pressure prevention efforts. Those with bigger legs may not need to worry as much about high blood pressure -- a contributor to heart attack and stroke."Distribution of fat matters.

Even though we think that fat is bad in all cases, it might be that leg fat is not as bad as we think," said the study's lead author, Aayush Visaria. He's a fourth year medical student at Rutgers New Jersey Medical School.Previous research has found that excess weight around the middle can increase the risk of heart disease and diabetes. "Middle fat is really important because that's where all the organs are -- liver, pancreas, intestines -- and they're all affected by fat.

Too much fat messes up the function of those organs," Visaria said.But that doesn't mean folks who gain more weight in their lower limbs don't need to maintain a healthy weight, too. "Regardless of where the fat is, a lot of fat isn't good. Having muscle is better than having fat.

Our study says, if you have fat, more fat in legs is better than having it in the abdomen," Visaria said.Dr. Vivek Bhalla is director of the Hypertension Center at Stanford University in California. "This study should not be interpreted to say that if a patient has higher leg fat that they will not develop high blood pressure.

It is an important study to motivate further research into the factors as to why blood pressure would be lower," he explained.Bhalla said it's not yet clear how fat distribution might affect your high blood pressure risk. He said it's possible that fat stored in different areas of the body may act in different ways.Visaria suggested that the difference may have something to do with triglyceride (a type of blood fat) levels. People in the study with more leg fat had decreased triglyceride levels, he noted.The new study included almost 6,000 adults participating in national health surveys between 2011 and 2016.

Their average age was 37. About half were female. Nearly one-quarter of the group had high blood pressure (defined in this study as blood pressure above 130/80 mm Hg).The researchers used special X-ray imaging to measure fat in the legs.

These measurements were compared to overall body fat measurements. Men who had 34% fat in their legs were defined as having high leg fat. For women, the cutoff was 39%.Those with higher leg fat were 61% less likely to have high blood pressure than their slim-legged counterparts.

The findings held even after the researchers adjusted for factors such as age, sex, race/ethnicity, smoking, alcohol use, cholesterol levels and levels of waist fat.The researchers noted that this study wasn't designed to prove a cause-and-effect relationship. It could only show an association between higher leg fat and lower blood pressure. Visaria said more research is necessary, particularly in older people.

He said the people in this study were between 20 and 59, so these findings may not be generalizable to people over 60.Bhalla pointed out that "obesity is a major epidemic in the United States and around the world, and like many things in medicine, it's not black or white, but rather there are shades of gray."In addition, Bhalla advised, "As we learn more about different types of fat, the distribution of fat, what factors are secreted by different types of fat and how that affects risk of common conditions -- [including] high blood pressure, heart disease and diabetes -- we need to keep in mind that we have to treat patients as individuals, measure their individual risk, and counsel patients appropriately." SLIDESHOW Low Blood Pressure (Hypotension). Symptoms, Signs, Causes See Slideshow He also said it's important to remember the things people can do right now to lower their blood pressure, including:The findings were scheduled for presentation Thursday at a virtual meeting of the American Heart Association. Findings presented at meetings should be viewed as preliminary until they've been published in a peer-reviewed journal.Copyright © 2020 HealthDay.

All rights reserved. From Heart Health Resources Featured Centers Health Solutions From Our Sponsors References SOURCES. Aayush Visaria, MPH, 4th year medical student, Rutgers New Jersey Medical School, Newark, N.J..

Vivek Bhalla, MD, associate professor, medicine and nephrology, and director, Stanford Hypertension Center, AHA-Certified Comprehensive Hypertension Center, Stanford University School of Medicine, Calif.. Sept. 10, 2020, presentation, American Heart Association virtual hypertension meetingLatest HIV News By Alan MozesHealthDay ReporterTHURSDAY, Sept.

10, 2020 (HealthDay News)The daily drug regimen known as PrEP is a nearly foolproof way to prevent HIV infection. But a new study suggests that many high-risk Americans may be giving the medication a pass because of cost.The warning stems from a pricing analysis that tracked about 2.6 million PrEP prescriptions filled between 2014 and 2018.The researchers found that during that time frame, PrEP prices -- which were already high -- shot up an average of 5% per year."Pre-exposure prophylaxis, or PrEP, is when people at risk for HIV take a medication to prevent HIV," explained study author Dr. Nathan Furukawa.

He is a medical officer in the division of HIV/AIDS prevention at the U.S. Centers for Disease Control and Prevention.First introduced back in 2012, the regimen entails taking a single pill once a day (brand name Truvada), though the pill actually combines two antiretroviral drugs. According to the CDC, a second option -- Descovy -- is also approved for PrEP, though it is not yet clear whether it specifically protects women during vaginal intercourse.But the bottom line is that "PrEP is highly effective at preventing HIV from sex or injection drug use when taken consistently," stressed Furukawa.

"If enough people at risk of HIV take PrEP, we can stop new HIV infections and end the HIV epidemic."Furukawa and his colleagues describe the widespread adoption of PrEP as "a foundational pillar" of the effort to rein in HIV among those most vulnerable, including gay and bisexual men, and drug users.But only about 18% of Americans deemed at high-risk for HIV had embraced PrEP as of 2018. And the latest findings, said Furukawa, suggest that its prohibitive cost may be to blame.The conclusion follows an examination of PrEP orders and prices compiled by the IQVIA prescription database. The database tracks more than nine in 10 medications dispensed by retail pharmacies in the United States, and between 60% and 86% of medications obtained online.The investigators found that between 2014 and 2018, the number of Americans who began taking PrEP increased significantly, rising from about 20,000 to nearly 205,000.Yet, at the same time, the cost of a month's supply of 30 tablets rose from $1,350 to $1,638, the study authors noted.But isn't PrEP covered by insurance?.

Yes and no, Furukawa said."PrEP is covered by nearly all insurance plans, most state Medicaid plans and Medicare," he noted. "However, out-of-pocket costs for patients who have not met their deductible may still be prohibitive."Out-of-pocket costs shot up by nearly 15% a year during the study period. That translates to an average out-of-pocket rise from $54 a month in 2014 to $94 a month by 2018, the findings showed."Even though insurers cover about 94% of PrEP medication costs, patients may still have difficulties paying the remaining out-of-pocket costs, since the cost of the medication is large," Furukawa explained.As for those who are underinsured or lack insurance altogether, state medication assistance programs and the manufacturer of Truvada (Gilead) may offer subsidies.

(Furukawa directed those interested to the nonprofit PrEPcost.org website.)Furukawa also acknowledged that there are other impediments to PrEP acceptance beyond cost, including lack of awareness and insufficient access to health care.Still, he suggested that lowering the cost of the medication could be an important factor in getting more people on board.That thought was seconded by Dr. Michael Horberg, associate medical director and director of HIV/AIDS and STD at the Kaiser Permanente Care Management Institute in Rockville, Md.While drug assistance programs are available, "pharmacy costs are always a big concern in preventive medicine," said Horberg.But PrEP cost is likely to fall in the coming years, he said, as a cheaper generic version of Truvada comes to market. And PrEP is also now classified as a level "A" recommended drug by the U.S.

Preventive Services Task Force. That designation should drive out-of-pocket costs down to as low as zero, Horberg added."Nonetheless, the pharmaceutical industry should aim to lower costs, and make this readily available to all U.S. Citizens who would benefit from PrEP," Horberg said.The findings were published Sept.

8 in the Annals of Internal Medicine.Copyright © 2020 HealthDay. All rights reserved. SLIDESHOW A Timeline of the HIV/AIDS Pandemic See Slideshow References SOURCES.

Nathan Furukawa, MD, MPH, medical officer, division of HIV/AIDS prevention, U.S. Centers for Disease Control and Prevention, Atlanta. Michael Horberg, MD, MAS, associate medical director and director, HIV/AIDS and STD, Kaiser Permanente Care Management Institute, Rockville, Md..

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Viagra doses 200mg

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A still unanswered question is what drives the small fraction of viagra doses 200mg activated germinal center (GC) B cells to become long-lived quiescent can viagra cause heart attack memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 viagra doses 200mg activity led to defects in formation of the CD38intBcl6hi/intEfnb1+ cells.

Conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 viagra doses 200mg and surface BCR that, in turn, contributed to their survival and development. We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR.

Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak help from T cells together with provision of an increased survival viagra doses 200mg signal are key for GC B cells to adopt a memory B cell fate. Memory B cells and long-lived plasma cells are responsible for effective long-term immunity against pathogens. The majority of these cells responding to T cell–dependent antigens are generated from viagra doses 200mg the germinal center (GC) reaction.

Indeed, memory B cells emerge from the GC as recirculating cells and, upon secondary antigen challenge, they are primed to elicit rapid antibody responses. GCs are viagra doses 200mg divided into two anatomical structures. The light zone (LZ) and the dark zone (DZ.

Allen et viagra doses 200mg al., 2007. Victora and Nussenzweig, 2012). B cells proliferate and undergo viagra doses 200mg somatic hypermutation in the DZ before entering the LZ, where they exit the cell cycle.

In the LZ, GC B cells expressing newly mutated B cell receptors (BCRs) capture antigen presented on follicular dendritic cells and internalize it for presentation to follicular helper T cells. Subsequently, antigen- and T cell–dependent selection takes place, whereby the “choice” of recycling to the DZ for further affinity maturation or of exiting the GC as viagra doses 200mg plasma or memory B cells is made. In regard to the selection mechanism, it has been postulated that precursor cells destined to become recycling GC, plasma, or memory B cells already become committed in the LZ, at least to some extent, thereafter entering the recycling DZ, plasma, or memory B cell pools (Inoue et al., 2018).

For instance, it has been demonstrated that a small fraction of LZ B cells expressing c-Myc, a key cell-cycle regulator, corresponds to precursor viagra doses 200mg cells for the recycling GC fate. C-Myc+ cells are enriched for high-affinity BCRs and ablation of c-Myc affects DZ reentry (Calado et al., 2012. Dominguez-Sola et viagra doses 200mg al., 2012.

Finkin et al., 2019). Bcl6loCD69hi LZ B cells expressing viagra doses 200mg IRF4, a critical transcription factor for plasma cell differentiation, were recently shown to be the precursors of plasma cells (Ise et al., 2018). In contrast to these insights into the precursor cells for recycling and plasma cell fates, studies of the memory fate decision have been hampered by the lack of a known master transcription factor for differentiation of memory B cells.

Hence, surrogate markers such as an S1PR2 reporter, CCR6 expression, or a cell cycle reporter have been recently employed for identification of memory precursor cells (Laidlaw et al., viagra doses 200mg 2017. Suan et al., 2017. Wang et al., 2017).

Although informative, these studies have not identified key features for development of the GC-derived precursor cells committed to the viagra doses 200mg long-lived memory B cell fate, or what signals regulate these key features. Here, after identifying a memory-prone population (CD38intBcl6hi/int Ephrin-B1 [Efnb1+]), we found that this small population exhibited lower mTORC1 activity than the recycling-prone population. Constitutive high mTORC1 activity led to defective development of CD38intBcl6hi/intEfnb1+ cells, whereas decreasing mTORC1 activity resulted in relative enrichment in this memory-prone cell population versus the viagra doses 200mg recycling-prone one.

Moreover, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR, thereby contributing to their survival and development. We also found that downregulation viagra doses 200mg of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity (Ersching et al., 2017), our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC cells to assume the memory B cell fate.

To clarify the initiating process for memory B cell differentiation occurring in the GC, viagra doses 200mg we wished to identify GC B cells destined to the memory fate. For this, we used Bcl6 protein reporter mice (Kitano et al., 2011). We immunized viagra doses 200mg these mice with 4-hydroxy-3-nitrophenylacetyl (NP)–chicken γ-globulin (CGG) in alum i.p.

And analyzed NP-specific IgG1+ splenic B cells at day 10. Since CD38 upregulation takes place during the transition from GC to memory B cells (Ridderstad and Tarlinton, 1998), we examined such CD38+ B cells that still maintained GC identity viagra doses 200mg to some extent, i.e., were Bcl6+, together with conventional CD38− GC B cells. By using a fractionation method described previously (Fig.

S1 A viagra doses 200mg. Ise et al., 2018), the LZ B cells were further separated based on their Bcl6 and CD69 expression pattern (upper right panel in Fig. 1 A) viagra doses 200mg.

Fraction (Fr.) 1 (CD38−Bcl6loCD69hi) and Fr.2 (CD38−Bcl6hiCD69hi) cells are plasma and recycling GC precursor cells, respectively (Ise et al., 2018). Characterization of Fr.3 (CD38−Bcl6hiCD69lo) cells is described viagra doses 200mg below. Efnb1 is expressed at a high level by almost all Fas+GL7+ cells, but is barely detectable on naive B cells (Laidlaw et al., 2017.

Lu et al., 2017 viagra doses 200mg. Wang et al., 2017), allowing us to identify transitional populations between GC and memory B cells. Hence, for CD38+ cells, by using Efnb1 and Bcl6, we further separated the NP+ IgG1+CD38+GL7−CD138− cells into Bcl6+Efnb1+ (Fr.5), Bcl6loEfnb1+ viagra doses 200mg (Fr.6), and Bcl6−Efnb1− (Fr.7.

Lower right panel in Fig. 1 A) viagra doses 200mg. Since expression level of Bcl6 in Fr.5 cells was slightly but significantly lower than that of Fr.3 cells, as shown by the left panel in Fig.

1 B, herein, we designated viagra doses 200mg Bcl6hi/int for Fr.5. CD38 expression levels on Fr.5, Fr.6, and Fr.7 cells were increased in that order (middle panel in Fig. 1 B.

Herein, indicated as CD38int, and CD38+ for Fr.5 and viagra doses 200mg 6/7, respectively). During the time course of the GC response, Fr.5 and Fr.6 cell numbers peaked at day 10 before declining, whereas Fr.7 cells peaked at day 12 and then slowly declined (Fig. S1 B) viagra doses 200mg.

These kinetic data suggest that Fr.5 and Fr.6 contain cells that are transient and intermediate, and that once cells enter the Fr.7 pool, they are stably maintained. The Fr.7 cells displayed a typical CD38+Bcl6−Efnb1− mature memory phenotype (Fig viagra doses 200mg. 1 B).

To assess the relationship between overall LZ B cells and Fr.5/6/7 cells, we performed viagra doses 200mg RNA sequencing (RNA-seq) analysis (Fig. S2 A). To obtain sufficient amounts of RNA for this analysis, we used transferred B1-8hi B viagra doses 200mg cells instead of non-BCR transgenic mice.

These NP-specific transgenic GC B cells were present in similar proportions in each fraction as in non-BCR transgenic mice (Fig. S1 C) viagra doses 200mg. The principal component analysis (PCA) for each fraction indicated that memory B cells (Fr.7) clustered most tightly with CD38+Bcl6loEfnb1+ (Fr.6) cells but differed greatly from total LZ GC B cells (Fig.

1 C) viagra doses 200mg. Fr.5 cells were intermediate between Fr.6 and LZ GC B cells. Fr.6 cells expressed viagra doses 200mg lower levels of S1pr2 and higher levels of Gpr183 (EBI2) mRNA compared with LZ B cells (Fig.

S1 D), implying that they are a cell population in the process of exiting the GC. Herein, we call Fr.6 “pre-memory B cells.” In contrast to Fr.6 and mature memory B cells (Fr.7), Fr.5 viagra doses 200mg cells seem to start the process of downregulating Bcl6. Fr.6 cells are most likely to correspond to the already identified GC-derived pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”.

Laidlaw et al., 2017), “LZ CCR6+” (Suan et viagra doses 200mg al., 2017), and “mKO2hi” (Wang et al., 2017) in that, like those cells, Fr.6 cells are Bcl6int/loBach2int (Fig. S3, A and B). The above data prompted us to consider that, among Fr.2, Fr.3, and Fr.5 cells, the CD38intBcl6hi/intEfnb1+ cells (Fr.5) could be potential GC-derived precursors of the pre-memory viagra doses 200mg B cells (Fr.6).

To test this possibility, we took the following three approaches. First, PCA of the RNA-seq data was performed, indicating that CD38intBcl6hi/intEfnb1+ cells (Fr.5) and pre-memory B viagra doses 200mg cells (Fr.6) clustered most closely together (Fig. 1 D).

Second, to monitor cellular quiescence, we employed mVenus-p27K− transgenic mice, in which mainly G0 phase cells are labeled (Oki et al., 2014), demonstrating that in contrast to Fr.2 and viagra doses 200mg Fr.3 cells, Fr.5 and Fr.6 cells had more mVenus-p27K− probe–positive, i.e., quiescent cells (Fig. 1 E). Finally, in order to assess the memory recall potential of the Fr.5 cells, we used a previously described adoptive transfer method (Wang et al., 2017).

As illustrated viagra doses 200mg in Fig. 1 F, Fr.2, Fr.3, Fr.5, or Fr.6 cells were isolated from NP-CGG/alum immunized mice and adoptively transferred (2 × 104 cells per mouse) into sublethally irradiated recipient mice together with CD4+ T cells isolated from CGG-immunized mice. The recipient mice were then challenged with NP-CGG viagra doses 200mg and analyzed on day 6 for NP-specific plasma cells.

Although less proficient than pre-memory B cells (Fr.6), the ability of the adoptively transferred CD38intBcl6hi/intEfnb1+ (Fr.5) cells to give rise to plasma cells was significantly superior to Fr.2 and Fr.3 cells (Fig. 1 G) viagra doses 200mg. To rule out the possibility that Fr.5 cells were cells that had reentered the GC reaction from already generated memory B cells, we stained them for Ki67 and observed lower expression in Fr.5 than in the pre-GC B cells, which are in the process of entering the GC (Fig.

S1 E) viagra doses 200mg. Together, CD38intBcl6hi/intEfnb1+ (Fr.5) cells are likely to be a precursor of pre-memory B cells, herein called Fr.5 “pro-memory B cells,” and to represent a precursor population of previously identified pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”. Laidlaw et al., 2017), “LZ CCR6+” (Suan et al., 2017), and “mKO2hi” (Wang viagra doses 200mg et al., 2017.

Fig. S3, A and viagra doses 200mg B). However, we do not exclude the possibility that the pro-memory B cell population (Fr.5) is heterogeneous in its origins and properties.

For instance, Fr.5 cells appear to overlap, to some extent, with LZ CCR6+ cells in that they are beginning to express viagra doses 200mg Ccr6 (Fig. S3 C). To gain insight into the specific viagra doses 200mg features of CD38intBcl6hi/intEfnb1+ (Fr.5) cells that promote their potential development and/or differentiation into memory cells, we compared their RNA-seq profile to that of the other LZ B cells (Fr.2 and Fr.3.

Fig. 2 A and Fig viagra doses 200mg. S2 A).

CD38−Bcl6hiCD69hi (Fr.2) viagra doses 200mg cells are destined to the recycling GC fate (Ise et al., 2018). Gene set enrichment analysis (GSEA) of Hallmark gene sets (Liberzon et al., 2015) revealed a strong enrichment in Fr.2 cells of c-Myc targets, E2F targets, and mTORC1 signaling genes (Fig. S4 A) viagra doses 200mg.

Consistent with the mRNA analysis, expression of c-Myc protein, mTORC1 activity (assessed by phospho-S6), and E2F activity (assessed by phospho-Rb) were significantly decreased in Fr.5 cells (Fig. S4 B) viagra doses 200mg. In support of this, when we produced anti-NP IgHV186.2 Igλ monoclonal antibodies cloned from single cell-sorted Fr.2 and Fr.5 NP+IgG1+ B cells and measured their relative affinity for NP29- or NP1-BSA, we found a significant overrepresentation of lower-affinity antibodies in CD38intBcl6hi/intEfnb1+ (Fr.5) cells (Fig.

2 B). Consistently, the frequency of canonical affinity–improving mutation (replacement of Trp33 with Leu33 viagra doses 200mg. W33L+) was lower in Fr.5 cells (Fig.

2 C) viagra doses 200mg. Hence, we conclude that, in contrast to CD38−Bcl6hiCD69hi (Fr.2) cells, most of the Fr.5 cells possess lower-affinity BCRs, an indication that they received less T cell help in the LZ (Victora et al., 2010). We next compared the RNA-seq profile viagra doses 200mg of Fr.3 to Fr.5 cells (Fig.

2 A and Fig. S2 A) viagra doses 200mg. Some differences were observed between these two fractions.

Particularly, expression viagra doses 200mg of some of mTORC1 signaling genes was higher in Fr.3 than Fr.5 cells (Fig. 2 D). Myc expression in Fr.3 cells viagra doses 200mg was somewhat higher compared with Fr.5 cells (Fig.

2 D). Reflecting these differences, GSEA showed an enrichment in Fr.3 of c-Myc viagra doses 200mg targets and mTORC1 signaling genes (Fig. 2 E), although the enrichment extent of Fr.3 to Fr.5 was much smaller than Fr.2 to Fr.5 cells (Fig.

S4 C) viagra doses 200mg. By flow cytometry analysis of c-Myc and pS6, however, we could not detect significant differences in both c-Myc protein expression and mTORC1 activity between Fr.3 and Fr.5 cells (Fig. S5 A) viagra doses 200mg.

These data suggest that our flow cytometry analysis might not have sufficed to detect small changes induced by differential mRNA levels between Fr.3 and Fr.5 cells. An alternative possibility is that, in addition to mRNA level, changes in translational/posttranslational regulation might take place between viagra doses 200mg Fr.3 and Fr.5 cells. The potential reason why Fr.5 but not Fr.3 cells can become pro-memory B cells, despite relatively small differences in RNA-seq profiles between these two populations, is described below.

To identify key properties for the development of Fr.5 cells and/or their activity, we considered that Bach2/Blimp1 double-deficient GC B cells could provide a clue, since these mutant cells are defective in generating GC-derived memory B cells (Shinnakasu viagra doses 200mg et al., 2016). To this end, we transferred B cells of three genotypes (Bach2f/fPrdm1f/fERT2cre B1-8hi, Bach2+/+Prdm1f/fERT2cre B1-8hi, and Bach2+/+Prdm1+/+ERT2cre B1-8hi) into recipient mice, treated them with tamoxifen, and then immunized them with NP-CGG/alum (Fig. 3 A) viagra doses 200mg.

In contrast to the control wild-type and Blimp1 single-deficient B cells, Bach2/Blimp1 double-deficient GC B cells showed an enrichment in DZ cells (Fig. 3 B) viagra doses 200mg. Moreover, the relatively small proportion of LZ B cells still contained Fr.2 and Fr.3 cells, whereas the numbers of Fr.5 and Fr.7 cells were robustly decreased in Bach2/Blimp1 double-deficient B cells (Fig.

3 B). Since Blimp1 single knockout did not significantly affect the numbers of viagra doses 200mg pro-memory (Fr.5) and mature memory B cells (Fr.7. Fig.

3 B), we conclude that Bach2 plays an important role in development of pro-memory cells viagra doses 200mg and subsequent mature memory B cells. To determine how Bach2 participates in this process, we performed RNA profiling of Bach2/Blimp1 double-deficient LZ B cells, together with Blimp1-deficient LZ B cells as a control (Fig. S2 B) viagra doses 200mg.

In Bach2/Blimp1 double-deficient LZ B cells, GSEA revealed a significant enrichment of c-Myc target genes, E2F target genes, and mTORC1 signaling genes, in that order (Fig. 3 C) viagra doses 200mg. This was also demonstrated by flow cytometry analysis (expression levels of c-Myc, pRb, and pS6.

Fig. 3 D). Moreover, as expected, the mutant GC B cells were hyperproliferative, as assessed by 5-ethynyl-2′-deoxyuridine (EdU) pulse labeling (Fig.

3 E). These results, considering the previous demonstration that c-Myc–overexpressing and hyper-mTORC1 GC B cells manifest a bias toward the DZ (Ersching et al., 2017. Finkin et al., 2019), like Bach2/Blimp1 double-deficient GC B cells, allowed us to hypothesize that the defective pro-memory in the mutant GC cells 5could result from anomalies of the mTORC1 and/or c-Myc pathways.

Here, we focused our analysis on the mTORC1 pathway. To test this hypothesis, we first asked whether normalizing mTORC1 activity in Bach2/Blimp1 double-deficient GC cells could rescue development of pro-memory B cells and subsequent memory B cells. We transferred Bach2f/fPrdm1f/fERT2cre B1-8hi B cells into rapamycin-resistant (MtorF2108L/F2108L) hosts (Ersching et al., 2017), deleted Bach2 and Prdm1 with tamoxifen, and then immunized the mice with NP-CGG/alum (Fig.

4 A). After immunization, the mice were treated with rapamycin to decrease mTORC1 activity in a transferred B cell–intrinsic manner. As shown in Fig.

4 B, the dose of rapamycin used nearly normalized pS6 levels in the Bach2/Blimp1 double-deficient LZ B cells. The rapamycin treatment partially corrected the c-Myc overexpression and hyperproliferation observed in the Bach2/Blimp1 double-deficient B1-8hi B cells (Fig. 4 B), suggesting coexistence of mTORC1-dependent and -independent pathways to regulate c-Myc activities.

In contrast to control vehicle treatment of Bach2/Blimp1 double-deficient B1-8hi B cells, upon rapamycin treatment, those mutant cells generated threefold higher numbers of IgG1+ memory B cells. The numbers of IgG1+CD73+ memory B cells were similarly increased (Fig. 4 C, right).

Furthermore, the Fr.5:Fr.2 ratio was also increased upon rapamycin treatment (Fig. 4 D). However, the memory B cells number upon rapamycin treatment did not reach those from wild-type B1-8hi B cells upon control vehicle injection (Fig.

4 C). Hence, we conclude that hyper-mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells is one of the mechanisms that cause defective development of memory B cells, although there must be other, currently unknown ones, as well. In regard to GC B cells, the numbers were not significantly changed upon rapamycin treatment of Bach2/Blimp1 double-deficient B1-8hi B cells.

Skewing of Bach2/Blimp1 double-deficient GC B cells toward the DZ was decreased upon rapamycin treatment, although a small enrichment was still observed (Fig. 4 C). To further examine whether, in a wild-type setting, restraining mTORC1 activity could indeed facilitate differentiation of GC B cells to memory cells, we performed adoptive transfer experiments.

For this, we conducted experiments in which two types of congenically marked B cells, rapamycin-sensitive (Mtor+/+) and rapamycin-resistant (MtorF2108L/F2108L) B1-8ge B cells, were cotransferred as a 1:1 mixture into rapamycin-resistant hosts (MtorF2108L/F2108L), which were immunized with NP-CGG/alum and then administered with rapamycin. As expected, rapamycin treatment led to a decrease in S6 phosphorylation in the transferred rapamycin-sensitive, but not rapamycin-resistant, B1-8ge GC B cells (Fig. 5 A).

Upon rapamycin treatment, the number of rapamycin-sensitive NP+ GC B cells was decreased while the number of NP+ memory B cells was increased compared with their rapamycin-resistant counterparts, assessed by conventional flow cytometry analysis (Fig. 5 B). To more directly demonstrate the transition from GC B cells to Fr.7 cells, we treated the immunized mice with EdU for 3 d (days 10–13) before analysis.

In this setting, incorporation of EdU marks GC cells that divided during the treatment period and the resultant quiescent memory B cells (Fig. 5 C). We previously confirmed that during this period, the majority of proliferating cells (>95%) are GC B cells and plasmablasts (Shinnakasu et al., 2016).

Upon rapamycin treatment, the frequency of EdU+IgG1+ Fr.7 cells compared with GC cells was higher among the rapamycin-sensitive B1-8ge cells than the rapamycin-resistant ones, demonstrating rapamycin-mediated facilitation of the transition from GC to Fr.7 cells (Fig. 5 D). Moreover, upon rapamycin treatment, the numbers of CD38−Bcl6hiCD69hi (Fr.2) and CD38intBcl6hi/intEfnb1+ (Fr.5) rapamycin-sensitive B1-8ge IgG1+ B cells were decreased and maintained, respectively.

Thus, the ratio of Fr.5 to Fr.2 was increased (Fig. 5 E). Together, we conclude that a relative enrichment in Fr.5 over Fr.2 cells is induced by rapamycin treatment, thereby facilitating the overall transition from GC B cells to memory B cells.

The memory B cells generated in the presence of rapamycin were able to induce similar recall antibody responses to those generated in the absence of rapamycin, as assessed by adoptive transfer experiments (Fig. 5 F). We next wished to examine why Fr.5, but not Fr.3 cells, can become pro-memory B cells.

Since there were almost no differences in mTORC1 activity between Fr.5 and Fr.3 cells (Fig. S5 A), it appears that an mTORC1lo state is necessary but not sufficient for development of pro-memory B cells (Fr.5). Thus, additional key properties must be required for development of these cells.

Since one of crucial features of mature memory B cells is longevity, one straightforward possibility is that Fr.5 cells begin to acquire more survival activity. Supporting this idea, CD38intBcl6hi/intEfnb1+ (Fr.5) cells were less apoptotic compared with CD38−Bcl6hiCD69lo (Fr.3) cells as assessed by active caspase-3 staining (Fig. 6 A).

Transcript data (Fig. 6 B) together with protein expression data (Fig. 6 C) demonstrated that Bcl2 expression was upregulated in Fr.5 cells compared with Fr.3 cells, and even more in pre-memory B cells (Fr.6).

Similarly, we found that the cell surface BCR expression level was increased stepwise from Fr.3 to Fr.6 cells (Fig. 6 D). We also observed a slight increase of IgG1 and Igα/β mRNA expression in Fr.5 over Fr.3 cells.

Thus, regulation of both mRNA and protein levels seems to be operative. To examine whether Bcl2 family protein–mediated survival activity could impact the development of Fr.5 cells, we employed GC B cells with haploinsufficiency of Bim (Bcl2l11. See Materials and methods), a counteracting factor against anti-apoptotic Bcl2-family members (O’Connor et al., 1998).

Bcl2l11+/+ ERT2cre B1-8ge B cells and Bcl2l11f/+ERT2cre B1-8ge B cells were cotransferred as a 1:1 mixture into wild-type recipient mice, which were then immunized with NP-CGG/alum and treated with tamoxifen on day 8 (Fig. 6 E). Bim mRNA expression was decreased to almost 50% of control levels after tamoxifen treatment in Bcl2l11f/+ GC B cells (Fig.

6 F). In this competitive setting, among the Fr.2/3/5/6 cells, the frequency was most significantly increased in Fr.5 and Fr.6 cells upon Bim haploinsufficiency (Fig. 6 G), although there was also a modest increase of Fr.3 cells.

Consequently, the frequency of Bcl2l11f/+ NP+IgG1+CD73+ memory B cells was also increased (Fig. S5 B). To examine the effects of surface BCR expression on survival, B1-8ge-flox/+ ERT2cre B cells were employed.

For these particular experiments, we mixed these B cells and control B1-8ge/+ ERT2cre B cells at a 7:3 ratio and adoptively cotransferred them into recipient mice, which were then immunized with NP-CGG/alum (Fig. 6 H) viagra after prostate removal. We injected tamoxifen on day 10 and examined surface BCR expression on day 12, demonstrating a significant decrease on Fr.5 cells derived from B1-8ge-flox/+ ERT2cre B cells (Fig.

6 I). To detect apoptotic cells in this experiment, we analyzed mixtures of Fr.5 and Fr.6 cells (CD38+Efnb1+) to acquire a sufficient number of cells for the assay. As demonstrated in Fig.

6 J, concomitant with decreased surface BCR expression, there was a higher frequency of apoptotic (aCasp3+) cells among pro/pre-memory cells derived from B1-8ge-flox/+ ERT2cre B cells. Similarly, frequency of apoptotic cells among total LZ GC cells was enhanced upon BCR downregulation (Fig. S5 C).

A control experiment using Prdm1f/+B1-8ge/+ ERT2cre B cells showed that a nonspecific effect on apoptosis induced simply by Cre-mediated double-strand breaks was negligible (Fig. S5 D). Together, stepwise increases of Bcl2 and surface BCR expression from pro-memory (Fr.5) cells to pre-memory (Fr.6) toward mature memory B cells are likely to contribute to their survival.

It is still unclear what signals and processes in LZ GC cells initiate their differentiation toward long-lived memory B cells. Here, by focusing on key features for development of GC-derived memory precursors, we show that an mTORC1lo state is necessary to develop pro-memory B cells. Since mTORC1lo LZ B cells receive weak T cell help and, as a result, have been thought to undergo apoptosis, this raises the question of how such pro-memory B cells are prevented from dying and able to differentiate into mature memory B cells.

Our experiments suggest that the memory precursor B cells express higher levels of Bcl2 and surface BCR, thereby acquiring a survival advantage. We have already shown that Bach2hi LZ GC B cells are predisposed to differentiate into memory B cells (Shinnakasu et al., 2016), indicating that memory cell commitment already begins in a subset of GC B cells. This memory-prone subset most likely corresponds to the Fr.5 (CD38intBcl6hi/intEfnb1+ pro-memory) cells.

Indeed, expression of Bach2 in Fr.5 is higher than in Fr.2 cells (Fig. 2 A and Fig. S3 B).

Fr.6 (pre-memory B) cells appear to be undergoing a further developmental step toward mature memory B cells, manifested by further downregulation of Bcl6 (Fig. 1 B). We found that mTORC1 has a marked effect on the ratio of memory-prone (Fr.5) to recycling-prone (Fr.2) GC B cell formation.

Rapamycin treatment increased the proportion of Fr.5 cells and, conversely, hyperactivation of mTORC1 in the Bach2/Blimp1 double-deficient setting led to a relative increase in Fr.2 cells. Several nonmutually exclusive possibilities can be envisaged to explain why lower mTORC1 activity contributes to development of memory-prone cells. Decay in mTORC1 activity as GC B cells proliferate in the DZ appears to be required for their timely return to the LZ (Ersching et al., 2017).

Given the importance of LZ residency for memory differentiation (Bannard et al., 2013), one possibility is that LZ residency imposed by mTORC1lo could allow development of pro-memory B cells. Second, apart from this spatial requirement mediated through modulation of mTORC1, inhibition of mTORC1, as is seen during the generation of natural killer cell memory (O’Sullivan et al., 2015), may stimulate autophagy, thereby enhancing pro-memory B cell survival. Finally, it is also well known that mTORC1 activity is suppressed in memory B cells (Boothby and Rickert, 2017).

Such metabolic changes as the cells progress toward mature memory B cells thus appear to be initiated already in pro-memory cells, and this might be a necessary first step for generating mature memory B cells. The partial restoration of memory B cells by rapamycin treatment in Bach2/Blimp1 double-deficient GC cells suggests that, in addition to hyper-mTORC1 activity, other anomalies occur in mutant GC B cells in regard to memory differentiation. One of them is likely the c-Myc overexpression, because of the following.

First, indeed, in rapamycin-treated Bach2/Blimp1 double-deficient GC cells, overexpression of c-Myc and hyperproliferation were still observed to a significant extent (Fig. 4 B). Second, c-Myc–overexpressing GC cells were reported to have a significant bias toward the DZ (Finkin et al., 2019).

Considering the importance of LZ residency for memory differentiation (Bannard et al., 2013), overexpression of c-Myc is assumed to be detrimental to memory differentiation. Hence, we would propose that restraining both mTORC1-mediated metabolism and c-Myc–mediated cell-cycle progression is required to develop pro-memory B cells and that Bach2 is one of the critical regulators for suppressing both pathways. Functionally, Bach2 is well known to act as a repressive guardian transcription factor (Igarashi et al., 2017).

In regard to relationship between signaling and Bach2 expression, the mTORC1 activity and Bach2 expression appear to be mutually exclusive, because the BCR-induced AKT-mTORC1 inhibits Bach2 expression (Kometani et al., 2013), and Bach2 represses transcription of mTORC1 signaling molecules. Such a negative feedback loop is characteristic of “bistable” signal transduction circuits, which can operate in two stable formats. This might take place between Fr.5 and Fr.2 cells.

It should be mentioned that, from mTORC1 signaling molecule side, Bach2 is one of the transcription factors, and probably additional factors participate in transcriptional regulation on mTORC1 signaling genes. In addition to the connection between BCR signal and Bach2, considering the T cell data showing that ICOS and integrin αE are upregulated in Bach2lo T cells (Grant et al., 2020. Sidwell et al., 2020), Bach2 might be involved in connecting the BCR signal to T cell help.

For instance, Bach2lo LZ GC cells with high-affinity BCRs might modulate T/B interactions through adhesion status and coreceptor expression and affect the strength of T cell help. This might further downregulate Bach2, because we previously showed that strong T cell help depresses Bach2 expression (Shinnakasu et al., 2016). After moving back into the LZ, apoptosis is generally thought to be the default pathway for LZ GC B cells.

However, high-affinity cells are spared and positively selected after they encounter sufficient cognate T cell help (Allen et al., 2007. Victora and Nussenzweig, 2012). These spared high-affinity cells correspond to Fr.2 cells, whereas the defaulting apoptotic LZ cells are likely to be Fr.3 cells.

Indeed, among LZ GC cells, Fr.3 cells were most apoptotic. Here, we show that a small population of pro-memory B cells exists in the LZ and, despite apparently receiving weak T cell help, they are relatively resistant to apoptosis. The inability of prior studies to detect such apoptosis-resistant LZ B cells is most likely due to the fact that the numbers of pro-memory cells are so limited (Mayer et al., 2017).

Previous data using B cell–specific Bcl2-tg mice (Smith et al., 1994) or Bim knockout mice (Fischer et al., 2007) showed that such mice develop an enlarged memory B cell compartment. Recently, more detailed analysis using the same Bcl2-tg mice (Stewart et al., 2018) provided mechanistic insights into the above phenomenon. First, in these mice, aberrant populations of seemingly quiescent cells arise that express markers of memory precursor cells.

Second, overexpression of Bcl2 is not sufficient for DZ GC B cells with damaged BCRs to reach the LZ. Hence, in a physiological setting, it is reasonable to speculate that, after returning to the LZ in a Bcl2-independent manner, if Bcl2 expression is upregulated in some of the LZ GC B cells, they are better able to be rescued from apoptosis in the late G1 phase and to begin to differentiate into memory B cells. Supporting this idea, we show here that among LZ GC cells, small numbers of pro-memory B cells (Fr.5), but not Fr.3 cells, begin to upregulate Bcl2, and that development of pro-memory B cells is facilitated by Bim haploinsufficiency.

Because Bach2 expression in Fr.5 cells is similar to Fr.3 cells (Fig. S3 B), Bach2 appears not to be involved in such differential survival activity between Fr.5 and Fr.3 cells. Rather, a Bach2-independent mechanism such as Bcl6 downregulation (discussed below) is likely to be operated, thereby allowing Fr.5 cells to survive enough to begin to differentiate into memory precursor cells.

In contrast to Fr.5 cells (pro-memory), Fr.6 cells (pre-memory) apparently possess more survival activity (Fig. 6 A), possibly explaining the generation kinetics between Fr.5 and Fr.6 cells. Fr.6 cells were more accumulated at later phases (days 14 and 20) during immune responses (Fig.

S1 B). Induced downregulation of surface BCR expression in pro/pre-memory B cells resulted in increased apoptosis in the pro-memory B cells. These results, together with the evidence that pro-memory B cells express higher surface BCR levels, lead us to propose that the BCR-mediated survival signal also plays a role in the development of pro-memory B cells.

Based on the previous report that BCR ablation leads to cell death, which can be delayed by constitutive Bcl2 expression (Lam et al., 1997), we considered the possibility that downregulation of the BCR might decrease Bcl2 expression in pro/pre-memory B cells. However, we could not detect such a connection (data not shown). In naive B cells, the constitutive PI3 kinase–Foxo1 pathway is known to replace the missing BCR-mediated survival signals (Srinivasan et al., 2009).

Therefore, a question arises of how pro-memory B cells, despite being mTORC1lo (reflecting lower Akt activity), generate such a survival signal. Given that there is no enlarged GC phenotype in PTEN or Foxo1 knockout mice (Dominguez-Sola et al., 2015. Inoue et al., 2017.

Sander et al., 2015. Suzuki et al., 2003), one straightforward explanation might be that the quality and/or quantity of BCR-mediated survival signals differ between naive B cells and GC-derived memory B cells. We provide evidence that downregulation of Bcl6 in pro-memory B cells could be one of the mechanisms for upregulation of Bcl2 and surface BCR.

However, since the extent of Bcl6 downregulation in pro-memory B cells is small, such a slight change might not account for the observed upregulation of Bcl2 and surface BCR. Hence, our data cannot completely exclude the possibility that, particularly at the pro-memory B cell stage, other mechanisms might operate to initiate upregulation of Bcl2 and BCR. In this case, it is likely that downregulation of Bcl6 acts as an amplification pathway for further upregulation of Bcl2 and surface BCR during differentiation toward mature memory B cells.

In regard to this differentiation pathway, our data using Bcl6 haploinsufficiency are highly complementary to previous in vitro data that ectopic expression of Bcl6 in B cell cultures blocks the GC B cells from differentiating into memory B cells (Kuo et al., 2007). Together, it is likely that stepwise decreases in Bcl6 expression (pro-memory >. Pre-memory >.

Mature memory B cells) play a key role in memory B cell development. This raises the question of how is Bcl6 downregulated. Three possibilities have already been reported.

(1) upon strong BCR engagement, Erk-mediated degradation of Bcl6 (Niu et al., 1998). (2) transcriptional downregulation of Bcl6, mediated by CD40-activated IRF4 (Saito et al., 2007). And (3) downregulation of Bcl6 by defective IL-21 signaling (Linterman et al., 2010).

Among these, in regard to differentiation from GC to memory B cells, the final possibility seems to best fit with our observation that pro-memory B cells possess lower-affinity BCRs, thereby receiving less T cell help. In addition, as a transcriptional circuit–type regulation, the transcription factor Hhex, critical for memory B cell differentiation, has been recently reported to participate in downregulation of Bcl6 (Laidlaw et al., 2020). In summary, this study provides important insights into the initial events for the fate decisions from GC to memory B cells.

The modulation of cellular metabolism and survival play fundamental roles. Given the importance of GC-derived memory B cells for protection against heterologous virus reinfection (Leach et al., 2019. Purtha et al., 2011), our findings may contribute to the development of efficient vaccination strategies.

Single-cell suspensions of splenocytes were analyzed and sorted on a FACSCanto II (BD Biosciences) or a FACSAria II (BD Biosciences). Alexa647-active caspase-3, V500-B220, V450-Bcl6, BV786-CD138, BV510-CD38, V500-CD45.2, BV510-IgG1, PE-IgG1 antibodies, and BV786-streptavidin were purchased from BD Biosciences. APC-eFluor780-B220, FITC-CD45.1, PE-CD45.1, APC-eFluor780-CD45.1, FITC-CD45.2, APC-eFluor780-CD45.2, APC-CD69, APC-eFluor780-CD69, eFluor450-CD73, PE-CD86, PerCP-Cy5.5-GL7 antibodies, PerCP-Cy5.5-streptavidin, PE-streptavidin, and APC-eFluor780-streptavidin were purchased from eBioscience.

PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend. PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. C-Myc antibody was purchased from Abcam.

PE-Bcl2 antibody was purchased from Miltenyi Biotec. Biotin-Efnb1 antibody was purchased from R&D Systems. Alexa488-goat anti-rabbit IgG antibody was purchased from Thermo Fisher Scientific.

For intracellular staining, the cells were fixed and permeabilized using a Foxp3 staining kit (eBioscience) for Bcl6, Bcl2, and pRb, a BD Cytofix/Cytoperm solution (BD Biosciences) for pS6 and active caspase-3, or a True-Nuclear Transcription Factor Staining Buffer Set (BioLegend) for c-Myc. APC-conjugated NP was prepared as described previously (Shinnakasu et al., 2016). Incorporation of EdU was detected using a Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.

We thank M.C. Nussenzweig (The Rockefeller University, New York, NY) for B1-8hi mice, T. Okada (RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan) for Bcl6-YFP mice, G.D.

Victora (The Rockefeller University) for MtorF2108L mice, and P.D. Burrows for critical reading of the manuscript. This work was supported by grants from JSPS KAKENHI (JP17K08882 to T.

Inoue. JP26221306 and JP19H01028 to T. Kurosaki), the SENSHIN Medical Research Foundation (to T.

Inoue), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. Inoue), and a research grant from Astellas Foundation for Research on Metabolic Disorders (to T. Inoue).

Kawai performed the experiments. T. Inoue and T.

Kurosaki designed the experiments. R. Shinnakasu, W.

Fukuyama provided essential reagents. E. Kawakami, N.

Sax, and K. Yamashita performed bioinformatics analyses. T.

Inoue and T. Kurosaki wrote the manuscript.GRIP1 is a broadly acting transcriptional coregulator whose role in MS/EAE or in MG at any state has never been investigated. To begin to identify the GRIP1-dependent transcriptome changes leading to neuroinflammation, we performed bulk RNAseq analysis on CD45+Cd11b+ myeloid cells isolated from spinal cords of WT and GRIP1-cKO mice.

Consistent with a lack of overt phenotype in our conditional GRIP1-deficient mice (Coppo et al., 2016. Rollins et al., 2017), at homeostasis, a CD45+Cd11b+ CNS myeloid cell population composed principally of MG displayed no significant transcriptomic differences between WT and GRIP1-cKO mice (Fig. 5 A, upper panel.

GRIP1 deletion efficiency is shown on the right as normalized read counts across “floxed” exon 11 of the Ncoa2 gene). In contrast, more heterogeneous activated CD45+Cd11b+ cells during EAE (Fig. S2 C and Fig.

2 C) presented distinct transcriptomic signatures in WT versus GRIP1-cKO mice. Indeed, genes upregulated in WT (Fig. 5 A, lower panel, and Fig.

5 B), such as chemokines and chemokine receptors (Ccl22, Ccr7), antigen presentation molecule (H2-q10), components of complement (C3, C1ra), and type I IFN (Trim12c, Oas3) pathways, are indicative of inflammation and EAE pathogenesis (Belikan et al., 2018. Salter and Stevens, 2017. Scheu et al., 2017).

Interestingly, a pool of genes downregulated in WT mice during EAE but persisting in GRIP1-cKO mice (e.g., Gpr34, P2ry12. Fig. 5 A, lower panel, and Fig.

5 B) are homeostatic genes referred to as the MG “sensome” (Hickman et al., 2013), which controls chemotaxis and tissue repair (Lou et al., 2016). To identify physiologically relevant pathways differentially active in myeloid cells from WT and GRIP1-cKO spinal cords during EAE, we performed quantitative set analysis of gene expression (QuSAGE. Yaari et al., 2013), a gene set enrichment analysis–like Bayesian method that provides better accounts for intergene correlations than classic gene set enrichment analysis.

QuSAGE determines pathway-wide expression (pathway activity) by combining probability density functions for individual gene expression using numerical convolution. Several pathways were expressed at higher levels in the WT CNS myeloid cells, including NODE-like receptor signaling pathways (Kyoto Encyclopedia of Genes and Genomes) and a nuclear receptor transcription pathway (REACTOME), including Nr4a2 (Nurr1) and Nr4a3 (Nor-1. Fig.

5 C). Remarkably, several key genes of the IFN axis (IFN signaling pathway [REACTOME]), including Irf4, Irf1, Ifng, Ifitm1, Gbp5, and Oas3, were also expressed at higher levels in the WT (Fig. 5 C), in accord with whole-brain and spinal cord quantitative PCR (qPCR) data (Fig.

3 A and Fig. S5 A) and with a demonstrated coactivator role for GRIP1 in type I IFN network in MФ (Flammer et al., 2010. Reily et al., 2006).

Collectively, these data demonstrate a failure to upregulate inflammatory and type I IFN pathways and persistence of homeostatic signature in GRIP1-cKO myeloid cells. However, it could potentially stem from the role of GRIP1 in MG, MФ, or both. To dissect the contribution of resident versus infiltrating myeloid cells to EAE pathogenesis, we performed single-cell RNAseq (scRNAseq) analysis of all myeloid CD45+CD11b+ cells from WT and GRIP1-cKO spinal cords at the peak of EAE (DPI20).

After filtering out low-quality barcodes (see Materials and methods), we analyzed 20,376 cells (6,427 WT and 11,949 cKO) expressing 11,093 genes. Automated cell type assignment with singleR yielded four major clusters—“monocytes,” “MФ,” “dendritic cells,” and “neutrophils” (Fig. 6 A and Table S1)—and a large number of minor clusters composed predominately of lymphoid cell impurities that were collected during the cell sorting and had the same location in uniform manifold approximation and projection (UMAP) coordinates (Fig.

6 A). Because of an unbalanced group size, we performed a bootstrapping analysis to determine the associations between genotype and singleR cell types. We counted cell types of 2,000 cells that were sampled with the replacement from each genotype with 500 repeats (Fig.

6 B and Fig. S4 A). This analysis indicated that singleR monocytes and neutrophils were more common in the cKO, whereas MФ were overrepresented in the WT.

There was a substantial overlap between singleR cell types, suggesting either the presence of cell subpopulations or different differentiation/activation states. To separate these states, we performed Louvain graph–based community clustering that yielded nine clusters (Fig. 6 C and Table S2).

Cluster 8 corresponded to singleR lymphoid cell–enriched group (Fig. 6 A. €œOthers,” “T cells”), whereas cluster 6 was highly enriched with canonical neutrophilic markers (Fig.

6, A, D, and E). Cluster 3 is enriched in proliferation markers (Fig. 6 E, Fig.

S4 B, and Table S4). Slingshot trajectory analyses anchored on cluster 3 (see Materials and methods) identified two main trajectories (3-5-9-7-1 and 3-5-9-2-4-6) bifurcating at cluster 9 (Fig. 6 C and Fig.

S4 C). The analysis of genes differentially expressed along trajectories suggested that the 3-5-9-7-1 trajectory likely corresponds to monocyte-to-MФ transitions. Conversely, clusters 2-4-6 exhibit an increasing gradient of expression of neutrophilic markers (Fig.

6 E. S100a8, S100a9), suggesting that clusters 4 and 2 contain a decreasing admixture of neutrophils from cluster 6. Cluster 3 expresses monocytic markers at high levels (Fig.

6 F. Ly6c2, F13a1, Stmn1) and activated MФ/MG markers at low levels (Fig. 6, F and G.

Cd74, Fth1, Fcgr2b, H2-Aa, Il1b) that reciprocally change along the trajectories. MФ-like clusters (1, 7, and 2) contain either different proportions of MФ/MG, different activation states, or an admixture of other cell types (e.g., oligodendrocyte precursors. Table S3).

Although expression distributions for activated MФ/MG markers are broadly comparable in these clusters (Fig. S4 D), differential expression analysis between WT and cKO stratified by Louvain clusters revealed that clusters 1, 7, 2, and 4 expressed markers of homeostatic MG at higher levels in the cells from cKO mice (Fig. 6 H, Fig.

S4 E, and Table S5. Sparc, Siglech, Olfml3, and Tmem119). Cluster 2 contained the largest percentage of cells expressing homeostatic MG markers.

Conversely, many markers of activated inflammatory MФ were upregulated in these clusters in the WT cells including Il1a, Il1r2, Il7r, Ifng, Ctla2s, and Nos2 (Fig. 6 I and Table S5)..

A still unanswered question male viagra pill walmart is viagra online no prescription what drives the small fraction of activated germinal center (GC) B cells to become long-lived quiescent memory B cells. We found here that a small population of GC-derived CD38intBcl6hi/intEfnb1+ cells with lower mTORC1 activity favored the memory B cell fate. Constitutively high mTORC1 activity led to defects in formation viagra online no prescription of the CD38intBcl6hi/intEfnb1+ cells. Conversely, decreasing mTORC1 activity resulted in relative enrichment of this memory-prone population over the recycling-prone one. Furthermore, the viagra online no prescription CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR that, in turn, contributed to their survival and development.

We also found that downregulation of Bcl6 resulted in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity, our data suggest a model in which weak viagra online no prescription help from T cells together with provision of an increased survival signal are key for GC B cells to adopt a memory B cell fate. Memory B cells and long-lived plasma cells are responsible for effective long-term immunity against pathogens. The majority of these cells responding to T cell–dependent viagra online no prescription antigens are generated from the germinal center (GC) reaction. Indeed, memory B cells emerge from the GC as recirculating cells and, upon secondary antigen challenge, they are primed to elicit rapid antibody responses.

GCs are divided into two viagra online no prescription anatomical structures. The light zone (LZ) and the dark zone (DZ. Allen et al., 2007 viagra online no prescription. Victora and Nussenzweig, 2012). B cells proliferate and undergo somatic hypermutation in the DZ before entering viagra online no prescription the LZ, where they exit the cell cycle.

In the LZ, GC B cells expressing newly mutated B cell receptors (BCRs) capture antigen presented on follicular dendritic cells and internalize it for presentation to follicular helper T cells. Subsequently, antigen- and T cell–dependent selection takes place, whereby the “choice” of recycling to the DZ for further viagra online no prescription affinity maturation or of exiting the GC as plasma or memory B cells is made. In regard to the selection mechanism, it has been postulated that precursor cells destined to become recycling GC, plasma, or memory B cells already become committed in the LZ, at least to some extent, thereafter entering the recycling DZ, plasma, or memory B cell pools (Inoue et al., 2018). For instance, it has been demonstrated that a small fraction of LZ B viagra online no prescription cells expressing c-Myc, a key cell-cycle regulator, corresponds to precursor cells for the recycling GC fate. C-Myc+ cells are enriched for high-affinity BCRs and ablation of c-Myc affects DZ reentry (Calado et al., 2012.

Dominguez-Sola et al., viagra online no prescription 2012. Finkin et al., 2019). Bcl6loCD69hi LZ B cells expressing IRF4, viagra online no prescription a critical transcription factor for plasma cell differentiation, were recently shown to be the precursors of plasma cells (Ise et al., 2018). In contrast to these insights into the precursor cells for recycling and plasma cell fates, studies of the memory fate decision have been hampered by the lack of a known master transcription factor for differentiation of memory B cells. Hence, surrogate markers such as an viagra online no prescription S1PR2 reporter, CCR6 expression, or a cell cycle reporter have been recently employed for identification of memory precursor cells (Laidlaw et al., 2017.

Suan et al., 2017. Wang et al., 2017). Although informative, these studies have not identified key features for development of the GC-derived precursor cells viagra online no prescription committed to the long-lived memory B cell fate, or what signals regulate these key features. Here, after identifying a memory-prone population (CD38intBcl6hi/int Ephrin-B1 [Efnb1+]), we found that this small population exhibited lower mTORC1 activity than the recycling-prone population. Constitutive high mTORC1 activity viagra online no prescription led to defective development of CD38intBcl6hi/intEfnb1+ cells, whereas decreasing mTORC1 activity resulted in relative enrichment in this memory-prone cell population versus the recycling-prone one.

Moreover, the CD38intBcl6hi/intEfnb1+ cells had higher levels of Bcl2 and surface BCR, thereby contributing to their survival and development. We also found that downregulation of Bcl6 resulted viagra online no prescription in increased expression of both Bcl2 and BCR. Given the positive correlation between the strength of T cell help and mTORC1 activity (Ersching et al., 2017), our data suggest a model in which weak help from T cells together with provision of an increased survival signal are key for GC cells to assume the memory B cell fate. To clarify the initiating process for memory B cell differentiation occurring in the GC, we wished to identify GC B cells destined to the viagra online no prescription memory fate. For this, we used Bcl6 protein reporter mice (Kitano et al., 2011).

We immunized these mice with 4-hydroxy-3-nitrophenylacetyl (NP)–chicken γ-globulin (CGG) in viagra online no prescription alum i.p. And analyzed NP-specific IgG1+ splenic B cells at day 10. Since CD38 upregulation takes place during the transition from GC to memory B cells (Ridderstad and Tarlinton, 1998), we examined such viagra online no prescription CD38+ B cells that still maintained GC identity to some extent, i.e., were Bcl6+, together with conventional CD38− GC B cells. By using a fractionation method described previously (Fig. S1 A viagra online no prescription.

Ise et al., 2018), the LZ B cells were further separated based on their Bcl6 and CD69 expression pattern (upper right panel in Fig. 1 A) viagra online no prescription. Fraction (Fr.) 1 (CD38−Bcl6loCD69hi) and Fr.2 (CD38−Bcl6hiCD69hi) cells are plasma and recycling GC precursor cells, respectively (Ise et al., 2018). Characterization of Fr.3 (CD38−Bcl6hiCD69lo) cells is described below viagra online no prescription. Efnb1 is expressed at a high level by almost all Fas+GL7+ cells, but is barely detectable on naive B cells (Laidlaw et al., 2017.

Lu et viagra online no prescription al., 2017. Wang et al., 2017), allowing us to identify transitional populations between GC and memory B cells. Hence, for CD38+ cells, by using Efnb1 and Bcl6, we further separated the NP+ IgG1+CD38+GL7−CD138− cells into Bcl6+Efnb1+ (Fr.5), Bcl6loEfnb1+ (Fr.6), and Bcl6−Efnb1− (Fr.7 viagra online no prescription. Lower right panel in Fig. 1 A) viagra online no prescription.

Since expression level of Bcl6 in Fr.5 cells was slightly but significantly lower than that of Fr.3 cells, as shown by the left panel in Fig. 1 B, herein, we designated Bcl6hi/int viagra online no prescription for Fr.5. CD38 expression levels on Fr.5, Fr.6, and Fr.7 cells were increased in that order (middle panel in Fig. 1 B. Herein, indicated as CD38int, viagra online no prescription and CD38+ for Fr.5 and 6/7, respectively).

During the time course of the GC response, Fr.5 and Fr.6 cell numbers peaked at day 10 before declining, whereas Fr.7 cells peaked at day 12 and then slowly declined (Fig. S1 B) viagra online no prescription. These kinetic data suggest that Fr.5 and Fr.6 contain cells that are transient and intermediate, and that once cells enter the Fr.7 pool, they are stably maintained. The Fr.7 cells displayed a typical CD38+Bcl6−Efnb1− mature viagra online no prescription memory phenotype (Fig. 1 B).

To assess the relationship between overall LZ B cells and Fr.5/6/7 cells, we performed RNA sequencing viagra online no prescription (RNA-seq) analysis (Fig. S2 A). To obtain sufficient amounts of RNA for this analysis, we used transferred B1-8hi B cells instead of non-BCR transgenic mice viagra online no prescription. These NP-specific transgenic GC B cells were present in similar proportions in each fraction as in non-BCR transgenic mice (Fig. S1 C) viagra online no prescription.

The principal component analysis (PCA) for each fraction indicated that memory B cells (Fr.7) clustered most tightly with CD38+Bcl6loEfnb1+ (Fr.6) cells but differed greatly from total LZ GC B cells (Fig. 1 C) viagra online no prescription. Fr.5 cells were intermediate between Fr.6 and LZ GC B cells. Fr.6 cells expressed lower levels of S1pr2 and higher levels of Gpr183 (EBI2) mRNA viagra online no prescription compared with LZ B cells (Fig. S1 D), implying that they are a cell population in the process of exiting the GC.

Herein, we call Fr.6 “pre-memory B cells.” In contrast to viagra online no prescription Fr.6 and mature memory B cells (Fr.7), Fr.5 cells seem to start the process of downregulating Bcl6. Fr.6 cells are most likely to correspond to the already identified GC-derived pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”. Laidlaw et al., 2017), “LZ CCR6+” (Suan et al., 2017), and “mKO2hi” (Wang et al., 2017) in that, like those cells, Fr.6 viagra online no prescription cells are Bcl6int/loBach2int (Fig. S3, A and B). The above data prompted us to consider that, among Fr.2, Fr.3, and Fr.5 cells, the CD38intBcl6hi/intEfnb1+ cells (Fr.5) could be potential GC-derived precursors viagra online no prescription of the pre-memory B cells (Fr.6).

To test this possibility, we took the following three approaches. First, PCA of the RNA-seq data was performed, indicating that CD38intBcl6hi/intEfnb1+ cells (Fr.5) viagra online no prescription and pre-memory B cells (Fr.6) clustered most closely together (Fig. 1 D). Second, to monitor cellular quiescence, we employed mVenus-p27K− transgenic mice, in which mainly G0 phase cells are labeled (Oki et al., 2014), demonstrating that in contrast to viagra online no prescription Fr.2 and Fr.3 cells, Fr.5 and Fr.6 cells had more mVenus-p27K− probe–positive, i.e., quiescent cells (Fig. 1 E).

Finally, in order to assess the memory recall potential of the Fr.5 cells, we used a previously described adoptive transfer method (Wang et al., 2017). As illustrated in Fig viagra online no prescription. 1 F, Fr.2, Fr.3, Fr.5, or Fr.6 cells were isolated from NP-CGG/alum immunized mice and adoptively transferred (2 × 104 cells per mouse) into sublethally irradiated recipient mice together with CD4+ T cells isolated from CGG-immunized mice. The recipient mice were then challenged with NP-CGG and viagra online no prescription analyzed on day 6 for NP-specific plasma cells. Although less proficient than pre-memory B cells (Fr.6), the ability of the adoptively transferred CD38intBcl6hi/intEfnb1+ (Fr.5) cells to give rise to plasma cells was significantly superior to Fr.2 and Fr.3 cells (Fig.

1 G) viagra online no prescription. To rule out the possibility that Fr.5 cells were cells that had reentered the GC reaction from already generated memory B cells, we stained them for Ki67 and observed lower expression in Fr.5 than in the pre-GC B cells, which are in the process of entering the GC (Fig. S1 E) viagra online no prescription. Together, CD38intBcl6hi/intEfnb1+ (Fr.5) cells are likely to be a precursor of pre-memory B cells, herein called Fr.5 “pro-memory B cells,” and to represent a precursor population of previously identified pre-memory B cells (“Efnb1+S1pr2lo [Pop 4]”. Laidlaw et al., 2017), “LZ CCR6+” (Suan viagra online no prescription et al., 2017), and “mKO2hi” (Wang et al., 2017.

Fig. S3, A and viagra online no prescription B). However, we do not exclude the possibility that the pro-memory B cell population (Fr.5) is heterogeneous in its origins and properties. For instance, Fr.5 cells appear to overlap, to some extent, with LZ CCR6+ cells in that they are beginning to viagra online no prescription express Ccr6 (Fig. S3 C).

To gain insight into the specific features of CD38intBcl6hi/intEfnb1+ (Fr.5) cells that promote their potential development and/or differentiation into memory viagra online no prescription cells, we compared their RNA-seq profile to that of the other LZ B cells (Fr.2 and Fr.3. Fig. 2 A and viagra online no prescription Fig. S2 A). CD38−Bcl6hiCD69hi (Fr.2) viagra online no prescription cells are destined to the recycling GC fate (Ise et al., 2018).

Gene set enrichment analysis (GSEA) of Hallmark gene sets (Liberzon et al., 2015) revealed a strong enrichment in Fr.2 cells of c-Myc targets, E2F targets, and mTORC1 signaling genes (Fig. S4 A) viagra online no prescription. Consistent with the mRNA analysis, expression of c-Myc protein, mTORC1 activity (assessed by phospho-S6), and E2F activity (assessed by phospho-Rb) were significantly decreased in Fr.5 cells (Fig. S4 B) viagra online no prescription. In support of this, when we produced anti-NP IgHV186.2 Igλ monoclonal antibodies cloned from single cell-sorted Fr.2 and Fr.5 NP+IgG1+ B cells and measured their relative affinity for NP29- or NP1-BSA, we found a significant overrepresentation of lower-affinity antibodies in CD38intBcl6hi/intEfnb1+ (Fr.5) cells (Fig.

2 B). Consistently, the frequency of canonical affinity–improving mutation (replacement viagra online no prescription of Trp33 with Leu33. W33L+) was lower in Fr.5 cells (Fig. 2 C) viagra online no prescription. Hence, we conclude that, in contrast to CD38−Bcl6hiCD69hi (Fr.2) cells, most of the Fr.5 cells possess lower-affinity BCRs, an indication that they received less T cell help in the LZ (Victora et al., 2010).

We next compared viagra online no prescription the RNA-seq profile of Fr.3 to Fr.5 cells (Fig. 2 A and Fig. S2 A) viagra online no prescription. Some differences were observed between these two fractions. Particularly, expression of some of mTORC1 signaling genes was higher in Fr.3 than viagra online no prescription Fr.5 cells (Fig.

2 D). Myc expression in Fr.3 cells was somewhat higher compared with Fr.5 cells viagra online no prescription (Fig. 2 D). Reflecting these viagra online no prescription differences, GSEA showed an enrichment in Fr.3 of c-Myc targets and mTORC1 signaling genes (Fig. 2 E), although the enrichment extent of Fr.3 to Fr.5 was much smaller than Fr.2 to Fr.5 cells (Fig.

S4 C) viagra online no prescription. By flow cytometry analysis of c-Myc and pS6, however, we could not detect significant differences in both c-Myc protein expression and mTORC1 activity between Fr.3 and Fr.5 cells (Fig. S5 A) viagra online no prescription. These data suggest that our flow cytometry analysis might not have sufficed to detect small changes induced by differential mRNA levels between Fr.3 and Fr.5 cells. An alternative possibility is that, in addition to mRNA level, changes in translational/posttranslational regulation might take place between Fr.3 and viagra online no prescription Fr.5 cells.

The potential reason why Fr.5 but not Fr.3 cells can become pro-memory B cells, despite relatively small differences in RNA-seq profiles between these two populations, is described below. To identify key properties viagra online no prescription for the development of Fr.5 cells and/or their activity, we considered that Bach2/Blimp1 double-deficient GC B cells could provide a clue, since these mutant cells are defective in generating GC-derived memory B cells (Shinnakasu et al., 2016). To this end, we transferred B cells of three genotypes (Bach2f/fPrdm1f/fERT2cre B1-8hi, Bach2+/+Prdm1f/fERT2cre B1-8hi, and Bach2+/+Prdm1+/+ERT2cre B1-8hi) into recipient mice, treated them with tamoxifen, and then immunized them with NP-CGG/alum (Fig. 3 A) viagra online no prescription. In contrast to the control wild-type and Blimp1 single-deficient B cells, Bach2/Blimp1 double-deficient GC B cells showed an enrichment in DZ cells (Fig.

3 B) viagra online no prescription. Moreover, the relatively small proportion of LZ B cells still contained Fr.2 and Fr.3 cells, whereas the numbers of Fr.5 and Fr.7 cells were robustly decreased in Bach2/Blimp1 double-deficient B cells (Fig. 3 B). Since Blimp1 single knockout did not significantly affect the numbers of pro-memory (Fr.5) and mature viagra online no prescription memory B cells (Fr.7. Fig.

3 B), we conclude that Bach2 plays an important role in development of pro-memory cells and viagra online no prescription subsequent mature memory B cells. To determine how Bach2 participates in this process, we performed RNA profiling of Bach2/Blimp1 double-deficient LZ B cells, together with Blimp1-deficient LZ B cells as a control (Fig. S2 B) viagra online no prescription. In Bach2/Blimp1 double-deficient LZ B cells, GSEA revealed a significant enrichment of c-Myc target genes, E2F target genes, and mTORC1 signaling genes, in that order (Fig. 3 C) viagra online no prescription.

This was also demonstrated by flow cytometry analysis (expression levels of c-Myc, pRb, and pS6. Fig. 3 D). Moreover, as expected, the mutant GC B cells were hyperproliferative, as assessed by 5-ethynyl-2′-deoxyuridine (EdU) pulse labeling (Fig. 3 E).

These results, considering the previous demonstration that c-Myc–overexpressing and hyper-mTORC1 GC B cells manifest a bias toward the DZ (Ersching et al., 2017. Finkin et al., 2019), like Bach2/Blimp1 double-deficient GC B cells, allowed us to hypothesize that the defective pro-memory in the mutant GC cells 5could result from anomalies of the mTORC1 and/or c-Myc pathways. Here, we focused our analysis on the mTORC1 pathway. To test this hypothesis, we first asked whether normalizing mTORC1 activity in Bach2/Blimp1 double-deficient GC cells could rescue development of pro-memory B cells and subsequent memory B cells. We transferred Bach2f/fPrdm1f/fERT2cre B1-8hi B cells into rapamycin-resistant (MtorF2108L/F2108L) hosts (Ersching et al., 2017), deleted Bach2 and Prdm1 with tamoxifen, and then immunized the mice with NP-CGG/alum (Fig.

4 A). After immunization, the mice were treated with rapamycin to decrease mTORC1 activity in a transferred B cell–intrinsic manner. As shown in Fig. 4 B, the dose of rapamycin used nearly normalized pS6 levels in the Bach2/Blimp1 double-deficient LZ B cells. The rapamycin treatment partially corrected the c-Myc overexpression and hyperproliferation observed in the Bach2/Blimp1 double-deficient B1-8hi B cells (Fig.

4 B), suggesting coexistence of mTORC1-dependent and -independent pathways to regulate c-Myc activities. In contrast to control vehicle treatment of Bach2/Blimp1 double-deficient B1-8hi B cells, upon rapamycin treatment, those mutant cells generated threefold higher numbers of IgG1+ memory B cells. The numbers of IgG1+CD73+ memory B cells were similarly increased (Fig. 4 C, right). Furthermore, the Fr.5:Fr.2 ratio was also increased upon rapamycin treatment (Fig.

4 D). However, the memory B cells number upon rapamycin treatment did not reach those from wild-type B1-8hi B cells upon control vehicle injection (Fig. 4 C). Hence, we conclude that hyper-mTORC1 activity in Bach2/Blimp1 double-deficient GC B cells is one of the mechanisms that cause defective development of memory B cells, although there must be other, currently unknown ones, as well. In regard to GC B cells, the numbers were not significantly changed upon rapamycin treatment of Bach2/Blimp1 double-deficient B1-8hi B cells.

Skewing of Bach2/Blimp1 double-deficient GC B cells toward the DZ was decreased upon rapamycin treatment, although a small enrichment was still observed (Fig. 4 C). To further examine whether, in a wild-type setting, restraining mTORC1 activity could indeed facilitate differentiation of GC B cells to memory cells, we performed adoptive transfer experiments. For this, we conducted experiments in which two types of congenically marked B cells, rapamycin-sensitive (Mtor+/+) and rapamycin-resistant (MtorF2108L/F2108L) B1-8ge B cells, were cotransferred as a 1:1 mixture into rapamycin-resistant hosts (MtorF2108L/F2108L), which were immunized with NP-CGG/alum and then administered with rapamycin. As expected, rapamycin treatment led to a decrease in S6 phosphorylation in the transferred rapamycin-sensitive, but not rapamycin-resistant, B1-8ge GC B cells (Fig.

5 A). Upon rapamycin treatment, the number of rapamycin-sensitive NP+ GC B cells was decreased while the number of NP+ memory B cells was increased compared with their rapamycin-resistant counterparts, assessed by conventional flow cytometry analysis (Fig. 5 B). To more directly demonstrate the transition from GC B cells to Fr.7 cells, we treated the immunized mice with EdU for 3 d (days 10–13) before analysis. In this setting, incorporation of EdU marks GC cells that divided during the treatment period and the resultant quiescent memory B cells (Fig.

5 C). We previously confirmed that during this period, the majority of proliferating cells (>95%) are GC B cells and plasmablasts (Shinnakasu et al., 2016). Upon rapamycin treatment, the frequency of EdU+IgG1+ Fr.7 cells compared with GC cells was higher among the rapamycin-sensitive B1-8ge cells than the rapamycin-resistant ones, demonstrating rapamycin-mediated facilitation of the transition from GC to Fr.7 cells (Fig. 5 D). Moreover, upon rapamycin treatment, the numbers of CD38−Bcl6hiCD69hi (Fr.2) and CD38intBcl6hi/intEfnb1+ (Fr.5) rapamycin-sensitive B1-8ge IgG1+ B cells were decreased and maintained, respectively.

Thus, the ratio of Fr.5 to Fr.2 was increased (Fig. 5 E). Together, we conclude that a relative enrichment in Fr.5 over Fr.2 cells is induced by rapamycin treatment, thereby facilitating the overall transition from GC B cells to memory B cells. The memory B cells generated in the presence of rapamycin were able to induce similar recall antibody responses to those generated in the absence of rapamycin, as assessed by adoptive transfer experiments (Fig. 5 F).

We next wished to examine why Fr.5, but not Fr.3 cells, can become pro-memory B cells. Since there were almost no differences in mTORC1 activity between Fr.5 and Fr.3 cells (Fig. S5 A), it appears that an mTORC1lo state is necessary but not sufficient for development of pro-memory B cells (Fr.5). Thus, additional key properties must be required for development of these cells. Since one of crucial features of mature memory B cells is longevity, one straightforward possibility is that Fr.5 cells begin to acquire more survival activity.

Supporting this idea, CD38intBcl6hi/intEfnb1+ (Fr.5) cells were less apoptotic compared with CD38−Bcl6hiCD69lo (Fr.3) cells as assessed by active caspase-3 staining (Fig. 6 A). Transcript data (Fig. 6 B) together with protein expression data (Fig. 6 C) demonstrated that Bcl2 expression was upregulated in Fr.5 cells compared with Fr.3 cells, and even more in pre-memory B cells (Fr.6).

Similarly, we found that the cell surface BCR expression level was increased stepwise from Fr.3 to Fr.6 cells (Fig. 6 D). We also observed a slight increase of IgG1 and Igα/β mRNA expression in Fr.5 over Fr.3 cells. Thus, regulation of both mRNA and protein levels seems to be operative. To examine whether Bcl2 family protein–mediated survival activity could impact the development of Fr.5 cells, we employed GC B cells with haploinsufficiency of Bim (Bcl2l11.

See Materials and methods), a counteracting factor against anti-apoptotic Bcl2-family members (O’Connor et al., 1998). Bcl2l11+/+ ERT2cre B1-8ge B cells and Bcl2l11f/+ERT2cre B1-8ge B cells were cotransferred as a 1:1 mixture into wild-type recipient mice, which were then immunized with NP-CGG/alum and treated with tamoxifen on day 8 (Fig. 6 E). Bim mRNA expression was decreased to almost 50% of control levels after tamoxifen treatment in Bcl2l11f/+ GC B cells (Fig. 6 F).

In this competitive setting, among the Fr.2/3/5/6 cells, the frequency was most significantly increased in Fr.5 and Fr.6 cells upon Bim haploinsufficiency (Fig. 6 G), although there was also a modest increase of Fr.3 cells. Consequently, the frequency of Bcl2l11f/+ NP+IgG1+CD73+ memory B cells was also increased (Fig. S5 B). To examine the effects of surface BCR expression on survival, B1-8ge-flox/+ ERT2cre B cells were employed.

For these particular experiments, we mixed these B cells and control B1-8ge/+ ERT2cre B cells at a 7:3 ratio and adoptively cotransferred them into recipient mice, which were then immunized with NP-CGG/alum (Fig. 6 H). We injected tamoxifen on day 10 and examined surface BCR expression on day 12, demonstrating a significant decrease on Fr.5 cells derived from B1-8ge-flox/+ ERT2cre B cells (Fig. 6 I). To detect apoptotic cells in this experiment, we analyzed mixtures of Fr.5 and Fr.6 cells (CD38+Efnb1+) to acquire a sufficient number of cells for the assay.

As demonstrated in Fig. 6 J, concomitant with decreased surface BCR expression, there was a higher frequency of apoptotic (aCasp3+) cells among pro/pre-memory cells derived from B1-8ge-flox/+ ERT2cre B cells. Similarly, frequency of apoptotic cells among total LZ GC cells was enhanced upon BCR downregulation (Fig. S5 C). A control experiment using Prdm1f/+B1-8ge/+ ERT2cre B cells showed that a nonspecific effect on apoptosis induced simply by Cre-mediated double-strand breaks was negligible (Fig.

S5 D). Together, stepwise increases of Bcl2 and surface BCR expression from pro-memory (Fr.5) cells to pre-memory (Fr.6) toward mature memory B cells are likely to contribute to their survival. It is still unclear what signals and processes in LZ GC cells initiate their differentiation toward long-lived memory B cells. Here, by focusing on key features for development of GC-derived memory precursors, we show that an mTORC1lo state is necessary to develop pro-memory B cells. Since mTORC1lo LZ B cells receive weak T cell help and, as a result, have been thought to undergo apoptosis, this raises the question of how such pro-memory B cells are prevented from dying and able to differentiate into mature memory B cells.

Our experiments suggest that the memory precursor B cells express higher levels of Bcl2 and surface BCR, thereby acquiring a survival advantage. We have already shown that Bach2hi LZ GC B cells are predisposed to differentiate into memory B cells (Shinnakasu et al., 2016), indicating that memory cell commitment already begins in a subset of GC B cells. This memory-prone subset most likely corresponds to the Fr.5 (CD38intBcl6hi/intEfnb1+ pro-memory) cells. Indeed, expression of Bach2 in Fr.5 is higher than in Fr.2 cells (Fig. 2 A and Fig.

S3 B). Fr.6 (pre-memory B) cells appear to be undergoing a further developmental step toward mature memory B cells, manifested by further downregulation of Bcl6 (Fig. 1 B). We found that mTORC1 has a marked effect on the ratio of memory-prone (Fr.5) to recycling-prone (Fr.2) GC B cell formation. Rapamycin treatment increased the proportion of Fr.5 cells and, conversely, hyperactivation of mTORC1 in the Bach2/Blimp1 double-deficient setting led to a relative increase in Fr.2 cells.

Several nonmutually exclusive possibilities can be envisaged to explain why lower mTORC1 activity contributes to development of memory-prone cells. Decay in mTORC1 activity as GC B cells proliferate in the DZ appears to be required for their timely return to the LZ (Ersching et al., 2017). Given the importance of LZ residency for memory differentiation (Bannard et al., 2013), one possibility is that LZ residency imposed by mTORC1lo could allow development of pro-memory B cells. Second, apart from this spatial requirement mediated through modulation of mTORC1, inhibition of mTORC1, as is seen during the generation of natural killer cell memory (O’Sullivan et al., 2015), may stimulate autophagy, thereby enhancing pro-memory B cell survival. Finally, it is also well known that mTORC1 activity is suppressed in memory B cells (Boothby and Rickert, 2017).

Such metabolic changes as the cells progress toward mature memory B cells thus appear to be initiated already in pro-memory cells, and this might be a necessary first step for generating mature memory B cells. The partial restoration of memory B cells by rapamycin treatment in Bach2/Blimp1 double-deficient GC cells suggests that, in addition to hyper-mTORC1 activity, other anomalies occur in mutant GC B cells in regard to memory differentiation. One of them is likely the c-Myc overexpression, because of the following. First, indeed, in rapamycin-treated Bach2/Blimp1 double-deficient GC cells, overexpression of c-Myc and hyperproliferation were still observed to a significant extent (Fig. 4 B).

Second, c-Myc–overexpressing GC cells were reported to have a significant bias toward the DZ (Finkin et al., 2019). Considering the importance of LZ residency for memory differentiation (Bannard et al., 2013), overexpression of c-Myc is assumed to be detrimental to memory differentiation. Hence, we would propose that restraining both mTORC1-mediated metabolism and c-Myc–mediated cell-cycle progression is required to develop pro-memory B cells and that Bach2 is one of the critical regulators for suppressing both pathways. Functionally, Bach2 is well known to act as a repressive guardian transcription factor (Igarashi et al., 2017). In regard to relationship between signaling and Bach2 expression, the mTORC1 activity and Bach2 expression appear to be mutually exclusive, because the BCR-induced AKT-mTORC1 inhibits Bach2 expression (Kometani et al., 2013), and Bach2 represses transcription of mTORC1 signaling molecules.

Such a negative feedback loop is characteristic of “bistable” signal transduction circuits, which can operate in two stable formats. This might take place between Fr.5 and Fr.2 cells. It should be mentioned that, from mTORC1 signaling molecule side, Bach2 is one of the transcription factors, and probably additional factors participate in transcriptional regulation on mTORC1 signaling genes. In addition to the connection between BCR signal and Bach2, considering the T cell data showing that ICOS and integrin αE are upregulated in Bach2lo T cells (Grant et al., 2020. Sidwell et al., 2020), Bach2 might be involved in connecting the BCR signal to T cell help.

For instance, Bach2lo LZ GC cells with high-affinity BCRs might modulate T/B interactions through adhesion status and coreceptor expression and affect the strength of T cell help. This might further downregulate Bach2, because we previously showed that strong T cell help depresses Bach2 expression (Shinnakasu et al., 2016). After moving back into the LZ, apoptosis is generally thought to be the default pathway for LZ GC B cells. However, high-affinity cells are spared and positively selected after they encounter sufficient cognate T cell help (Allen et al., 2007. Victora and Nussenzweig, 2012).

These spared high-affinity cells correspond to Fr.2 cells, whereas the defaulting apoptotic LZ cells are likely to be Fr.3 cells. Indeed, among LZ GC cells, Fr.3 cells were most apoptotic. Here, we show that a small population of pro-memory B cells exists in the LZ and, despite apparently receiving weak T cell help, they are relatively resistant to apoptosis. The inability of prior studies to detect such apoptosis-resistant LZ B cells is most likely due to the fact that the numbers of pro-memory cells are so limited (Mayer et al., 2017). Previous data using B cell–specific Bcl2-tg mice (Smith et al., 1994) or Bim knockout mice (Fischer et al., 2007) showed that such mice develop an enlarged memory B cell compartment.

Recently, more detailed analysis using the same Bcl2-tg mice (Stewart et al., 2018) provided mechanistic insights into the above phenomenon. First, in these mice, aberrant populations of seemingly quiescent cells arise that express markers of memory precursor cells. Second, overexpression of Bcl2 is not sufficient for DZ GC B cells with damaged BCRs to reach the LZ. Hence, in a physiological setting, it is reasonable to speculate that, after returning to the LZ in a Bcl2-independent manner, if Bcl2 expression is upregulated in some of the LZ GC B cells, they are better able to be rescued from apoptosis in the late G1 phase and to begin to differentiate into memory B cells. Supporting this idea, we show here that among LZ GC cells, small numbers of pro-memory B cells (Fr.5), but not Fr.3 cells, begin to upregulate Bcl2, and that development of pro-memory B cells is facilitated by Bim haploinsufficiency.

Because Bach2 expression in Fr.5 cells is similar to Fr.3 cells (Fig. S3 B), Bach2 appears not to be involved in such differential survival activity between Fr.5 and Fr.3 cells. Rather, a Bach2-independent mechanism such as Bcl6 downregulation (discussed below) is likely to be operated, thereby allowing Fr.5 cells to survive enough to begin to differentiate into memory precursor cells. In contrast to Fr.5 cells (pro-memory), Fr.6 cells (pre-memory) apparently possess more survival activity (Fig. 6 A), possibly explaining the generation kinetics between Fr.5 and Fr.6 cells.

Fr.6 cells were more accumulated at later phases (days 14 and 20) during immune responses (Fig. S1 B). Induced downregulation of surface BCR expression in pro/pre-memory B cells resulted in increased apoptosis in the pro-memory B cells. These results, together with the evidence that pro-memory B cells express higher surface BCR levels, lead us to propose that the BCR-mediated survival signal also plays a role in the development of pro-memory B cells. Based on the previous report that BCR ablation leads to cell death, which can be delayed by constitutive Bcl2 expression (Lam et al., 1997), we considered the possibility that downregulation of the BCR might decrease Bcl2 expression in pro/pre-memory B cells.

However, we could not detect such a connection (data not shown). In naive B cells, the constitutive PI3 kinase–Foxo1 pathway is known to replace the missing BCR-mediated survival signals (Srinivasan et al., 2009). Therefore, a question arises of how pro-memory B cells, despite being mTORC1lo (reflecting lower Akt activity), generate such a survival signal. Given that there is no enlarged GC phenotype in PTEN or Foxo1 knockout mice (Dominguez-Sola et al., 2015. Inoue et al., 2017.

Sander et al., 2015. Suzuki et al., 2003), one straightforward explanation might be that the quality and/or quantity of BCR-mediated survival signals differ between naive B cells and GC-derived memory B cells. We provide evidence that downregulation of Bcl6 in pro-memory B cells could be one of the mechanisms for upregulation of Bcl2 and surface BCR. However, since the extent of Bcl6 downregulation in pro-memory B cells is small, such a slight change might not account for the observed upregulation of Bcl2 and surface BCR. Hence, our data cannot completely exclude the possibility that, particularly at the pro-memory B cell stage, other mechanisms might operate to initiate upregulation of Bcl2 and BCR.

In this case, it is likely that downregulation of Bcl6 acts as an amplification pathway for further upregulation of Bcl2 and surface BCR during differentiation toward mature memory B cells. In regard to this differentiation pathway, our data using Bcl6 haploinsufficiency are highly complementary to previous in vitro data that ectopic expression of Bcl6 in B cell cultures blocks the GC B cells from differentiating into memory B cells (Kuo et al., 2007). Together, it is likely that stepwise decreases in Bcl6 expression (pro-memory >. Pre-memory >. Mature memory B cells) play a key role in memory B cell development.

This raises the question of how is Bcl6 downregulated. Three possibilities have already been reported. (1) upon strong BCR engagement, Erk-mediated degradation of Bcl6 (Niu et al., 1998). (2) transcriptional downregulation of Bcl6, mediated by CD40-activated IRF4 (Saito et al., 2007). And (3) downregulation of Bcl6 by defective IL-21 signaling (Linterman et al., 2010).

Among these, in regard to differentiation from GC to memory B cells, the final possibility seems to best fit with our observation that pro-memory B cells possess lower-affinity BCRs, thereby receiving less T cell help. In addition, as a transcriptional circuit–type regulation, the transcription factor Hhex, critical for memory B cell differentiation, has been recently reported to participate in downregulation of Bcl6 (Laidlaw et al., 2020). In summary, this study provides important insights into the initial events for the fate decisions from GC to memory B cells. The modulation of cellular metabolism and survival play fundamental roles. Given the importance of GC-derived memory B cells for protection against heterologous virus reinfection (Leach et al., 2019.

Purtha et al., 2011), our findings may contribute to the development of efficient vaccination strategies. Single-cell suspensions of splenocytes were analyzed and sorted on a FACSCanto II (BD Biosciences) or a FACSAria II (BD Biosciences). Alexa647-active caspase-3, V500-B220, V450-Bcl6, BV786-CD138, BV510-CD38, V500-CD45.2, BV510-IgG1, PE-IgG1 antibodies, and BV786-streptavidin were purchased from BD Biosciences. APC-eFluor780-B220, FITC-CD45.1, PE-CD45.1, APC-eFluor780-CD45.1, FITC-CD45.2, APC-eFluor780-CD45.2, APC-CD69, APC-eFluor780-CD69, eFluor450-CD73, PE-CD86, PerCP-Cy5.5-GL7 antibodies, PerCP-Cy5.5-streptavidin, PE-streptavidin, and APC-eFluor780-streptavidin were purchased from eBioscience. PE-B220, PE-Cy7-CD138, PE-Cy7-CD38, PacificBlue-CD45.1, PE-CD45.2, biotin-CD69, PerCP-Cy5.5-CD86, BV421-CXCR4, V450-Ki67 antibodies, and BV510-streptavidin were purchased from BioLegend.

PE-pRb and PE-pS6 antibodies were purchased from Cell Signaling. C-Myc antibody was purchased from Abcam. PE-Bcl2 antibody was purchased from Miltenyi Biotec. Biotin-Efnb1 antibody was purchased from R&D Systems. Alexa488-goat anti-rabbit IgG antibody was purchased from Thermo Fisher Scientific.

For intracellular staining, the cells were fixed and permeabilized using a Foxp3 staining kit (eBioscience) for Bcl6, Bcl2, and pRb, a BD Cytofix/Cytoperm solution (BD Biosciences) for pS6 and active caspase-3, or a True-Nuclear Transcription Factor Staining Buffer Set (BioLegend) for c-Myc. APC-conjugated NP was prepared as described previously (Shinnakasu et al., 2016). Incorporation of EdU was detected using a Click-iT Plus EdU Flow Cytometry Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. We thank M.C. Nussenzweig (The Rockefeller University, New York, NY) for B1-8hi mice, T.

Okada (RIKEN Center for Integrative Medical Sciences, Kanagawa, Japan) for Bcl6-YFP mice, G.D. Victora (The Rockefeller University) for MtorF2108L mice, and P.D. Burrows for critical reading of the manuscript. This work was supported by grants from JSPS KAKENHI (JP17K08882 to T. Inoue.

JP26221306 and JP19H01028 to T. Kurosaki), the SENSHIN Medical Research Foundation (to T. Inoue), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T. Inoue), and a research grant from Astellas Foundation for Research on Metabolic Disorders (to T. Inoue).

Author contributions. T. Inoue and C. Kawai performed the experiments. T.

Inoue and T. Kurosaki designed the experiments. R. Shinnakasu, W. Ise, T.

Oki, T. Kitamura, and H. Fukuyama provided essential reagents. E. Kawakami, N.

Sax, and K. Yamashita performed bioinformatics analyses. T. Inoue and T. Kurosaki wrote the manuscript.GRIP1 is a broadly acting transcriptional coregulator whose role in MS/EAE or in MG at any state has never been investigated.

To begin to identify the GRIP1-dependent transcriptome changes leading to neuroinflammation, we performed bulk RNAseq analysis on CD45+Cd11b+ myeloid cells isolated from spinal cords of WT and GRIP1-cKO mice. Consistent with a lack of overt phenotype in our conditional GRIP1-deficient mice (Coppo et al., 2016. Rollins et al., 2017), at homeostasis, a CD45+Cd11b+ CNS myeloid cell population composed principally of MG displayed no significant transcriptomic differences between WT and GRIP1-cKO mice (Fig. 5 A, upper panel. GRIP1 deletion efficiency is shown on the right as normalized read counts across “floxed” exon 11 of the Ncoa2 gene).

In contrast, more heterogeneous activated CD45+Cd11b+ cells during EAE (Fig. S2 C and Fig. 2 C) presented distinct transcriptomic signatures in WT versus GRIP1-cKO mice. Indeed, genes upregulated in WT (Fig. 5 A, lower panel, and Fig.

5 B), such as chemokines and chemokine receptors (Ccl22, Ccr7), antigen presentation molecule (H2-q10), components of complement (C3, C1ra), and type I IFN (Trim12c, Oas3) pathways, are indicative of inflammation and EAE pathogenesis (Belikan et al., 2018. Salter and Stevens, 2017. Scheu et al., 2017). Interestingly, a pool of genes downregulated in WT mice during EAE but persisting in GRIP1-cKO mice (e.g., Gpr34, P2ry12. Fig.

5 A, lower panel, and Fig. 5 B) are homeostatic genes referred to as the MG “sensome” (Hickman et al., 2013), which controls chemotaxis and tissue repair (Lou et al., 2016). To identify physiologically relevant pathways differentially active in myeloid cells from WT and GRIP1-cKO spinal cords during EAE, we performed quantitative set analysis of gene expression (QuSAGE. Yaari et al., 2013), a gene set enrichment analysis–like Bayesian method that provides better accounts for intergene correlations than classic gene set enrichment analysis. QuSAGE determines pathway-wide expression (pathway activity) by combining probability density functions for individual gene expression using numerical convolution.

Several pathways were expressed at higher levels in the WT CNS myeloid cells, including NODE-like receptor signaling pathways (Kyoto Encyclopedia of Genes and Genomes) and a nuclear receptor transcription pathway (REACTOME), including Nr4a2 (Nurr1) and Nr4a3 (Nor-1. Fig. 5 C). Remarkably, several key genes of the IFN axis (IFN signaling pathway [REACTOME]), including Irf4, Irf1, Ifng, Ifitm1, Gbp5, and Oas3, were also expressed at higher levels in the WT (Fig. 5 C), in accord with whole-brain and spinal cord quantitative PCR (qPCR) data (Fig.

3 A and Fig. S5 A) and with a demonstrated coactivator role for GRIP1 in type I IFN network in MФ (Flammer et al., 2010. Reily et al., 2006). Collectively, these data demonstrate a failure to upregulate inflammatory and type I IFN pathways and persistence of homeostatic signature in GRIP1-cKO myeloid cells. However, it could potentially stem from the role of GRIP1 in MG, MФ, or both.

To dissect the contribution of resident versus infiltrating myeloid cells to EAE pathogenesis, we performed single-cell RNAseq (scRNAseq) analysis of all myeloid CD45+CD11b+ cells from WT and GRIP1-cKO spinal cords at the peak of EAE (DPI20). After filtering out low-quality barcodes (see Materials and methods), we analyzed 20,376 cells (6,427 WT and 11,949 cKO) expressing 11,093 genes. Automated cell type assignment with singleR yielded four major clusters—“monocytes,” “MФ,” “dendritic cells,” and “neutrophils” (Fig. 6 A and Table S1)—and a large number of minor clusters composed predominately of lymphoid cell impurities that were collected during the cell sorting and had the same location in uniform manifold approximation and projection (UMAP) coordinates (Fig. 6 A).

Because of an unbalanced group size, we performed a bootstrapping analysis to determine the associations between genotype and singleR cell types. We counted cell types of 2,000 cells that were sampled with the replacement from each genotype with 500 repeats (Fig. 6 B and Fig. S4 A). This analysis indicated that singleR monocytes and neutrophils were more common in the cKO, whereas MФ were overrepresented in the WT.

There was a substantial overlap between singleR cell types, suggesting either the presence of cell subpopulations or different differentiation/activation states. To separate these states, we performed Louvain graph–based community clustering that yielded nine clusters (Fig. 6 C and Table S2). Cluster 8 corresponded to singleR lymphoid cell–enriched group (Fig. 6 A.

€œOthers,” “T cells”), whereas cluster 6 was highly enriched with canonical neutrophilic markers (Fig. 6, A, D, and E). Cluster 3 is enriched in proliferation markers (Fig. 6 E, Fig. S4 B, and Table S4).

Slingshot trajectory analyses anchored on cluster 3 (see Materials and methods) identified two main trajectories (3-5-9-7-1 and 3-5-9-2-4-6) bifurcating at cluster 9 (Fig. 6 C and Fig. S4 C). The analysis of genes differentially expressed along trajectories suggested that the 3-5-9-7-1 trajectory likely corresponds to monocyte-to-MФ transitions. Conversely, clusters 2-4-6 exhibit an increasing gradient of expression of neutrophilic markers (Fig.

6 E. S100a8, S100a9), suggesting that clusters 4 and 2 contain a decreasing admixture of neutrophils from cluster 6. Cluster 3 expresses monocytic markers at high levels (Fig. 6 F. Ly6c2, F13a1, Stmn1) and activated MФ/MG markers at low levels (Fig.

6, F and G. Cd74, Fth1, Fcgr2b, H2-Aa, Il1b) that reciprocally change along the trajectories. MФ-like clusters (1, 7, and 2) contain either different proportions of MФ/MG, different activation states, or an admixture of other cell types (e.g., oligodendrocyte precursors. Table S3). Although expression distributions for activated MФ/MG markers are broadly comparable in these clusters (Fig.

S4 D), differential expression analysis between WT and cKO stratified by Louvain clusters revealed that clusters 1, 7, 2, and 4 expressed markers of homeostatic MG at higher levels in the cells from cKO mice (Fig. 6 H, Fig. S4 E, and Table S5. Sparc, Siglech, Olfml3, and Tmem119). Cluster 2 contained the largest percentage of cells expressing homeostatic MG markers.

Conversely, many markers of activated inflammatory MФ were upregulated in these clusters in the WT cells including Il1a, Il1r2, Il7r, Ifng, Ctla2s, and Nos2 (Fig. 6 I and Table S5)..

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The coronavirus pandemic has accelerated that shift, with the federal government relaxing regulations to make it easier for doctors to get paid for an online visit.In September, Amazon Care announced that it had expanded its service from its headquarters in the Seattle area to all of its offices throughout Washington State.An Amazon spokesperson declined to comment.The Trump administration on Friday announced a deal with CVS Health and Walgreens to administer coronavirus vaccines to the elderly and staff in long-term care facilities.The vaccine will be free of charge and available for residents in all long-term care settings, including skilled nursing facilities, nursing homes, assisted living facilities, residential care homes and adult family homes, the Department of Health and Human Services said in a press release.There are about 15,000 long-term care facilities as well as an additional 35,000 assisted living facilities in the U.S., the Centers for Disease Control and Prevention's deputy director for infectious diseases, Jay Butler, told reporters on a call Friday. The eligibility requirements for facilities are "very broad," he added.Starting Monday, long-term care facilities will be able to select which pharmacy viagra uk they prefer to have on-site. The facilities can opt-in to the viagra uk program through the CDC's National Healthcare Safety Network, Butler said. If a facility chooses to not opt-in, there will be the possibility of being able to administer vaccines through other sources, including from local pharmacies, he added.CVS and Walgreens will schedule and viagra uk coordinate on-site clinic dates directly with each facility, HHS said.

The companies anticipate that three total visits over two months are likely to be viagra uk needed to administer both doses of vaccine to residents and staff, the agency said. Long-term care facilities are not mandated to participate, HHS added."Protecting the vulnerable has been the number one priority of the Trump Administration's response to COVID-19, and that commitment will continue through distributing a safe and effective vaccine earliest to those who need it most," HHS Secretary Alex Azar said in a statement.Centers for Medicare and Medicaid Services Administrator Seema Verma said the deal will ensure that nursing homes, which have been hit hard by the virus, "are at the front of the line for the COVID vaccine and will bring their grueling trial to a close as swiftly as possible."The announcement comes the same day states must submit their draft plans to the federal government on how they will distribute a coronavirus vaccine if and when one is approved for public use.Once a vaccine is approved, it will likely be released in stages, with the elderly and health-care workers getting it first. There are four potential vaccines viagra uk backed by the U.S. Currently in viagra uk late-stage testing.

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Amazon employees are told they can get health care on callAmazon Care, the company's online medical clinic for its own employees, is trying to hire half viagra online no prescription a dozen people in business development roles to "build and grow relationships with commercial and public sector enterprises." Most of the roles were posted in the past month.By building a business development team, the company is signaling an intention to go resource broader than its own employees. Amazon has a history of developing products that it tests out on its own workforce before expanding to a broader population.Moreover, a person familiar with the business unit's plans told CNBC that Amazon Care has started reaching out to health plans and employers in the Washington area to discuss viagra online no prescription opportunities to expand beyond its own employees. The plans are in an exploratory phase and may not result in expansion.Amazon Care, which launched viagra online no prescription as a pilot in the fall of 2019, offers a virtual medical clinic for employees and their dependents in Washington state. The goal is to viagra online no prescription make it easier to access high-quality primary care online, and at-home visits are also available in some areas.Amazon has increasingly moved into the medical sector in recent years.

In 2018, it acquired PillPack, which offers at-home medication delivery, and has built a pharmacy team under that division. It also has a health and wellness unit focused on voice applications viagra online no prescription within its Alexa team. The company moved into the wearables market in August with a device known as Halo to track its users' health and fitness.Telemedicine viagra online no prescription represents a sizable market opportunity. It is expected to be worth more than $17 billion by 2026 as more viagra online no prescription people opt to engage with their doctors online.

The coronavirus pandemic has accelerated that shift, with the federal government relaxing regulations to make it easier for doctors to get paid for an online visit.In September, Amazon Care announced that it had expanded its service from its headquarters in the Seattle area to all of its offices throughout Washington State.An Amazon spokesperson declined to comment.The Trump administration on Friday announced a deal with CVS Health and Walgreens to administer coronavirus vaccines to the elderly and staff in long-term care facilities.The vaccine will be free of charge and available for residents in all long-term care settings, including skilled nursing facilities, nursing homes, assisted living facilities, residential care homes and adult family homes, the Department of Health and Human Services said in a press release.There are about 15,000 long-term care facilities as well as an additional 35,000 assisted living facilities in the U.S., the Centers for Disease Control and Prevention's deputy director for infectious diseases, Jay Butler, told reporters on a call Friday. The eligibility requirements for facilities are "very broad," he added.Starting viagra online no prescription Monday, long-term care facilities will be able to select which pharmacy they prefer to have on-site. The facilities can opt-in to the program through viagra online no prescription the CDC's National Healthcare Safety Network, Butler said. If a facility chooses to viagra online no prescription not opt-in, there will be the possibility of being able to administer vaccines through other sources, including from local pharmacies, he added.CVS and Walgreens will schedule and coordinate on-site clinic dates directly with each facility, HHS said hop over to this site.

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Most of the potential vaccines require viagra online no prescription two doses, although Johnson &. Johnson's requires just one shot, and some of them need to be transported and stored at varying and specific temperatures.Paul Mango, deputy chief of staff for policy at HHS, told reporters on an Oct. 9 call viagra online no prescription that the federal government is in the process of "actively engaging tens of thousands of provider outlets for these vaccines." Mango added that the U.S. Currently has assembled 40 million-plus vaccination kits with the bottles, needles and other items needed for the shots."All of viagra online no prescription those are in warehouses ready to go, so that's a big logistical task or undertaking," he said.On a call with reporters Friday, Mango said the federal government was in touch with other pharmacies besides CVS and Walgreens but said that they could not "deliver on these capabilities and our executional requirements.""These two players have been doing this for a while," he said.

"They have demonstrated the ability from an IT perspective, from a training and personnel perspective, viagra online no prescription from an asset perspective."HHS said Friday that CVS and Walgreens will receive and manage vaccines and also ensure they are stored at the proper temperatures. The companies will also be responsible for on-site administration of the vaccine."Ensuring access to COVID-19 vaccines, particularly among our most vulnerable populations, will be critical to saving lives and helping our nation recover from the pandemic," Walgreens president John Standley said in a statement.Troy Brennan, chief medical officer at CVS Health, said in a statement, "CVS Health has been on the frontlines of the fight against COVID-19, working across the health care spectrum in all the communities we serve and that will continue to be the case when we have a vaccine to dispense."HHS also said that it is "using multiple authorities" to ensure appropriate reimbursement for the vaccinations and that no American will have to pay out of pocket.Mango said Friday the government has also put aside a fund to cover vaccinations for the uninsured similar to the one used to cover coronavirus testing..