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Wealthy nations must do much more, much faster.The United Nations General Assembly in September 2021 will bring countries together at a critical time for marshalling collective action to tackle the global environmental crisis buy zithromax in usa. They will meet again at the biodiversity summit in Kunming, China, and the climate buy zithromax in usa conference (Conference of the Parties (COP)26) in Glasgow, UK. Ahead of these pivotal meetings, we—the editors of health journals worldwide—call for urgent action to keep average global temperature increases below 1.5°C, halt the destruction of nature and protect health.Health is already being harmed by global temperature increases and the destruction of the natural world, a state of affairs health professionals have been bringing attention to for decades.1 The science is unequivocal. A global increase of 1.5°C above the preindustrial average and the continued loss of biodiversity risk catastrophic harm to health that will be impossible to reverse.2 3 Despite the world’s necessary preoccupation with buy antibiotics, we cannot wait for the zithromax to pass to rapidly reduce emissions.Reflecting the severity of the buy zithromax in usa moment, this editorial appears in health journals across the world. We are united in recognising that only fundamental and buy zithromax in usa equitable changes to societies will reverse our current trajectory.The risks to health of increases above 1.5°C are now well established.2 Indeed, no temperature rise is ‘safe’.

In the past 20 years, heat-related mortality among people aged over 65 has increased by more than 50%.4 Higher temperatures have brought increased dehydration and renal function loss, dermatological malignancies, tropical s, adverse mental health outcomes, pregnancy complications, allergies, and cardiovascular and pulmonary morbidity and mortality.5 6 Harms disproportionately affect the most vulnerable, including children, older populations, ethnic minorities, poorer communities and those with underlying health problems.2 4Global heating is also contributing to the decline in global yield potential for major crops, falling by 1.8%–5.6% since 1981. This, together with the effects of extreme weather and soil depletion, is hampering efforts to reduce undernutrition.4 Thriving ecosystems are essential to human health, and the widespread destruction of nature, including habitats and species, is eroding water and food security and increasing the chance of zithromaxs.3 7 8The consequences of the environmental crisis fall buy zithromax in usa disproportionately on those countries and communities that have contributed least to the problem and are least able to mitigate the harms. Yet no country, no matter how wealthy, can shield itself from these impacts. Allowing the consequences to fall disproportionately on the buy zithromax in usa most vulnerable will breed more conflict, food insecurity, forced displacement and zoonotic disease, with severe implications for all countries and communities. As with the buy zithromax in usa buy antibiotics zithromax, we are globally as strong as our weakest member.Rises above 1.5°C increase the chance of reaching tipping points in natural systems that could lock the world into an acutely unstable state.

This would critically impair our ability to mitigate harms and to prevent catastrophic, runaway environmental change.9 10Global targets are not enoughEncouragingly, many governments, financial institutions and businesses are setting targets to reach net-zero emissions, including targets for 2030. The cost of renewable energy is dropping buy zithromax in usa rapidly. Many countries are aiming to protect at least 30% of the world’s land and oceans by 2030.11These promises buy zithromax in usa are not enough. Targets are easy to set and hard to achieve. They are yet to be matched with credible short-term and longer-term plans to accelerate cleaner technologies buy zithromax in usa and transform societies.

Emissions reduction plans do not adequately incorporate health considerations.12 Concern is growing that temperature rises above 1.5°C are beginning to be seen as inevitable, or even acceptable, to powerful members of the global community.13 Relatedly, current strategies for reducing emissions to net zero by the middle of the century implausibly assume that the world will acquire great capabilities to remove greenhouse gases from the atmosphere.14 15This insufficient action means that temperature increases are likely to be well in excess of 2°C,16 a catastrophic outcome for health and environmental stability. Critically, the destruction of nature does not have parity of esteem with the climate element of the crisis, and every single global target to restore biodiversity loss by 2020 was missed.17 This buy zithromax in usa is an overall environmental crisis.18Health professionals are united with environmental scientists, businesses and many others in rejecting that this outcome is inevitable. More can and must be done now—in Glasgow and Kunming—and in the immediate years that buy zithromax in usa follow. We join health professionals worldwide who have already supported calls for rapid action.1 19Equity must be at the centre of the global response. Contributing a fair share to the global effort means that reduction commitments must account for the cumulative, historical contribution each country has made to emissions, as well buy zithromax in usa as its current emissions and capacity to respond.

Wealthier countries will have to cut emissions more quickly, making reductions by 2030 beyond those currently proposed20 21 and reaching net-zero emissions before 2050. Similar targets and emergency action are needed for biodiversity loss and the wider destruction of the natural world.To achieve these targets, governments must make fundamental changes to how our societies and economies are organised buy zithromax in usa and how we live. The current strategy of encouraging markets to swap buy zithromax in usa dirty for cleaner technologies is not enough. Governments must intervene to support the redesign of transport systems, cities, production and distribution of food, markets for financial investments, health systems, and much more. Global coordination is needed to ensure that the rush for cleaner technologies does not come at the cost of more environmental destruction and buy zithromax in usa human exploitation.Many governments met the threat of the buy antibiotics zithromax with unprecedented funding.

The environmental crisis buy zithromax in usa demands a similar emergency response. Huge investment will be needed, beyond what is being considered or delivered anywhere in the world. But such investments will produce huge positive health and economic outcomes buy zithromax in usa. These include high-quality jobs, reduced air pollution, increased physical activity, and improved housing and diet. Better air quality alone would realise health benefits that easily offset the global costs of emissions reductions.22These measures will also improve the social and economic determinants of health, the poor state of which may have made populations more vulnerable to the buy antibiotics zithromax.23 But the changes cannot be achieved through a return to damaging austerity policies or the continuation of the large inequalities of wealth and power within and between countries.Cooperation hinges on wealthy nations doing moreIn particular, buy zithromax in usa countries that have disproportionately created the environmental crisis must do more to support low-income and middle-income countries to build cleaner, healthier and more resilient societies.

High-income countries must meet and go beyond their outstanding commitment buy zithromax in usa to provide $100 billion a year, making up for any shortfall in 2020 and increasing contributions to and beyond 2025. Funding must be equally split between mitigation and adaptation, including improving the resilience of health systems.Financing should be through grants rather than loans, building local capabilities and truly empowering communities, and should come alongside forgiving large debts, which constrain the agency of so many low-income countries. Additional funding must be marshalled to compensate for inevitable loss and damage caused by the consequences of the environmental crisis.As health professionals, we must do all we can to aid the transition to buy zithromax in usa a sustainable, fairer, resilient and healthier world. Alongside acting to reduce the harm from the environmental crisis, we should proactively contribute to global prevention of further damage and action on the root causes of the crisis. We must buy zithromax in usa hold global leaders to account and continue to educate others about the health risks of the crisis.

We must join in the work to achieve environmentally sustainable health systems before 2040, recognising that this will mean changing clinical buy zithromax in usa practice. Health institutions have already divested more than $42 billion of assets from fossil fuels. Others should join them.4The greatest threat to global public health is the continued failure of world leaders to keep buy zithromax in usa the global temperature rise below 1.5°C and to restore nature. Urgent, society-wide changes must be made and will lead to buy zithromax in usa a fairer and healthier world. We, as editors of health journals, call for governments and other leaders to act, marking 2021 as the year that the world finally changes course.Ethics statementsPatient consent for publicationNot required.AbstractPhenome-wide association study (PheWAS) has been increasingly used to identify novel genetic associations across a wide spectrum of phenotypes.

This systematic review aims to summarise the PheWAS methodology, discuss the advantages and challenges of PheWAS, and provide potential buy zithromax in usa implications for future PheWAS studies. Medical Literature Analysis and Retrieval System Online (MEDLINE) and Excerpta Medica Database (EMBASE) databases were searched to identify all published PheWAS studies up until 24 April 2021. The PheWAS methodology buy zithromax in usa incorporating how to perform PheWAS analysis and which software/tool could be used, were summarised based on the extracted information. A total of 1035 buy zithromax in usa studies were identified and 195 eligible articles were finally included. Among them, 137 (77.0%) contained 10 000 or more study participants, 164 (92.1%) defined the phenome based on electronic medical records data, 140 (78.7%) used genetic variants as predictors, and 73 (41.0%) conducted replication analysis to validate PheWAS findings and almost all of them (94.5%) received consistent results.

The methodology buy zithromax in usa applied in these PheWAS studies was dissected into several critical steps, including quality control of the phenome, selecting predictors, phenotyping, statistical analysis, interpretation and visualisation of PheWAS results, and the workflow for performing a PheWAS was established with detailed instructions on each step. This study provides a comprehensive overview of PheWAS methodology to help practitioners achieve a better understanding of the PheWAS design, to detect understudied or overstudied outcomes, and to direct their research by applying the most appropriate software and online tools for their study data structure.genetic association studiesmolecular epidemiologypublic health.

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A fourth wave of the opioid epidemic is coming, a national expert on drug use and policy said during a virtual panel discussion this week zithromax antibiotico 500 hosted by the Berkshire County, Massachusetts, District Attorney’s Office and the Berkshire Opioid read the article Addiction Prevention Collaborative.Dr. Daniel Ciccarone, a professor of family and community medicine at the University of California, San Francisco (UCSF) School of Medicine, said the next wave in the country’s opioid health emergency will focus on stimulants like methamphetamine and cocaine, and drug combinations where stimulants are used zithromax antibiotico 500 in conjunction with opioids.“The use of methamphetamines is back and it’s back big time,” said Ciccarone, whose most recent research has focused on heroin use.Previously, officials had said there were three waves of the opioid epidemic – the first being prescription pills, the second being heroin, and the third being synthetic drugs, like fentanyl.Now, Ciccarone said, what federal law enforcement and medical experts are seeing is an increase in the use of stimulants, especially methamphetamines.The increase in deaths due to stimulants may be attributed to a number of causes. The increase in supply, both imported and domestically produced, as well as the increase of the drugs’ potency.“Meth’s purity and potency has gone up to historical levels,” he said.

€œAs of 2018, we’ve reached unseen zithromax antibiotico 500 heights of 97 percent potency and 97 percent purity. In a prohibitionist world, we should not be seeing such high quality. This is almost pharmaceutical quality.”Additionally, zithromax antibiotico 500 law enforcement and public health experts like Ciccarone are seeing an increase in the co-use of stimulants with opioids, he said.

Speedballs, cocaine mixed with heroin, and goofballs, methamphetamines used with heroin or fentanyl, are becoming more common from the Midwest into Appalachia and up through New England, he said.Federal law enforcement officials are recommending local communities prepare for the oncoming rise in illegal drugs coming into their communities.“Some people will use them both at the same time, but some may use them in some combination regularly,” he said. €œThey may use meth in the morning to zithromax antibiotico 500 go to work, and use heroin at night to come down.”The co-use, he said, was an organic response to the fentanyl overdose epidemic.“Some of the things that we heard … is that meth is popularly construed as helping to decrease heroin and fentanyl use. Helping with heroin withdraw symptoms and helping with heroin overdoses,” he said.

€œWe debated this for many years that people were using stimulants to reverse overdoses – we’re hearing it again.”“Supply is up, purity is up, price is down,” he zithromax antibiotico 500 said. €œWe know from economics that when drug patterns go in that direction, use is going up.”Ciccarone said that there should not be deaths because of stimulants, but that heroin/fentanyl is the deadly element in the equation.His recommendations to communities were not to panic, but to lower the stigma surrounding drug use in order to affect change. Additionally, he zithromax antibiotico 500 said, policies should focus on reduction.

supply reduction, demand reduction and harm reduction. But not focus on only one single drug.Additionally, he said that by addressing issues within communities and by healing communities socially, economically and zithromax antibiotico 500 spiritually, communities can begin to reduce demand.“We’ve got to fix the cracks in our society, because drugs fall into the cracks,” he said.Shutterstock U.S. Rep.

Annie Kuster (D-NH) recently held two virtual roundtables addressing how buy antibiotics has affected New Hampshire’s healthcare industry.“The health and economic crisis caused by buy antibiotics has created significant challenges for Granite State healthcare, mental health, and substance use treatment providers — at the same time, we are seeing increases in substance abuse and mental illness zithromax antibiotico 500 across New Hampshire,” Kuster said. €œFrom the transition to telehealth care and cancellations of elective procedures to a lack of personal protective equipment and increasing health needs zithromax antibiotico 500 of our communities – providers have overcome a multitude of obstacles due to buy antibiotics in recent months. I was glad to hear from these hard-working Granite Staters, whose insights will continue to guide my work in Congress as we respond to this zithromax.

I’m committed zithromax antibiotico 500 to ensuring that communities across New Hampshire can safely access the care and treatment they deserve.”The first roundtable addressed substance-use disorder (SUD) and mental health.The second virtual roundtable was an opportunity for health care providers to speak about their workplace challenges during the zithromax. Kuster is the founder and co-chairwoman of the Bipartisan Opioid Task Force, which held a virtual discussion in June on the opioid crisis and the zithromax.Shutterstock Opioid prescription rates for outpatient knee surgery vary nationwide, according to a study recently published in BMJ Open. €œWe found massive levels of variation in the proportion of patients who are prescribed opioids between states, even after adjusting for nuances of the procedure and differences in zithromax antibiotico 500 patient characteristics,” said Dr.

M. Kit Delgado, the study’s senior author and an assistant professor of Emergency Medicine and Epidemiology zithromax antibiotico 500 in the Perelman School of Medicine at the University of Pennsylvania. €œWe’ve also seen that the average number of pills prescribed was extremely high for outpatient procedures of this type, particularly for patients who had not been taking opioids prior to surgery.”Researchers examined insurance claims for nearly 100,000 patients who had arthroscopic knee surgery between 2015 and 2019 and had not used any opioid prescriptions in the six months before the surgery.Within three days of a procedure, 72 percent of patients filled an opioid prescription.

High prescription zithromax antibiotico 500 rates were found in the Midwest and the Rocky Mountain regions. The coasts had lower rates.Nationwide, the average prescription strength was equivalent to 250 milligrams of morphine over five days. This is the threshold for increased risk of opioid overdose death, zithromax antibiotico 500 according to the Centers for Disease Control and Prevention.Shutterstock U.S.

Secretary of Labor Eugene Scalia awarded nearly $20 million to four states significantly impacted by the opioid crisis, the Department of Labor announced Thursday. The Florida Department of Economic Opportunity, the Maryland Department zithromax antibiotico 500 of Labor, the Ohio Department of Job and Family Services, and the Wisconsin Department of Workforce Development were awarded the money as part of the DOL’s “Support to Communities. Fostering Opioid Recovery through Workforce Development” created after the passage of the SUPPORT for Patients and Communities Act of 2018.

The money will zithromax antibiotico 500 be used to retrain workers in areas with high rates of substance use disorders. At a press conference in Piketon, Ohio, Scalia said the DOL had awarded Ohio’s Department of Job and Family Services $5 million to help communities in southern Ohio combat the opioid zithromax antibiotico 500 crisis in that area. €œToday’s funding represents this Administration’s continued commitment to serving those most in need,” said Assistant Secretary for Employment and Training John Pallasch.

€œThe U.S zithromax antibiotico 500. Department of Labor is taking a strong stand to support individuals and communities impacted by the crisis.”Grantees will use the funds to collaborate with community partners, such as employers, local workforce development boards, treatment and recovery centers, law enforcement officials, faith-based community organizations, and others, to address the economic effects of substance misuse, opioid use, addiction, and overdose.Shutterstock CVS Health has completed the installation of time-delayed safe technology at all 446 Massachusetts locations as part of its initiatives aimed at reducing the misuse and diversion of prescription medications in Massachusetts, the company announced Thursday. The safes are intended to prevent robberies of controlled substance medications, such as oxycodone and hydrocodone, by electronically delaying the time it takes for pharmacy employees to open the safe where those drugs are stored.The company also announced that it had added 50 new medication disposal units in select stores zithromax antibiotico 500 throughout Massachusetts.

Those units join 106 secure disposal units previously installed at CVS locations across the state and another 43 units previously donated to Massachusetts law enforcement agencies. The company plans to install another six units in stores by the zithromax antibiotico 500 year’s end. €œWhile our nation and our company focus on buy antibiotics treatment, testing, and other measures to prevent community transmission of the zithromax, the misuse of prescription drugs remains an ongoing challenge in Massachusetts and elsewhere that warrants our continued attention,” said John Hering, Region Director for CVS Health.

€œThese steps to reduce the theft and diversion of zithromax antibiotico 500 opioid medications bring added security to our stores and more disposal options for our communities.”In 2015, CVS implemented time-delayed safe technology in CVS pharmacies across Indianapolis in response to the high volume of pharmacy robberies in that city. The company saw a 70 percent decline in pharmacy robberies in stores where the time-delayed safes were installed. Since then, the company has installed 4,760 time-delayed safes in 15 states and the District of Columbia and has seen a 50 percent decline in pharmacy robberies in those areas zithromax antibiotico 500.

The company said it would add an additional 1,000 in-store medication disposal units to the 2,500 units it currently has in CVS pharmacies nationwide. The units allow zithromax antibiotico 500 customers to drop unused prescriptions into a safe place for their disposal to prevent those drugs from being misused. CVS stores that do not offer medication disposal units offer all customers filling opioid prescriptions for the first time with DisposeRX packets that effectively and efficiently breakdown unused drugs into a biodegradable gel for safe disposal in the trash at home..

A fourth wave of the opioid epidemic buy zithromax in usa is coming, a national expert on drug use and policy said during a virtual panel discussion this week hosted by the Berkshire County, Massachusetts, District Attorney’s Office and the Cialis online canada Berkshire Opioid Addiction Prevention Collaborative.Dr. Daniel Ciccarone, a professor of family and community medicine at buy zithromax in usa the University of California, San Francisco (UCSF) School of Medicine, said the next wave in the country’s opioid health emergency will focus on stimulants like methamphetamine and cocaine, and drug combinations where stimulants are used in conjunction with opioids.“The use of methamphetamines is back and it’s back big time,” said Ciccarone, whose most recent research has focused on heroin use.Previously, officials had said there were three waves of the opioid epidemic – the first being prescription pills, the second being heroin, and the third being synthetic drugs, like fentanyl.Now, Ciccarone said, what federal law enforcement and medical experts are seeing is an increase in the use of stimulants, especially methamphetamines.The increase in deaths due to stimulants may be attributed to a number of causes. The increase in supply, both imported and domestically produced, as well as the increase of the drugs’ potency.“Meth’s purity and potency has gone up to historical levels,” he said. €œAs of 2018, we’ve reached unseen heights of 97 percent potency and 97 percent purity buy zithromax in usa.

In a prohibitionist world, we should not be seeing such high quality. This is almost pharmaceutical quality.”Additionally, law enforcement and public health experts like Ciccarone are seeing an increase in the co-use of stimulants with buy zithromax in usa opioids, he said. Speedballs, cocaine mixed with heroin, and goofballs, methamphetamines used with heroin or fentanyl, are becoming more common from the Midwest into Appalachia and up through New England, he said.Federal law enforcement officials are recommending local communities prepare for the oncoming rise in illegal drugs coming into their communities.“Some people will use them both at the same time, but some may use them in some combination regularly,” he said. €œThey may use meth in the morning to go to work, and buy zithromax in usa use heroin at night to come down.”The co-use, he said, was an organic response to the fentanyl overdose epidemic.“Some of the things that we heard … is that meth is popularly construed as helping to decrease heroin and fentanyl use.

Helping with heroin withdraw symptoms and helping with heroin overdoses,” he said. €œWe debated this for many years that people were using stimulants to reverse overdoses – we’re hearing it again.”“Supply is up, purity is up, price buy zithromax in usa is down,” he said. €œWe know from economics that when drug patterns go in that direction, use is going up.”Ciccarone said that there should not be deaths because of stimulants, but that heroin/fentanyl is the deadly element in the equation.His recommendations to communities were not to panic, but to lower the stigma surrounding drug use in order to affect change. Additionally, he said, policies should focus on buy zithromax in usa reduction.

supply reduction, demand reduction and harm reduction. But not focus on only one single drug.Additionally, he said that by addressing issues within communities and by healing communities socially, economically and spiritually, communities can begin to reduce demand.“We’ve got to fix the cracks in our society, because buy zithromax in usa drugs fall into the cracks,” he said.Shutterstock U.S. Rep. Annie Kuster (D-NH) recently held two buy zithromax in usa virtual roundtables addressing how buy antibiotics has affected New Hampshire’s healthcare industry.“The health and economic crisis caused by buy antibiotics has created significant challenges for Granite State healthcare, mental health, and substance use treatment providers — at the same time, we are seeing increases in substance abuse and mental illness across New Hampshire,” Kuster said.

€œFrom the transition to telehealth care and cancellations of elective procedures to a buy zithromax in usa lack of personal protective equipment and increasing health needs of our communities – providers have overcome a multitude of obstacles due to buy antibiotics in recent months. I was glad to hear from these hard-working Granite Staters, whose insights will continue to guide my work in Congress as we respond to this zithromax. I’m committed to ensuring that communities across New Hampshire can safely access the care and treatment they deserve.”The first roundtable addressed substance-use disorder (SUD) and mental health.The second virtual roundtable was an opportunity buy zithromax in usa for health care providers to speak about their workplace challenges during the zithromax. Kuster is the founder and co-chairwoman of the Bipartisan Opioid Task Force, which held a virtual discussion in June on the opioid crisis and the zithromax.Shutterstock Opioid prescription rates for outpatient knee surgery vary nationwide, according to a study recently published in BMJ Open.

€œWe found massive levels of variation in the proportion of patients who are prescribed opioids between states, even after adjusting for nuances of the procedure and differences in patient characteristics,” buy zithromax in usa said Dr. M. Kit Delgado, the study’s senior author and an assistant professor of Emergency Medicine and Epidemiology in the Perelman School of Medicine at buy zithromax in usa the University of Pennsylvania. €œWe’ve also seen that the average number of pills prescribed was extremely high for outpatient procedures of this type, particularly for patients who had not been taking opioids prior to surgery.”Researchers examined insurance claims for nearly 100,000 patients who had arthroscopic knee surgery between 2015 and 2019 and had not used any opioid prescriptions in the six months before the surgery.Within three days of a procedure, 72 percent of patients filled an opioid prescription.

High prescription rates were found in the Midwest and the Rocky Mountain regions buy zithromax in usa. The coasts had lower rates.Nationwide, the average prescription strength was equivalent to 250 milligrams of morphine over five days. This is the threshold for increased risk of opioid overdose death, according to the Centers for Disease Control and buy zithromax in usa Prevention.Shutterstock U.S. Secretary of Labor Eugene Scalia awarded nearly $20 million to four states significantly impacted by the opioid crisis, the Department of Labor announced Thursday.

The Florida Department of Economic Opportunity, the Maryland Department of Labor, the Ohio Department of Job and Family Services, and the Wisconsin Department of Workforce buy zithromax in usa Development were awarded the money as part of the DOL’s “Support to Communities. Fostering Opioid Recovery through Workforce Development” created after the passage of the SUPPORT for Patients and Communities Act of 2018. The money will be used to retrain workers in areas buy zithromax in usa with high rates of substance use disorders. At a press conference in Piketon, Ohio, Scalia said the DOL had awarded Ohio’s Department of Job and Family Services $5 million to help communities in southern Ohio combat the opioid crisis in buy zithromax in usa that area.

€œToday’s funding represents this Administration’s continued commitment to serving those most in need,” said Assistant Secretary for Employment and Training John Pallasch. €œThe U.S buy zithromax in usa. Department of Labor is taking a strong stand to support individuals and communities impacted by the crisis.”Grantees will use the funds to collaborate with community partners, such as employers, local workforce development boards, treatment and recovery centers, law enforcement officials, faith-based community organizations, and others, to address the economic effects of substance misuse, opioid use, addiction, and overdose.Shutterstock CVS Health has completed the installation of time-delayed safe technology at all 446 Massachusetts locations as part of its initiatives aimed at reducing the misuse and diversion of prescription medications in Massachusetts, the company announced Thursday. The safes are intended to prevent robberies of controlled substance medications, such as oxycodone and hydrocodone, by electronically delaying the time it takes for pharmacy employees to open the safe where those drugs are stored.The company also announced that it had added 50 new buy zithromax in usa medication disposal units in select stores throughout Massachusetts.

Those units join 106 secure disposal units previously installed at CVS locations across the state and another 43 units previously donated to Massachusetts law enforcement agencies. The company plans to install another buy zithromax in usa six units in stores by the year’s end. €œWhile our nation and our company focus on buy antibiotics treatment, testing, and other measures to prevent community transmission of the zithromax, the misuse of prescription drugs remains an ongoing challenge in Massachusetts and elsewhere that warrants our continued attention,” said John Hering, Region Director for CVS Health. €œThese steps to reduce the theft and diversion of opioid medications bring added security to our stores and more disposal options for our communities.”In 2015, CVS implemented time-delayed safe technology in CVS pharmacies across Indianapolis in response to the high volume of buy zithromax in usa pharmacy robberies in that city.

The company saw a 70 percent decline in pharmacy robberies in stores where the time-delayed safes were installed. Since then, the company has installed 4,760 time-delayed buy zithromax in usa safes in 15 states and the District of Columbia and has seen a 50 percent decline in pharmacy robberies in those areas. The company said it would add an additional 1,000 in-store medication disposal units to the 2,500 units it currently has in CVS pharmacies nationwide. The units allow customers to drop unused prescriptions into a safe place for their buy zithromax in usa disposal to prevent those drugs from being misused.

CVS stores that do not offer medication disposal units offer all customers filling opioid prescriptions for the first time with DisposeRX packets that effectively and efficiently breakdown unused drugs into a biodegradable gel for safe disposal in the trash at home..

What should I watch for while taking Zithromax?

Tell your prescriber or health care professional if your symptoms do not improve in 2 to 3 days. Contact your prescriber or health care professional as soon as you can if you get an allergic reaction to azithromycin, such as rash, itching, difficulty swallowing, or swelling of the face, lips or tongue. Keep out of the sun, or wear protective clothing outdoors and use a sunscreen. Do not use sun lamps or sun tanning beds or booths. If you get severe or watery diarrhea, do not treat yourself. Call your prescriber or health care professional for advice. Antacids can stop azithromycin from working. If you get an upset stomach and want to take an antacid, make sure there is an interval of at least 2 hours since you last took azithromycin, or 4 hours before your next dose. If you are going to have surgery, tell your prescriber or health care professional that you are taking azithromycin.

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One of the priority actions in the New Zealand Healthy Ageing Strategy (2016) was to improve models zithromax side effects of care for Home and community Support Services (HCSS) in response to the multiple and growing demands on HCSS. The National Framework for HCSS provides guidance for district health boards for future commissioning, developing, delivering and evaluating HCSS to improve national consistency and quality of care. The National zithromax side effects Framework for HCSS was developed in collaboration with key stakeholders in the HCSS sector, including older people and their whānau. It includes. a vision and principles to guide service design core (essential) components of services that could be expected anywhere in the country a draft outcomes framework describing the outcomes sought from HCSS at individual, population and system levels.

The National Framework for zithromax side effects HCSS covers DHB-funded services for. people aged 65 years and over who have an assessed need in response to an interRAI assessment and meet criteria for funding people considered to be alike in age and interest – for example, Pacific peoples and Māori, aged over 55 years, and others aged over 60 years, with age-related disabilities older people receiving HCSS who require increased support following an acute health episode who have required hospitalisation HCSS that may continue concurrently with short-term Accident Compensation Corporation (ACC) services. Three additional initiatives are linked with developing the National Framework to help achieve consistency in service commissioning, provision and resource allocation. First, a zithromax side effects National Service Specification for HCSS. This service specification will become the nationally mandated specification describing in detail the services and service approaches required of DHBs and providers.

This National Service Specification will be implemented by July 2022, in line with DHB service commissioning timetables. This approach aims to achieve the best balance between national consistency and flexibility for DHBs in meeting the zithromax side effects needs of their populations. Second, a nationally consistent case-mix methodology will be developed for all DHBs to use as a way of improving targeting of resources according to need. Some DHBs are already applying case-mix methods to resource allocation or use. However, different versions of the methodology are being used, resulting in some inconsistency in resource allocation and lack of transparency across DHBs zithromax side effects.

This indicates the need for a single, nationally consistent case-mix method which will also be implemented across all DHBs by July 2022. Third, a nationally consistent outcomes and measurement framework will be developed for use in HCSS and is expected to be completed by July 2021.The Historical mortality web tool presents mortality data (numbers and age-standardised rates) by sex for certain causes of death from 1948 to 2016. Mortality data zithromax side effects by sex, age group and ethnicity (Māori and non-Māori) is presented from 1996 to 2016.The web tool enables you to explore trends over time using interactive graphs and tables. Filtered results and the full data set can be downloaded from within the web tool. The causes of death included are.

All cancer Ischaemic heart disease Cerebrovascular disease Chronic lower respiratory diseases Other forms of heart disease Influenza and Pneumonia Diabetes mellitus Motor vehicle accidents Intentional self-harm zithromax side effects Assault All deaths. The full data set presented in the web tool is available for you to download in text file format. A technical document accompanies the web tool. This document contains zithromax side effects information about the data source and analytical methods used to produce summary data, and a data dictionary for variables used in the web tool. About the data used in this edition Data from 1948 to 1995 presented in these tables was sourced from publications in the Ministry of Health Mortality data and stats series.

Data from 1996 to 2016 was extracted from the New Zealand Mortality Collection records on 07 June 2019. At the time of extraction, there were zithromax side effects 606,450 deaths registered from 1996 to 2016. Included in this data were 641 deaths provisionally coded awaiting coroners’ findings and 41 deaths awaiting coroners’ findings with no known cause. Ethnic breakdowns of mortality data are only shown from 1996 onwards because there was a significant change in the way ethnicity was defined, and in the way ethnicity data was collected in 1995. For more information please refer to the Ministry of Health report, zithromax side effects Mortality and Demographic Data 1996.

Disclaimer In this edition, data for causes of death was extracted and recalculated for the years 1996–2016 to reflect ongoing updates to data in the New Zealand Mortality Collection (for example, following the release of coroners’ findings) and the revision of population estimates and projections following each census. For this reason there may be small changes to some numbers and rates from those presented in previous publications and tables. We have quality checked the collection, extraction, and reporting of the data presented here. However errors can occur. Contact the Ministry of Health if you have any concerns regarding any of the data or analyses presented here, at data-enquires@health.govt.nz.

One of the priority actions in buy zithromax in usa the New Zealand Healthy Ageing Strategy (2016) was to improve models of care for Home and community Support Services (HCSS) in response to the zithromax prescription online multiple and growing demands on HCSS. The National Framework for HCSS provides guidance for district health boards for future commissioning, developing, delivering and evaluating HCSS to improve national consistency and quality of care. The National Framework for HCSS was developed in collaboration with key stakeholders in the HCSS sector, buy zithromax in usa including older people and their whānau. It includes.

a vision and principles to guide service design core (essential) components of services that could be expected anywhere in the country a draft outcomes framework describing the outcomes sought from HCSS at individual, population and system levels. The National Framework buy zithromax in usa for HCSS covers DHB-funded services for. people aged 65 years and over who have an assessed need in response to an interRAI assessment and meet criteria for funding people considered to be alike in age and interest – for example, Pacific peoples and Māori, aged over 55 years, and others aged over 60 years, with age-related disabilities older people receiving HCSS who require increased support following an acute health episode who have required hospitalisation HCSS that may continue concurrently with short-term Accident Compensation Corporation (ACC) services. Three additional initiatives are linked with developing the National Framework to help achieve consistency in service commissioning, provision and resource allocation.

First, a National buy zithromax in usa Service Specification for HCSS. This service specification will become the nationally mandated specification describing in detail the services and service approaches required of DHBs and providers. This National Service Specification will be implemented by July 2022, in line with DHB service commissioning timetables. This approach aims to achieve the best balance between national consistency and flexibility for buy zithromax in usa DHBs in meeting the needs of their populations.

Second, a nationally consistent case-mix methodology will be developed for all DHBs to use as a way of improving targeting of resources according to need. Some DHBs are already applying case-mix methods to resource allocation or use. However, different versions of the methodology are being used, resulting in some inconsistency in resource allocation and lack buy zithromax in usa of transparency across DHBs. This indicates the need for a single, nationally consistent case-mix method which will also be implemented across all DHBs by July 2022.

Third, a nationally consistent outcomes and measurement framework will be developed for use in HCSS and is expected to be completed by July 2021.The Historical mortality web tool presents mortality data (numbers and age-standardised rates) by sex for certain causes of death from 1948 to 2016. Mortality data by sex, http://protoolmanufacturing.com/portfolio-item/dyson/ age group and ethnicity (Māori and non-Māori) is presented from 1996 to 2016.The web tool enables you to explore trends over time using interactive graphs buy zithromax in usa and tables. Filtered results and the full data set can be downloaded from within the web tool. The causes of death included are.

All cancer Ischaemic heart disease Cerebrovascular disease Chronic lower respiratory buy zithromax in usa diseases Other forms of heart disease Influenza and Pneumonia Diabetes mellitus Motor vehicle accidents Intentional self-harm Assault All deaths. The full data set presented in the web tool is available for you to download in text file format. A technical document accompanies the web tool. This document contains information buy zithromax in usa about the data source and analytical methods used to produce summary data, and a data dictionary for variables used in the web tool.

About the data used in this edition Data from 1948 to 1995 presented in these tables was sourced from publications in the Ministry of Health Mortality data and stats series. Data from 1996 to 2016 was extracted from the New Zealand Mortality Collection records on 07 June 2019. At the time of extraction, there were 606,450 deaths registered buy zithromax in usa from 1996 to 2016. Included in this data were 641 deaths provisionally coded awaiting coroners’ findings and 41 deaths awaiting coroners’ findings with no known cause.

Ethnic breakdowns of mortality data are only shown from 1996 onwards because there was a significant change in the way ethnicity was defined, and in the way ethnicity data was collected in 1995. For more information please refer to the Ministry buy zithromax in usa of Health report, Mortality and Demographic Data 1996. Disclaimer In this edition, data for causes of death was extracted and recalculated for the years 1996–2016 to reflect ongoing updates to data in the New Zealand Mortality Collection (for example, following the release of coroners’ findings) and the revision of population estimates and projections following each census. For this reason there may be small changes to some numbers and rates from those presented in previous publications and tables.

We have quality checked the buy zithromax in usa collection, extraction, and reporting of the data presented here. However errors can occur. Contact the Ministry of Health if you have any concerns regarding any of the data or analyses presented here, at data-enquires@health.govt.nz.

Zithromax 500mg oral tablet

How to cite this article:Singh OP zithromax 500mg oral tablet site link. The National Commission for Allied and Healthcare Professions Act, 2020 and its implication for mental health. Indian J Psychiatry 2021;63:119-20The National Commission for Allied and Healthcare Professions Act, 2020 has been notified on March 28, 2021, by the Gazette of India published by the Ministry of Law zithromax 500mg oral tablet and Justice. This bill aims to “provide for regulation and maintenance of standards of education and services by allied and healthcare professionals, assessment of institutions, maintenance of a Central Register and State Register and creation of a system to improve access, research and development and adoption of latest scientific advancement and for matters connected therewith or incidental thereto.”[1]This act has created a category of Health Care Professionals which is defined as.

€œhealthcare professional” includes a scientist, therapist, or other professional who studies, advises, researches, supervises or provides preventive, curative, rehabilitative, therapeutic or promotional health services and who has obtained any qualification of degree under this Act, the duration of which shall not be <3600 h spread over a period of 3 years to 6 years divided into specific semesters.[1]According to the act, “Allied health professional” includes an associate, technician, or technologist who is trained to perform any technical and practical task to support diagnosis and treatment of illness, disease, injury or impairment, and to support implementation of any healthcare treatment and referral plan recommended by a medical, nursing, or any other healthcare professional, and who has obtained any qualification of diploma or degree under this Act, the duration of which shall not be less than 2000 h spread over a period of 2 years to 4 years divided into specific semesters.”[1]It is noticeable that while the term “Health Care Professionals” does not include doctors who are registered under National Medical Council, Mental Health Care Act (MHCA), 2017 includes psychiatrists under the ambit of Mental Health Care Professionals.[2] This discrepancy needs to be corrected - psychiasts, being another group of medical specialists, should be kept out of the broad umbrella of “Mental Healthcare Professionals.”The category of Behavioural Health Sciences Professional has been included and defined as “a person who undertakes scientific study of the emotions, behaviours and biology relating to a person's mental well-being, their ability to function in everyday life and their concept of self. €œBehavioural health” is the preferred term to “mental health” and includes professionals such as counselors, analysts, psychologists, educators and support workers, who provide counseling, therapy, and mediation services to individuals, families, groups, and communities in response to social and personal difficulties.”[1]This is a welcome step to the extent that it creates a diverse category of trained workforce in the field of Mental Health (Behavioural Health Science Professionals) and tries to regulate their training although it mainly aims to promote zithromax 500mg oral tablet mental wellbeing. However there is a huge lacuna in the term of “Mental Illness” as defined by MHCA, 2017. Only severe disorders are included as per definition and there is no clarity regarding inclusion of other psychiatric disorders, namely “common mental disorders” such as anxiety and depression.

This leaves a strong possibility of concept of “psychiatric illnesses” being zithromax 500mg oral tablet limited to only “severe psychiatric disorders” (major psychoses) thus perpetuating the stigma and alienation associated with psychiatric patients for centuries. Psychiatrists being restricted to treating severe mental disorders as per MHCA, 2017, there is a strong possibility that the care of common mental disorders may gradually pass on under the care of “behavioural health professionals” as per the new act!. There is need to look into this aspect by zithromax 500mg oral tablet the leadership in psychiatry, both organizational and academic psychiatry, and reduce the contradictions between the MHCA, 2017 and this nascent act. All disorders classified in ICD 10 and DSM 5 should be classified as “Psychiatric Disorders” or “Mental Illness.” This will not only help in fighting the stigma associated with psychiatric illnesses but also promote the integration of psychiatry with other specialties.

References 1.The National browse around here Commission for Allied and Healthcare Professions Act, 2021. The Gazette of India zithromax 500mg oral tablet. Published by Ministry of Law and Justice. 28 March, 2021 zithromax 500mg oral tablet.

2.The Mental Healthcare Act, 2017. The Gazette of India. Published by Ministry of Law and zithromax 500mg oral tablet Justice. April 7, 2017.

Correspondence Address:Om Prakash SinghAA 304, Ashabari Apartments, O/31, Baishnabghata, Patuli Township, Kolkata - 700 094, West Bengal IndiaSource of Support. None, Conflict of zithromax 500mg oral tablet Interest. NoneDOI. 10.4103/indianjpsychiatry.indianjpsychiatry_268_21.

How to buy zithromax in usa http://ribbonebrewingcompany.com/?p=84 cite this article:Singh OP. The National Commission for Allied and Healthcare Professions Act, 2020 and its implication for mental health. Indian J Psychiatry 2021;63:119-20The National Commission for Allied and Healthcare Professions Act, 2020 has been notified on March 28, 2021, by the Gazette of India published by the Ministry buy zithromax in usa of Law and Justice. This bill aims to “provide for regulation and maintenance of standards of education and services by allied and healthcare professionals, assessment of institutions, maintenance of a Central Register and State Register and creation of a system to improve access, research and development and adoption of latest scientific advancement and for matters connected therewith or incidental thereto.”[1]This act has created a category of Health Care Professionals which is defined as. €œhealthcare professional” includes a scientist, therapist, or other professional who studies, advises, researches, supervises or provides preventive, curative, rehabilitative, therapeutic or promotional health services and who has obtained any qualification of degree under this Act, the duration of which shall not be <3600 h spread over a period of 3 years to 6 years divided into specific semesters.[1]According to the act, “Allied health professional” includes an associate, technician, or technologist who is trained to perform any technical and practical task to support diagnosis and treatment of illness, disease, injury or impairment, and to support implementation of any healthcare treatment and referral plan recommended by a medical, nursing, or any other healthcare professional, and who has obtained any qualification of diploma or degree under this Act, the duration of which shall not be less than 2000 h spread over a period of 2 years to 4 years divided into specific semesters.”[1]It is noticeable that while the term “Health Care Professionals” does not include doctors who are registered under National Medical Council, Mental Health Care Act (MHCA), 2017 includes psychiatrists under the ambit of Mental Health Care Professionals.[2] This discrepancy needs to be corrected - psychiasts, being another group of medical specialists, should be kept out of the broad umbrella of “Mental Healthcare Professionals.”The category of Behavioural Health Sciences Professional has been included and defined as “a person who undertakes scientific study of the emotions, behaviours and biology relating to a person's mental well-being, their ability to function in everyday life and their concept of self.

€œBehavioural health” is the preferred term to “mental health” and includes professionals such as counselors, analysts, psychologists, educators and support workers, who provide counseling, therapy, and mediation services to individuals, families, buy zithromax in usa groups, and communities in response to social and personal difficulties.”[1]This is a welcome step to the extent that it creates a diverse category of trained workforce in the field of Mental Health (Behavioural Health Science Professionals) and tries to regulate their training although it mainly aims to promote mental wellbeing. However there is a huge lacuna in the term of “Mental Illness” as defined by MHCA, 2017. Only severe disorders are included as per definition and there is no clarity regarding inclusion of other psychiatric disorders, namely “common mental disorders” such as anxiety and depression. This leaves a strong possibility of concept of “psychiatric illnesses” being limited to only “severe psychiatric disorders” (major psychoses) thus perpetuating the buy zithromax in usa stigma and alienation associated with psychiatric patients for centuries. Psychiatrists being restricted to treating severe mental disorders as per MHCA, 2017, there is a strong possibility that the care of common mental disorders may gradually pass on under the care of “behavioural health professionals” as per the new act!.

There is need to look into this aspect by the leadership in psychiatry, both organizational and academic psychiatry, and reduce the contradictions buy zithromax in usa between the MHCA, 2017 and this nascent act. All disorders classified in ICD 10 and DSM 5 should be classified as “Psychiatric Disorders” or “Mental Illness.” This will not only help in fighting the stigma associated with psychiatric illnesses but also promote the integration of psychiatry with other specialties. References 1.The National Commission for Allied and Healthcare Professions Act, 2021. The Gazette buy zithromax in usa of India. Published by Ministry of Law and Justice.

28 March, buy zithromax in usa 2021. 2.The Mental Healthcare Act, 2017. The Gazette of India. Published by Ministry of Law buy zithromax in usa and Justice. April 7, 2017.

Correspondence Address:Om Prakash SinghAA 304, Ashabari Apartments, O/31, Baishnabghata, Patuli Township, Kolkata - 700 094, West Bengal IndiaSource of Support. None, Conflict of buy zithromax in usa Interest. NoneDOI. 10.4103/indianjpsychiatry.indianjpsychiatry_268_21.

Zithromax for staph

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in zithromax for staph the tumor microenvironment (TME) for improved responses learn the facts here now. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion zithromax for staph of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages.

The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent zithromax for staph mice, an important step toward the acceleration of effective therapies reaching the clinic. The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al., 2019). Another approach to ACT involves the redirection of zithromax for staph peripheral blood T cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen binding.

CAR-T cell therapy against hematologic malignancies, by targeting the B cell lineage antigens CD19 or the B cell maturation antigen, has proven efficacious in the clinic, and there is optimism that similar success will be achieved for some solid tumors (Geyer and Brentjens, 2016. Irving et al., 2017). A range of physical (Lanitis et al., 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and effector function limit the potency zithromax for staph of CAR-T cell therapy against solid tumors (Brown et al., 2016. Louis et al., 2011).

Moreover, chronic antigen exposure and a lack of sufficient costimulation in the tumor microenvironment (TME) can cause CAR-T cell exhaustion (Irving et al., 2017). Coengineering of CAR-T cells may help zithromax for staph to overcome some of these obstacles (Lanitis et al., 2020). Genetic modifications, for example, can be made to enable better homing and tumor penetration or render CAR-T cells resistant to suppressive mechanisms in the TME (Stromnes et al., 2010). In addition, CAR-T cells can be armed with secretory molecules or additional receptors zithromax for staph to support CAR-T cell activity and/or harness endogenous immunity (Adachi et al., 2018.

Pegram et al., 2012). Preclinical evaluation of CAR-T cells has, for the most part, been performed with xenograft tumor models in immunodeficient mice (Lee et al., 2011. Mardiros et al., 2013 zithromax for staph . Lanitis et al., 2012).

Although this approach can be used to evaluate human CAR-T cell persistence, homing, tumor control, and survival following ACT, critical parameters, including potential toxicity against normal tissues (Tran et al., 2013), and the impact of endogenous immunity on both tumor control and escape are not addressed in such models (Spear et al., 2012. Avanzi et zithromax for staph al., 2018). As varying obstacles must be overcome to enhance CAR-T cell responses against different solid tumor types, comprehensive studies in immunocompetent syngeneic tumor models would enable more accurate screening of T cell engineering strategies and provide important insights into improving coengineering and combinatorial treatment approaches (Lanitis et al., 2020). A key limitation of CAR evaluation in syngeneic models stems from inadequate methodologies for efficient murine T cell transduction and zithromax for staph expansion.

Indeed, unless T cells derived from multiple donor spleens are transduced or the engineered T cells are restimulated for further expansion, which among other drawbacks are costly and can promote exhaustion and apoptosis (Bucks et al., 2009), respectively, current protocols yield insufficient numbers of CAR-T cells for ACT studies (Lee et al., 2009). The efficiency of cell-surface expression of second-generation (2G) CARs, comprising the endodomain (ED) of CD3ζ and one costimulatory ED (e.g., CD28 or 4-1BB), generally reaches 40–60% (Kochenderfer et al., 2010. Davila et zithromax for staph al., 2013. Wang et al., 2014.

Fu et al., 2013). Although retroviral transduction rates as high as 70–80% for murine T cells have been reported, this was assessed zithromax for staph at 2 to 3 d after transduction (Tran et al., 2013. Kuhn et al., 2019. Kusabuka et al., 2016) and thus may include false positives due to transient expression from zithromax for staph nonintegrated vector DNA (i.e., pseudo-transduction.

Case et al., 1999, Costello et al., 2000). Moreover, short-term transduction efficiency is often based on reporter genes like GFP, which may overestimate CAR expression levels (Kusabuka et al., 2016. Kuhn et zithromax for staph al., 2019. Davila et al., 2013).

Finally, while stable retroviral packaging and producer cell lines may enable transduction efficiencies zithromax for staph for 2G and third-generation (3G. I.e., a CAR having two or more costimulatory EDs) CARs of >60% (Fu et al., 2013), this is a laborious approach if multiple CAR designs are to be compared (Chinnasamy et al., 2010). Here, we report the development of an efficient and highly reproducible protocol for primary murine T cell retroviral transduction and expansion, yielding functional murine 2G-CAR-T cells, as well as fourth-generation (4G)-CAR-T cells coengineered to express murine IL-15 (mIL-15) for enhanced in vitro and in vivo function and TME reprogramming. Overall, our work provides important tools for zithromax for staph enabling the systematic evaluation of 4G-CAR-T cells in immunocompetent, syngeneic tumor-bearing mice, which we believe is critical for effective therapies reaching the clinic.

We sought to optimize murine T cell activation, transduction, and expansion methods for preclinical CAR therapy evaluation in immunocompetent, syngeneic tumor-bearing mice. The final protocol we developed is summarized in Fig. 1 and is described in zithromax for staph detail in Materials and methods. We used a 2G-CAR targeting vascular endothelial cell growth factor receptor 2 (VEGFR-2), comprising the well-characterized single-chain variable fragment (scFv) DC101 (Chinnasamy et al., 2010), a CD8α hinge and transmembrane domain, and the murine EDs of CD28 and CD3ζ.

The anti-VEGFR-2 CAR retroviral vector is abbreviated as DC101-28z zithromax for staph (Fig. 2 A). Because retrozithromaxes infect proliferating cells (Kusabuka et al., 2016. Chinnasamy et zithromax for staph al., 2010.

Hu et al., 2017), we first compared three commonly used methods for inducing T cell activation. (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab (αCD3/CD28 beads) plus recombinant human IL-2 (hIL-2), (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab (αCD3-plate/CD28) plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7. Stimulation with αCD3/CD28 beads consistently resulted in the highest frequency of CD44+ CD62L− (recently activated, memory), CD25+ or CD69+ zithromax for staph (activated), and Ki67+ (proliferating) CD3+ T cells (Fig. 2 B and Fig.

S1 A) zithromax for staph . We next found that concentration of viral particles through ultracentrifugation yielded higher viral titers (>3 × 107 transducing units/ml. Fig. 2 C) and enabled significantly higher transduction of primary activated primary murine T cells as compared zithromax for staph with unconcentrated retrozithromax (Fig.

2 D), reaching a plateau at a multiplicity of (MOI) of 5 (∼80% CAR expression. Fig. 2 E). A single transduction at 24 h after activation versus transduction at both 24 and 48 h did not affect the efficiency in terms of either percentage of cells transduced or CAR expression level per cell (i.e., mean fluorescence intensity [MFI].

Fig. 2, E and F). We observed, however, that the transduction efficiency at 48 h after activation was inferior to that obtained at 24 h after activation (Fig. 2, E and F).

A schema of the T cell activation and transduction approaches compared are depicted in Fig. 2 G. Finally, we observed highest CAR transduction efficiency in CD3+ lymphocytes activated with αCD3/CD28 beads in the presence of hIL-2 as compared with the other aforementioned activation methods (Fig. 2, H and I).

Similar results were observed for CD8+ T cells, while for CD4+ T cells, the percentage CAR expression was the same for both αCD3/CD28-bead and αCD3-plate/CD28 activation (Fig. S1 B). Thus, αCD3/CD28-bead activation was used for all further experiments. Notably, we also investigated concentrated lentiviral transduction of αCD3/CD28-bead–activated murine T cells using the same anti-VEGFR-2 CAR, and consistent with another study (Kerkar et al., 2011), we obtained very low transduction efficiency (∼10%, data not shown).

While long-term T cell culture in IL-2 drives terminal differentiation, the common γ-chain cytokines IL-7 and IL-15 have been reported to promote a central memory T cell (TCM cell) phenotype enabling superior persistence and in vivo tumor control upon ACT (Klebanoff et al., 2005). Thus, we next compared the expansion and functional properties of transduced murine CAR-T cells cultured in hIL-2 alone versus hIL-2 for the first 3 days, followed by hIL-7/IL-15 for the remainder of the culture period (Fig. 3 A). Both hIL-7 and hIL-15 have been previously demonstrated to act on murine T cells to promote homeostatic proliferation and survival (Eisenman et al., 2002.

Nanjappa et al., 2008). As for hIL-2–expanded CAR-T cells (Fig. 2 G), we observed that a single transduction of T cells at 24 h and subsequent expansion in hIL-7/IL-15 was sufficient to achieve a robust and stable transduction efficiency at a MOI as low as 5 (Fig. 3 B).

Both culture conditions (hIL-2 alone versus hIL-2 followed by hIL-7/IL-15) enabled high CAR expression on day 7 (Fig. 3 C). On day 9, however, we observed a 26-fold expansion of CAR-T cells exposed to hIL-7/IL-15 as compared with a 9-fold expansion in the presence of hIL-2 alone at a standard concentration of 50 IU/ml (Fig. 3 D).

Moreover, CAR-T cells cultured with hIL-7/IL-15 continued to expand for at least 14 d, while T cells cultured in hIL-2 alone reached a plateau after 1 wk (Fig. 3 D) and exhibited significantly higher levels of cell death starting early in the culture (Fig. 3 E). We also observed a significantly higher frequency of CD8+ T cells in the hIL-7/IL-15 culture (Fig.

3 F). Finally, transduced T cells expanded with hIL-7/IL-15 had a significantly higher proportion of TCM cells based on cell-surface expression of the hyaluronic acid receptor CD44 and the L-selectin CD62L from day 5 after cytokine addition (Fig. 3, G and H). We sought to evaluate the in vitro reactivity of hIL-2 only versus hIL-7/IL-15 expanded CAR-T cells against target antigen.

On day 7 after transduction, we co-cultured CAR-T cells with bEnd3 murine endothelial cells expressing VEGFR-2, as well as with control VEGFR-2− H5V murine endothelial cells (Fig. 3 I). HIL-7/IL-15 expanded CAR-T cells secreted significantly higher levels of IFN-γ, granzyme B, and IL-2 (Fig. 3 J) after bEnd3 target cell recognition in vitro.

Because CAR-T cell expansion with hIL-7/IL-15 results in a higher frequency of CD8+ T cells as compared with hIL-2 only, we next sorted CD8+ T cells on day 7 after transduction and performed a co-culture with bEnd3 and H5V cells. Higher levels of granzyme B, IL-2, and IFN-γ were secreted by hIL-7/IL-15–expanded CD8+ CAR-T cells than hIL-2–expanded ones (Fig. S2). Moreover, hIL-7/IL-15–expanded CAR-T cells exhibited significantly higher persistence (Fig.

3 K), division rates (Fig. 3 L), and numbers of proliferating CD8+ T cells after 4 d of co-culture (Fig. 3 M). Thus, as compared with hIL-2 alone, CAR-T cell expansion with hIL-7/IL-15 promotes higher viability and favors a TCM cell phenotype, more robust expansion, and superior secretion of cytokines and long-term proliferative capacity upon challenge with target cells.

The high transduction efficiency achieved with our optimized method encouraged us to evaluate the coexpression of transgenes and test the impact of additional cargo on CAR-T cell performance. Given the enhanced functional properties of CAR-T cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce mIL-15. Notably, hIL-15 has been previously demonstrated to significantly improve the antitumor activity of human CAR-T cells targeting glioblastoma (Krenciute et al., 2017). A bicistronic retroviral vector encoding mIL-15 and the DC101 CAR, both driven by the 5′ LTR of the retrozithromax (de Felipe et al., 1999) and separated by a self-cleaving 2A peptide sequence (T2A.

Liu et al., 2017), was built to express this 4G-CAR construct (Fig. 4 A). With a single round of transduction at a MOI as low as 5, we achieved a similarly high expression of the 4G- as the 2G-CAR (Fig. 4, B and C), as well as high intracellular expression of mIL-15 (Fig.

4 D). Significant mIL-15 was also detected by ELISA upon lysis of 4G-CAR-T cells (Fig. 4 E), but very low levels of mIL-15 were found in the culture supernatant (data not shown), presumably due to sequestration of the cytokine by cell-surface IL-15 receptor-α (IL-15-Rα), as has been previously observed for human T cells engineered to secrete hIL-15 (Markley and Sadelain, 2010). Our hypothesis was supported by the fact that we detected high levels of soluble mIL-15 in the supernatants of transfected human Phoenix Eco cells (i.e., the retrozithromax producer cell line.

Fig. 4 F). Moreover, 4G-CAR–transduced C1498 leukemia cells (which do not express IL-15-Rα. Fig.

S3 A) secreted high levels of mIL-15 (Fig. 4, G and H). Finally, we activated both 2G- and 4G-CAR-T cells with cognate antigen and found significant secretion of mIL-15 by the 4G-CAR-T cells (Fig. 4 I), as has similarly been reported in the context of engineered human T cells (Krenciute et al., 2017).

We next sought to investigate the impact of mIL-15 coexpression on CAR-T cell signaling and phenotype. In the absence of exogenous cytokine in the culture supernatant, we observed elevated pSTAT5 in the 4G- versus 2G-CAR-T cells both in terms of frequency and level per cell (Fig. 4, J and K). We further evaluated IL-15-Rα expression and detected lower levels on 4G-CAR-T cells (Fig.

4, L and M), presumably due to receptor internalization (Dubois et al., 2002) and/or mIL-15 occupancy blocking the Ab binding site. Subsequently, we assessed expression of the antiapoptotic protein Bcl-2, previously reported to enhance 2G- versus first-generation (1G)–CAR-T cell persistence (Song et al., 2012), and found higher expression levels on days 2 and 5 after transduction for 4G- as compared with 2G-CAR-T cells in the absence of exogenous cytokines (Fig. S3, B and C). In addition, we observed significantly higher frequencies of Ki67+ Bcl-2+ 4G-CAR-T cells on days 2 and 5 after transduction (Fig.

5, A and B). Thus, mIL-15 coexpression appears to augment both CAR-T cell survival and proliferation. We further assessed the phenotype of CAR-T cells in the absence of exogenous cytokines in the culture medium and found that on day 2 following transduction, 2G- and 4G-CAR-T cells displayed no differences in the proportion of naive (CD62Lhigh CD44low), central memory (CM. CD62Lhigh CD44high) and effector memory (EM.

CD62Llow CD44high) T cell phenotype populations. However, by day 5 after transduction, 4G-CAR-T cells had a higher proportion of naive and CM cells and fewer EM cells, as compared with 2G-CAR-T cells (Fig. 5, C and D). Notably, there were significantly lower levels of the inhibitory receptor programmed cell death 1 (PD-1.

Both percentage and MFI) on 4G- compared with 2G-CAR-T cells (Fig. 5, E and F). Consistent with the above findings, we observed that in the absence of exogenous cytokine the 4G-CAR-T cells exhibited increased expansion during the first 2 d after transduction as compared with the 2G-CAR-T cells (Fig. 5 G).

Both 2G- and 4G-CAR-T cells began to contract at a similar rate from day 2 after transduction, but there were significantly more 4G- than 2G-CAR-T cells on days 5 and 7 (Fig. 5 G). Finally, we observed higher viability of 4G-CAR-T cells over time (Fig. 5 H).

Thus, with our optimized protocol, we achieved a high rate of T cell transduction with retrozithromax coexpressing a CAR and mIL-15, and in the absence of exogenous cytokines, these 4G-CAR-T cells exhibit a less differentiated and inhibitory phenotype as well as enhanced expansion and viability in vitro. We next sought to evaluate the expansion of 4G- versus 2G-CAR-T cells in the presence of exogenous hIL-7/IL-15. We observed continuous expansion of 4G- and 2G-CAR-T cells for 2 wk but at a significantly higher rate for the 4G-CAR-T cells (Fig. 6 A).

Viability was similarly high for both over a 10-d period (Fig. 6 B). Notably, 4G-CAR-T cells cultured in hIL-2 demonstrated enhanced expansion at days 5 and 9 as compared with similarly cultured 2G-CAR-T cells (Fig. 6 C).

We subsequently sought to determine if increasing hIL-15 levels in the medium could augment 2G-CAR-T cell expansion. We demonstrated that 2G-CAR-T cells cultured in the presence of increasing concentrations of hIL-15 (while maintaining hIL-7 at 10 ng/ml) achieved significant increases in fold expansion, reaching or surpassing that of 4G-CAR-T cells (cultured in standard 10 ng/ml hIL-15) at day 9 after transduction in the presence of 50 ng/ml or 100 ng/ml hIL-15, respectively (Fig. 6 D and Fig. S3 D).

Notably, increasing the concentration of hIL-15 in the culture medium from 10 to 50 or 100 ng/ml significantly increased the expansion of 4G-CAR-T cells (Fig. 6 E), and the fold expansion of 4G-CAR-T cells was nearly double compared to that of 2G-CAR-T cells (cultured in equivalent increased hIL-15 concentrations) on day 9 after transduction (Fig. 6 E and Fig. S3 D).

We next tested the effector capacity of 4G- as compared with 2G-CAR-T cells against target cells. Significantly higher levels of IL-2 were produced by 4G- than 2G-CAR-T cells upon co-culture with VEGFR-2+ bEnd3 cells at 1 wk after transduction, while neither reacted against VEGFR-2− H5V cells (Fig. 6 F). We further observed mIL-15 secretion by 4G-CAR-T cells only upon co-culture with bEnd3 cells and not H5V cells (Fig.

6 G). In addition, there was significantly higher expansion of 4G- than 2G-CAR-T cells at day 4 after co-culture with bEnd3 cells, and neither expanded upon co-culture with H5V cells (Fig. 6, H and I). The 4G-CAR-T cells also exhibited significantly higher proliferation (Fig.

6 J) and numbers of dividing CD8+ T cells compared with 2G-CAR- or control T cells at day 4 of the co-culture (Fig. 6, K and L). The ability of 4G- and 2G-CAR-T cells to induce apoptosis of target cells was equivalent (Fig. 6 M, and N), in accordance with previous evaluation of hIL-15-CAR-T cells (Krenciute et al., 2017).

We further tested the 4G- and 2G-CAR-T cells in vivo against subcutaneous B16 melanoma tumors. Briefly, on day 8 after tumor cell injection, with tumors approaching 20–40 mm3 in volume, CD45.2+ C57BL/6 mice were lymphodepleted by sublethal total body irradiation and subsequently received two intravenous T cell injections (8–9 × 106 CD45.1+ cells at each injection. Fig. 7 A).

In mice treated with control T cells, the tumors grew rapidly, while modest tumor control was observed in mice that received 2G-CAR-T cells, similar to previous reports for this tumor vasculature targeting CAR (Chinnasamy et al., 2010, 2012). Mice treated with 4G-CAR-T cells, however, had significantly attenuated tumor growth (Fig. 7 B). Ex vivo analysis of transferred CD45.1+ T cells in the blood, spleen, and tumor on day 11 after ACT revealed significantly higher engraftment of 4G- than 2G-CAR-T cells and control T cells (Fig.

7, C–E). In addition, CAR expression levels were higher for 4G- than 2G-CAR-T cells in blood, spleen, and tumor (Fig. 7, C, D, and F). Notably, we observed sustained presence of the mIL-15 transgene in the spleens and tumors of mice treated with 4G-CAR-T cells (Fig.

7, D and F). Finally, in agreement with our in vitro data, 4G-CAR-T cells expressed significantly higher levels of the antiapoptotic protein Bcl-2 in vivo (Fig. 7 G. Flow cytometry gating strategy shown in Fig.

S4). Thus, mIL-15 coexpression by CAR-T cells enhances not only expansion and in vitro effector functions but also in vivo persistence and tumor control. Finally, we sought to comprehensively evaluate the effect of mIL-15 coexpression on CAR-T cells in vivo and to determine if endogenous immune cells are also impacted. Following the same ACT strategy as demonstrated above (Fig.

8 A), we observed that 4G-CAR-T cells in the spleen (Fig. 8, B and C) and tumor-draining lymph nodes (Fig. S5, A and B) exhibited a higher frequency of Ki67 (cellular marker for proliferation) than 2G-CAR-T cells. In the tumor, despite that Ki67 expression levels were similar for both 4G- and 2G-CAR-T cells (Fig.

8, D and E), the 4G-CAR-T cells displayed significantly lower levels of PD-1 (Fig. 8, F and G). Analysis of endogenous immune infiltrate revealed significantly higher coexpression of CD69 and Ki67 by natural killer (NK) cells in 4G- as compared with 2G-CAR-T cell–treated tumors (Fig. 8, H and I).

In addition, in 4G-CAR-T cell–treated mice there were lower levels of tumor-residing M2 (F4/80+ CD206+) macrophages, which are often associated with immunosuppression in the TME (Fig. 8 J, K). Both the activation of NK cells and lower levels of M2 macrophages may contribute to tumor control in the context of 4G-CAR-T cell transfer. Tumor-residing B cells (CD19+ MHC II+) were not detected (Fig.

S5, C and D), and there were no differences in splenic B cell frequency in any of the treated mice (Fig. S5, E and F). Finally, similar frequencies of tumor-residing dendritic cells (DCs. CD11b− CD11c+) were observed among the control and CAR-T cell–treated mice (Fig.

S5, G and H). The flow cytometry gating strategy for the ex vivo characterization of the different immune cell populations is shown in Fig. S4. Thus, 4G-CAR-T cells coexpressing mIL-15, in addition to conferring enhanced tumor control as compared with 2G-CAR-T cells, also reprogram the TME in favor of protective endogenous immunity.

CAR-T cell therapy has yielded unprecedented clinical responses against some hematological malignancies, but not against epithelial-derived solid tumors (Irving et al., 2017). Rational combinatorial treatments and innovative CAR-T cell coengineering strategies (Lanitis et al., 2020) offer solutions for overcoming obstacles in the solid TME, but these are best evaluated in immunocompetent mice to enable the interplay of the endogenous immune system. In this study, we have presented optimized conditions for murine T cell activation, retroviral transduction, and expansion that allowed us to achieve consistently high and stable transgene expression levels, as well as robust expansion of both 2G- and 4G-CAR-T cells having a predominantly TCM cell phenotype, which is favored for ACT (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005). We have also elucidated the beneficial impact of mIL-15 coexpression by murine CAR-T cells both in vitro and in vivo. Retroviral vectors, most commonly derived from the murine stem cell zithromax (MSCV), a derivative of the Moloney murine leukemia zithromax, have proven to be the most effective approach for stably introducing genes into murine T cells (Kerkar et al., 2011). Lentizithromax, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al., 2004.

Tsurutani et al., 2007). Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al., 2017. Zhang et al., 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al., 2009). Retrozithromax-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al., 2010.

Davila et al., 2013. Fu et al., 2013). Moreover, the long-term stability of CAR expression by murine T cells has not previously been thoroughly evaluated (Kusabuka et al., 2016. Kochenderfer et al., 2010).

Despite that it is common procedure to concentrate lentizithromax via ultracentrifugation, this is usually not performed for CAR-encoding retrozithromaxes. In this study, we demonstrated that retrozithromax can be efficiently concentrated, leading to significantly improved CAR transduction efficiencies. We further observed a correlation between CD8+ T cell activation levels (the highest level was achieved by αCD3/CD28 bead stimulation) and transduction efficiency. Previous studies have presented CAR expression early after transduction (2–3 d.

Tran et al., 2013. Kusabuka et al., 2016. Kochenderfer et al., 2010) and thus cannot distinguish from pseudo-transduction (Case et al., 1999. Costello et al., 2000).

In addition, some studies have applied antibiotic selection for enrichment of CAR-T cells (Kusabuka et al., 2016) or have measured GFP (or other markers) that can overestimate transduction efficiency. Here, we have demonstrated robust, long-term CAR expression in murine T cells by staining with recombinant target antigen and in the absence of any selection/enrichment method. In this study, we have also shown the utility of the common γ-chain cytokines hIL-7/IL-15 for enhanced CAR-T cell expansion and survival, as well as for promoting a TCM cell phenotype and ameliorating effector function. Others have reported superior tumor control by IL-7/IL-15 than IL-2–expanded T cells (Cha et al., 2010.

Gattinoni et al., 2005. Mueller et al., 2008). It has also been previously demonstrated that exposure of murine T cells to IL-2 can potentiate apoptosis by suppressing the inhibitor of Fas signaling, FLIP (FLICE-inhibitory protein), and enhancing the expression of the proapoptotic molecule Fas ligand (Lenardo, 1991. Refaeli et al., 1998).

In contrast, IL-7 and IL-15 inhibit activation-induced cell death, support the proliferation and survival of T cells (Waldmann, 2015. Jiang et al., 2004. Cha et al., 2010), promote a TCM cell phenotype characterized by longer telomeres, and elevate T cell persistence and antitumor efficacy (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005. Klebanoff et al., 2004. Le et al., 2009). Similarly, it has been shown that IL-7 and IL-15 enable enhanced human CAR-T cell effector function upon antigen recognition (Xu et al., 2014.

Zhou et al., 2019) and that exogenous IL-15 can expand anti-CD19 CAR-T cells in treated patients by up to 180-fold (Ramanayake et al., 2015). Contradictory reports of lower murine T cell function in vitro following culture in IL-7/IL-15 versus IL-2 alone are presumably due to the method of T cell stimulation used, differences in the concentration of IL-2 used, and the duration of expansion (Cha et al., 2010. Gattinoni et al., 2005. Mueller et al., 2008).

We further showed that our methodologies enable the efficient coexpression of mIL-15 and a CAR (encoded by a bicistronic vector) in murine T cells. Human CAR-T cells coexpressing hIL-15 as a fusion protein tethered to the cell surface, or in a secreted form, have previously demonstrated enhanced expansion and persistence upon antigen stimulation (both in vitro and in vivo), as well as increased tumor control (Hoyos et al., 2010. Markley and Sadelain, 2010). As such, there are high expectations for clinical efficacy of IL-15–CAR-T cells.

In nonactivated murine 4G-CAR-T cells, we observed very low levels of mIL-15 in the culture supernatant, but upon antigenic stimulation, significantly higher amounts were detected, in line with reports for hIL-15 CAR-T cells (Krenciute et al., 2017. Hoyos et al., 2010). Elevated levels of pSTAT5 in the 4G- versus 2G-CAR-T cells indicated active signaling by cytokine/receptor engagement. The functional integrity of the coexpressed mIL-15 was further supported by enhanced 4G-CAR-T cell proliferation and survival, possibly due to up-regulation of the antiapoptotic molecule Bcl-2 (Wu et al., 2002.

Shenoy et al., 2014). In addition, mIL-15 coexpression promoted a TCM cell phenotype, limited PD-1 up-regulation, and conferred superior effector function upon antigenic challenge. The culture methods presented herein comprising hIL-7/hIL-15 in the medium permitted efficient murine CAR-T cell expansion, which was significantly reinforced upon mIL-15 coexpression by CAR-T cells. This enabled us to further investigate the efficacy of 4G-CAR-T cells in vivo against B16 melanoma tumors.

We observed higher tumor control and persistence of 4G- as compared with the 2G-CAR-T cells and sustained expression of the mIL-15 transgene. Moreover 4G-CAR-T cells exhibited higher Bcl-2 levels, in line with our in vitro data, suggesting that mIL-15 can render CAR-T cells more resistant to apoptosis in vivo. The coexpression of mIL-15 was also associated with significantly lower up-regulation of PD-1, an inhibitory receptor that can impair T cell function in the TME (Ahmadzadeh et al., 2009). Finally, evaluation of endogenous tumor immune infiltrate revealed a significantly higher frequency of activated (CD69+ Ki67+) NK cells and fewer M2 (F4/80+ CD206+) macrophages upon 4G- versus 2G-CAR-T cell transfer.

As NK cells are associated with delayed melanoma tumor growth (Nath et al., 2019), and M2 macrophages have been shown to contribute to tumor progression and metastasis (Poh and Ernst, 2018), the observed TME remodeling upon 4G-CAR-T cell transfer is favorable for tumor control. Our findings are consistent with prior studies. For example, coadministration of IL-15 with tumor-directed monoclonal antibodies enhanced Ab-dependent cellular cytotoxicity by augmenting both NK cell and macrophage activation (Zhang et al., 2018). In another study, it was shown that the absence of IL-15 in immunocompetent mice promotes the formation of M2 macrophages (Gillgrass et al., 2014).

In summary, we have presented comprehensive and highly reproducible methods for efficient retroviral transduction and robust expansion of murine CAR-T cells endowed with favorable properties for ACT studies in immunocompetent mice. We further demonstrated that coexpression of mIL-15 directly promotes CAR-T cell fitness and function and remodels the TME to favor tumor control. As it is becoming apparent that endogenous immunity can play a critical role in either suppressing or supporting CAR-T cell function in the TME (Kuhn et al., 2019), comprehensive studies in immunocompetent mice are critical for accelerating the translation of effective CAR therapies to the clinic. The murine brain endothelioma cell line bEnd3, the murine immortalized heart endothelial cell line H5V, and the murine leukemia cell line C1498 were cultured in DMEM-GlutaMAX comprising 4,500 mg/liter glucose and 110 mg/liter sodium pyruvate and supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate.

The melanoma cell line B16-F10 was grown as a monolayer in DMEM-GlutaMAX supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin sulfate. Cells were passaged twice weekly to maintain them under exponential growth conditions and were routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Primary murine T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 10 mM nonessential amino acids (referred to as murine T cell culture medium).

Murine T cell culture medium was further supplemented with human cytokines as described in the method for T cell expansion. The retroviral vector pMSGV (murine stem cell zithromax [MSCV]–based splice-gag vector) comprising the MSCV LTR was used as the backbone for all CAR constructs. A 2G-CAR consisting of the anti-VEGFR-2 scFv, DC101, the CD8α hinge (H), and TM region, followed by the EDs of CD28 and CD3ζ (DC101-28-z), was kindly provided by Dr. Steven A.

Rosenberg (National Cancer Institute, Bethesda, MD. Chinnasamy et al., 2010). The DC101-28-z CAR was built by PCR amplification of a 362-bp fragment from the 2G construct with the primers. 5′-ACG​CGC​GGC​CGC​AAC​TAC​TAC​CAA​GC-3′ and 5′-ACG​CGT​CGA​CGG​GGC​GGT​ACG​CTG​CAA​AGT​CTC-3′ followed by NotI and SalI digestion of both the PCR product and the parental 2G vector, gel purification, and ligation.

To generate the 4G-CAR construct encoding both mIL-15 and the VEGFR-2–directed CAR (mIL-15-T2A-DC101-28-z), a gene-string encoding the murine Igκ leader sequence followed by codon-optimized mIL-15 and T2A, flanked by XhoI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, was synthesized. The DC101-28-z construct and fragment were then digested (XhoI and EcoRI), gel purified, and ligated together. All genes strings were synthesized by GeneArt AG, and all constructs were fully sequenced by Microsynth AG. High-titer, replication-defective retrozithromax was produced and concentrated as depicted in Fig.

1. Briefly, Phoenix Eco cells were seeded at 107 per T-150 tissue culture flask in 35 ml culture medium (Fig. 1 A, 1) 24 h before transfection with 14.4 µg pCL-Eco Retrozithromax Packaging Vector and 21.4 µg pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific. Fig.

1 A, 2). All plasmids were purified using HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Thermo Fisher Scientific). For the transfection mixture, a 3:1 ratio of Turbofect/plasmid was prepared in 2 ml Opti-MEM and incubated for 30 min at room temperature (RT. Fig.

1 A, 2). Medium was then removed from T-150 flasks bearing 80–90% confluent Phoenix Eco cells and the transfection mixture was applied and incubated for 1 min, followed by addition of 31 ml fresh medium (Fig. 1 A, 2). The viral supernatant was discarded 20–24 h after transfection and replaced with 33 ml fresh medium (Fig.

1 A, 3). At 48 (Fig. 1 A, 4) and 72 h (Fig. 1 A, 5) after transfection, the supernatant was harvested, and viral particles were concentrated by ultracentrifugation for 2 h at 24,000 g at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 0.4 ml murine T cell medium.

The retrozithromax was then used immediately, or aliquoted, frozen on dry ice, and stored at −80°C. As depicted in Fig. 1 B, murine T cells were isolated from single-cell suspensions of dissociated spleens from CD45.1+ congenic C57BL/6 mice bred in-house at the animal facility of the University of Lausanne (UNIL. Epalinges, Switzerland) using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies.

Fig. 1 B, 1.1). T cells were plated at 106/ml in 24- or 48-well plates in T cell medium (described above) and stimulated with αCD3/CD28 Ab-coated beads (Invitrogen) at a bead to cell ratio of 2:1 and 50 IU/ml hIL-2 (Glaxo. Fig.

1 B, 1.1). Non–treated cell-culture grade 48- or 24-well plates (Corning Falcon) were precoated with 0.25 ml or 0.5 ml, respectively, of recombinant RetroNectin (Takara Bio) at a final concentration of 20 μg/ml, overnight (O/N) at 4°C (Fig. 1 B, 1.2). 1 d after T cell activation, the retronectin-precoated plates were washed with PBS, blocked with 2% BSA in PBS for 30 min at RT (Fig.

1 B, 2.1). Subsequently, plates were washed once, retrozithromax was added at the MOI indicated in the figures, and plates were then spun at 2,000 g for 1.5 h at 32°C (Fig. 1 B, 2.2). The supernatants were then aspirated, and 0.5 to 106 of 24 h activated T cells were transferred to each coated well (48- or 24-well plates.

Fig. 1 B, 2.3). The plates were centrifuged for 10 min at 300 g and incubated O/N (Fig. 1 B, 2.3).

In some experiments the transduction procedure was performed at 48 h, or at both 24 and 48 h after activation. The cultures were maintained at a cell density of 0.5 to 106 cells/ml and replenished with fresh T cell medium (supplemented with hIL-2 alone or hIL-2 followed by hIL-7/IL-15 on day 2 after transduction) every other day (Fig. 1 B, 3). At day 7, CAR surface expression was assessed by flow cytometric analysis (as described below), and the rested engineered T cells were adjusted for equal expression before functional in vitro and in vivo assays (Fig.

1 B, 4). Murine C1498 leukemia cells were transduced as described above for primary murine T cells, except that they were not activated and were maintained afterwards in DMEM-GlutaMAX complete medium at a cell density of 3 × 105 viable cells/ml. For flow cytometric analysis, cells were surface stained using antibodies against CD3ε (145-2C11), CD4 (GK1.5, RM4-5), CD8α (53–6.7), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45 (30F/11), CD62L (MEL-14), CD69 (H1-2F3), IL-15-Rα (6B4C88), PD-1 (29F.1A12), Ly-6G (1A8), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD206 (C068C2), NK-1.1 (PK136), CD19 (6D5), and MHC class II (M5/114.15.2). Abs were purchased from eBioscience and BioLegend or produced in-house from hybridomas by the flow cytometry platform.

DC101-CAR expression by retrovirally transduced T cells was detected by incubation with soluble mouse VEGFR-2–hIgG-Fc fusion protein (R&D Systems) followed by staining with labeled goat anti-hIgG Fc (clone HP6017. Biolegend). Thy1.1-T cells were stained in parallel as a negative control. VEGFR-2 expression by mouse endothelial cell lines was evaluated by cell-surface staining with rat anti-VEGFR-2 Ab (clone Avas12.

BioLegend) and matched isotype control (Rat IgG2a κ isotype. Clone RTK2758. BioLegend). For detection of phosphorylated STAT5, cells were fixed with BD Cytofix Fixation Buffer at 4°C for 15 min and permeabilized with BD Phosflow Perm Buffer III for 30 min at 4°C.

Intracellular phospho-staining was performed for 1 h at RT in the dark with Ab against phospho-STAT5 (Tyr694. D47E7 XP Rabbit mAb 4322. Cell Signaling). For intracellular staining of mIL-15 (clone AIO.3.

EBioscience), Bcl-2 (clone 10C4. EBioscience), and Ki67 (clone SolA15. EBioscience), T cells were fixed and then permeabilized using the FoxP3 transcription factor staining buffer set (eBioscience) according to the manufacturer’s recommendations. For the detection of mIL-15, the cells were further washed and incubated for 30 min with anti-rat IgG2a.

To discriminate dead cells, 7-AAD (BioLegend) staining was performed. Live/dead fixable Aqua Dead cell staining was used to exclude dead cells in the ex vivo analysis of immune cells derived from the spleens, tumors, and tumor-draining lymph nodes according to the manufacturer’s instructions (Molecular Probes, Life Technologies). Data were acquired with a BD flow cytometer and analyzed using FlowJo software (Tree Star). Cells extracted from dissociated tumors were lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific).

Total RNA was isolated using the RNeasy Mini Kit (Qiagen). After treatment with RNase-free DNase I (Qiagen), 400 ng of total RNA was reverse transcribed using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), as indicated by the manufacturer. Quantitative real-time PCR was performed according to the commercial protocol using SYBR Green Fast PCR Master Mix (Thermo Fisher Scientific) and the 7500 Fast Real-Time PCR System (Applied Biosystems). Primers to specifically amplify regions of the DC101 scFv of the CAR cassette, or the mIL-15 transgene, were designed using the GenScript website and are as follows.

DC101 forward, 5′-GCA​ACC​CAA​ACT​CCT​CAT​CT-3′. DC101 reverse, 5′-TAT​CAT​CAG​CCT​CCA​CAG​GA-3′. IL-15 forward, 5′-CCA​GGA​TCT​ACA​GGC​GAC​AA-3′. IL-15 reverse, 5′-ATG​CTC​TGG​ATC​AGG​CTC​TC-3′.

PCR amplification of the housekeeping gene GAPDH was performed as a control, and to allow normalization of samples. The following primers were used for GAPDH. GAPDH forward, 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′. GAPDH reverse, 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′.

Each sample was run in triplicate, and each experiment included three nontemplate control wells. The relative mRNA levels (fold change) of each transgene among the different samples were quantified using the comparative 2−ΔΔCt method. We wish to thank members of the Flow Cytometry Platform and the Animal Care Facility of UNIL for their excellent support. We also kindly thank Dr.

Steven A. Rosenberg (National Cancer Institute, Bethesda, MD) for sharing a second generation anti-VEGFR-2 CAR construct comprising the scFv DC101. This work was generously supported by Ludwig Cancer Research, the European Research Council (advanced grant 1400206AdG-322875 to G. Coukos), and the Biltema Foundation.

P. Romero is supported in part by Oncosuisse (grant KFS-4404-02-2018). Author contributions. M.

Irving, G. Coukos, and E. Lanitis conceived, designed, developed, and supervised the study and wrote the manuscript. E.

Lanitis, G. Rota, P. Kosti, C. Ronet, and A.

Spill conducted experiments and acquired and analyzed data. A. Spill supported the in vivo and ex vivo studies. B.

Seijo built essential constructs. P. Romero and D. Dangaj reviewed the data and manuscript and provided suggestions.

All authors read and approved the manuscript.Christopher Mapperley Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review &. Editing 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK2Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for other works by this author on:.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate buy zithromax in usa so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we buy zithromax in usa describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype.

We present a bicistronic retroviral vector encoding both a tumor vasculature–targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching buy zithromax in usa the clinic.

The adoptive cell transfer (ACT) of ex vivo–expanded T lymphocytes has yielded robust and durable clinical responses against several cancer-types, such as tumor-infiltrating lymphocyte therapy of advanced melanoma (Mardiana et al., 2019). Another approach to ACT involves the redirection of peripheral blood T buy zithromax in usa cells to tumor antigens by engineering them to express a chimeric antigen receptor (CAR) that triggers cellular activation upon tumor antigen binding. CAR-T cell therapy against hematologic malignancies, by targeting the B cell lineage antigens CD19 or the B cell maturation antigen, has proven efficacious in the clinic, and there is optimism that similar success will be achieved for some solid tumors (Geyer and Brentjens, 2016.

Irving et al., 2017). A range of physical (Lanitis et al., 2015) and immunometabolic barriers that can prevent T cell homing, transendothelial migration across tumor blood vessels, engraftment/persistence, and effector function limit the potency of CAR-T buy zithromax in usa cell therapy against solid tumors (Brown et al., 2016. Louis et al., 2011).

Moreover, chronic antigen exposure and a lack of sufficient costimulation in the tumor microenvironment (TME) can cause CAR-T cell exhaustion (Irving et al., 2017). Coengineering of CAR-T cells may help buy zithromax in usa to overcome some of these obstacles (Lanitis et al., 2020). Genetic modifications, for example, can be made to enable better homing and tumor penetration or render CAR-T cells resistant to suppressive mechanisms in the TME (Stromnes et al., 2010).

In addition, CAR-T cells buy zithromax in usa can be armed with secretory molecules or additional receptors to support CAR-T cell activity and/or harness endogenous immunity (Adachi et al., 2018. Pegram et al., 2012). Preclinical evaluation of CAR-T cells has, for the most part, been performed with xenograft tumor models in immunodeficient mice (Lee et al., 2011.

Mardiros et buy zithromax in usa al., 2013. Lanitis et al., 2012). Although this approach can be used to evaluate human CAR-T cell persistence, homing, tumor control, and survival following ACT, critical parameters, including potential toxicity against normal tissues (Tran et al., 2013), and the impact of endogenous immunity on both tumor control and escape are not addressed in such models (Spear et al., 2012.

Avanzi et al., buy zithromax in usa 2018). As varying obstacles must be overcome to enhance CAR-T cell responses against different solid tumor types, comprehensive studies in immunocompetent syngeneic tumor models would enable more accurate screening of T cell engineering strategies and provide important insights into improving coengineering and combinatorial treatment approaches (Lanitis et al., 2020). A key limitation of CAR evaluation in syngeneic models stems from inadequate methodologies for efficient murine T cell transduction and buy zithromax in usa expansion.

Indeed, unless T cells derived from multiple donor spleens are transduced or the engineered T cells are restimulated for further expansion, which among other drawbacks are costly and can promote exhaustion and apoptosis (Bucks et al., 2009), respectively, current protocols yield insufficient numbers of CAR-T cells for ACT studies (Lee et al., 2009). The efficiency of cell-surface expression of second-generation (2G) CARs, comprising the endodomain (ED) of CD3ζ and one costimulatory ED (e.g., CD28 or 4-1BB), generally reaches 40–60% (Kochenderfer et al., 2010. Davila et al., 2013 buy zithromax in usa.

Wang et al., 2014. Fu et al., 2013). Although retroviral transduction rates as high as 70–80% for murine T cells have been reported, buy zithromax in usa this was assessed at 2 to 3 d after transduction (Tran et al., 2013.

Kuhn et al., 2019. Kusabuka et al., 2016) and thus may include false positives due to transient expression from nonintegrated vector DNA (i.e., pseudo-transduction buy zithromax in usa. Case et al., 1999, Costello et al., 2000).

Moreover, short-term transduction efficiency is often based on reporter genes like GFP, which may overestimate CAR expression levels (Kusabuka et al., 2016. Kuhn et al., 2019 buy zithromax in usa. Davila et al., 2013).

Finally, while stable retroviral packaging and producer cell lines may buy zithromax in usa enable transduction efficiencies for 2G and third-generation (3G. I.e., a CAR having two or more costimulatory EDs) CARs of >60% (Fu et al., 2013), this is a laborious approach if multiple CAR designs are to be compared (Chinnasamy et al., 2010). Here, we report the development of an efficient and highly reproducible protocol for primary murine T cell retroviral transduction and expansion, yielding functional murine 2G-CAR-T cells, as well as fourth-generation (4G)-CAR-T cells coengineered to express murine IL-15 (mIL-15) for enhanced in vitro and in vivo function and TME reprogramming.

Overall, our work buy zithromax in usa provides important tools for enabling the systematic evaluation of 4G-CAR-T cells in immunocompetent, syngeneic tumor-bearing mice, which we believe is critical for effective therapies reaching the clinic. We sought to optimize murine T cell activation, transduction, and expansion methods for preclinical CAR therapy evaluation in immunocompetent, syngeneic tumor-bearing mice. The final protocol we developed is summarized in Fig.

1 and is described buy zithromax in usa in detail in Materials and methods. We used a 2G-CAR targeting vascular endothelial cell growth factor receptor 2 (VEGFR-2), comprising the well-characterized single-chain variable fragment (scFv) DC101 (Chinnasamy et al., 2010), a CD8α hinge and transmembrane domain, and the murine EDs of CD28 and CD3ζ. The anti-VEGFR-2 buy zithromax in usa CAR retroviral vector is abbreviated as DC101-28z (Fig.

2 A). Because retrozithromaxes infect proliferating cells (Kusabuka et al., 2016. Chinnasamy et buy zithromax in usa al., 2010.

Hu et al., 2017), we first compared three commonly used methods for inducing T cell activation. (i) magnetic beads coated with anti-(α) CD3 antibody (Ab) and αCD28 Ab (αCD3/CD28 beads) plus recombinant human IL-2 (hIL-2), (ii) plate-immobilized αCD3 Ab along with soluble αCD28 Ab (αCD3-plate/CD28) plus hIL-2, and (iii) Concanavalin A plus hIL-2 and hIL-7. Stimulation with αCD3/CD28 beads consistently resulted in the highest buy zithromax in usa frequency of CD44+ CD62L− (recently activated, memory), CD25+ or CD69+ (activated), and Ki67+ (proliferating) CD3+ T cells (Fig.

2 B and Fig. S1 A) buy zithromax in usa. We next found that concentration of viral particles through ultracentrifugation yielded higher viral titers (>3 × 107 transducing units/ml.

Fig. 2 C) buy zithromax in usa and enabled significantly higher transduction of primary activated primary murine T cells as compared with unconcentrated retrozithromax (Fig. 2 D), reaching a plateau at a multiplicity of (MOI) of 5 (∼80% CAR expression.

Fig. 2 E). A single transduction at 24 h after activation versus transduction at both 24 and 48 h did not affect the efficiency in terms of either percentage of cells transduced or CAR expression level per cell (i.e., mean fluorescence intensity [MFI].

Fig. 2, E and F). We observed, however, that the transduction efficiency at 48 h after activation was inferior to that obtained at 24 h after activation (Fig.

2, E and F). A schema of the T cell activation and transduction approaches compared are depicted in Fig. 2 G.

Finally, we observed highest CAR transduction efficiency in CD3+ lymphocytes activated with αCD3/CD28 beads in the presence of hIL-2 as compared with the other aforementioned activation methods (Fig. 2, H and I). Similar results were observed for CD8+ T cells, while for CD4+ T cells, the percentage CAR expression was the same for both αCD3/CD28-bead and αCD3-plate/CD28 activation (Fig.

S1 B). Thus, αCD3/CD28-bead activation was used for all further experiments. Notably, we also investigated concentrated lentiviral transduction of αCD3/CD28-bead–activated murine T cells using the same anti-VEGFR-2 CAR, and consistent with another study (Kerkar et al., 2011), we obtained very low transduction efficiency (∼10%, data not shown).

While long-term T cell culture in IL-2 drives terminal differentiation, the common γ-chain cytokines IL-7 and IL-15 have been reported to promote a central memory T cell (TCM cell) phenotype enabling superior persistence and in vivo tumor control upon ACT (Klebanoff et al., 2005). Thus, we next compared the expansion and functional properties of transduced murine CAR-T cells cultured in hIL-2 alone versus hIL-2 for the first 3 days, followed by hIL-7/IL-15 for the remainder of the culture period (Fig. 3 A).

Both hIL-7 and hIL-15 have been previously demonstrated to act on murine T cells to promote homeostatic proliferation and survival (Eisenman et al., 2002. Nanjappa et al., 2008). As for hIL-2–expanded CAR-T cells (Fig.

2 G), we observed that a single transduction of T cells at 24 h and subsequent expansion in hIL-7/IL-15 was sufficient to achieve a robust and stable transduction efficiency at a MOI as low as 5 (Fig. 3 B). Both culture conditions (hIL-2 alone versus hIL-2 followed by hIL-7/IL-15) enabled high CAR expression on day 7 (Fig.

3 C). On day 9, however, we observed a 26-fold expansion of CAR-T cells exposed to hIL-7/IL-15 as compared with a 9-fold expansion in the presence of hIL-2 alone at a standard concentration of 50 IU/ml (Fig. 3 D).

Moreover, CAR-T cells cultured with hIL-7/IL-15 continued to expand for at least 14 d, while T cells cultured in hIL-2 alone reached a plateau after 1 wk (Fig. 3 D) and exhibited significantly higher levels of cell death starting early in the culture (Fig. 3 E).

We also observed a significantly higher frequency of CD8+ T cells in the hIL-7/IL-15 culture (Fig. 3 F). Finally, transduced T cells expanded with hIL-7/IL-15 had a significantly higher proportion of TCM cells based on cell-surface expression of the hyaluronic acid receptor CD44 and the L-selectin CD62L from day 5 after cytokine addition (Fig.

3, G and H). We sought to evaluate the in vitro reactivity of hIL-2 only versus hIL-7/IL-15 expanded CAR-T cells against target antigen. On day 7 after transduction, we co-cultured CAR-T cells with bEnd3 murine endothelial cells expressing VEGFR-2, as well as with control VEGFR-2− H5V murine endothelial cells (Fig.

3 I). HIL-7/IL-15 expanded CAR-T cells secreted significantly higher levels of IFN-γ, granzyme B, and IL-2 (Fig. 3 J) after bEnd3 target cell recognition in vitro.

Because CAR-T cell expansion with hIL-7/IL-15 results in a higher frequency of CD8+ T cells as compared with hIL-2 only, we next sorted CD8+ T cells on day 7 after transduction and performed a co-culture with bEnd3 and H5V cells. Higher levels of granzyme B, IL-2, and IFN-γ were secreted by hIL-7/IL-15–expanded CD8+ CAR-T cells than hIL-2–expanded ones (Fig. S2).

Moreover, hIL-7/IL-15–expanded CAR-T cells exhibited significantly higher persistence (Fig. 3 K), division rates (Fig. 3 L), and numbers of proliferating CD8+ T cells after 4 d of co-culture (Fig.

3 M). Thus, as compared with hIL-2 alone, CAR-T cell expansion with hIL-7/IL-15 promotes higher viability and favors a TCM cell phenotype, more robust expansion, and superior secretion of cytokines and long-term proliferative capacity upon challenge with target cells. The high transduction efficiency achieved with our optimized method encouraged us to evaluate the coexpression of transgenes and test the impact of additional cargo on CAR-T cell performance.

Given the enhanced functional properties of CAR-T cells exposed to hIL-7/IL-15 at 48 h after transduction as opposed to hIL-2 alone, we focused on coengineering T cells to constitutively produce mIL-15. Notably, hIL-15 has been previously demonstrated to significantly improve the antitumor activity of human CAR-T cells targeting glioblastoma (Krenciute et al., 2017). A bicistronic retroviral vector encoding mIL-15 and the DC101 CAR, both driven by the 5′ LTR of the retrozithromax (de Felipe et al., 1999) and separated by a self-cleaving 2A peptide sequence (T2A.

Liu et al., 2017), was built to express this 4G-CAR construct (Fig. 4 A). With a single round of transduction at a MOI as low as 5, we achieved a similarly high expression of the 4G- as the 2G-CAR (Fig.

4, B and C), as well as high intracellular expression of mIL-15 (Fig. 4 D). Significant mIL-15 was also detected by ELISA upon lysis of 4G-CAR-T cells (Fig.

4 E), but very low levels of mIL-15 were found in the culture supernatant (data not shown), presumably due to sequestration of the cytokine by cell-surface IL-15 receptor-α (IL-15-Rα), as has been previously observed for human T cells engineered to secrete hIL-15 (Markley and Sadelain, 2010). Our hypothesis was supported by the fact that we detected high levels of soluble mIL-15 in the supernatants of transfected human Phoenix Eco cells (i.e., the retrozithromax producer cell line. Fig.

4 F). Moreover, 4G-CAR–transduced C1498 leukemia cells (which do not express IL-15-Rα. Fig.

S3 A) secreted high levels of mIL-15 (Fig. 4, G and H). Finally, we activated both 2G- and 4G-CAR-T cells with cognate antigen and found significant secretion of mIL-15 by the 4G-CAR-T cells (Fig.

4 I), as has similarly been reported in the context of engineered human T cells (Krenciute et al., 2017). We next sought to investigate the impact of mIL-15 coexpression on CAR-T cell signaling and phenotype. In the absence of exogenous cytokine in the culture supernatant, we observed elevated pSTAT5 in the 4G- versus 2G-CAR-T cells both in terms of frequency and level per cell (Fig.

4, J and K). We further evaluated IL-15-Rα expression and detected lower levels on 4G-CAR-T cells (Fig. 4, L and M), presumably due to receptor internalization (Dubois et al., 2002) and/or mIL-15 occupancy blocking the Ab binding site.

Subsequently, we assessed expression of the antiapoptotic protein Bcl-2, previously reported to enhance 2G- versus first-generation (1G)–CAR-T cell persistence (Song et al., 2012), and found higher expression levels on days 2 and 5 after transduction for 4G- as compared with 2G-CAR-T cells in the absence of exogenous cytokines (Fig. S3, B and C). In addition, we observed significantly higher frequencies of Ki67+ Bcl-2+ 4G-CAR-T cells on days 2 and 5 after transduction (Fig.

5, A and B). Thus, mIL-15 coexpression appears to augment both CAR-T cell survival and proliferation. We further assessed the phenotype of CAR-T cells in the absence of exogenous cytokines in the culture medium and found that on day 2 following transduction, 2G- and 4G-CAR-T cells displayed no differences in the proportion of naive (CD62Lhigh CD44low), central memory (CM.

CD62Lhigh CD44high) and effector memory (EM. CD62Llow CD44high) T cell phenotype populations. However, by day 5 after transduction, 4G-CAR-T cells had a higher proportion of naive and CM cells and fewer EM cells, as compared with 2G-CAR-T cells (Fig.

5, C and D). Notably, there were significantly lower levels of the inhibitory receptor programmed cell death 1 (PD-1. Both percentage and MFI) on 4G- compared with 2G-CAR-T cells (Fig.

5, E and F). Consistent with the above findings, we observed that in the absence of exogenous cytokine the 4G-CAR-T cells exhibited increased expansion during the first 2 d after transduction as compared with the 2G-CAR-T cells (Fig. 5 G).

Both 2G- and 4G-CAR-T cells began to contract at a similar rate from day 2 after transduction, but there were significantly more 4G- than 2G-CAR-T cells on days 5 and 7 (Fig. 5 G). Finally, we observed higher viability of 4G-CAR-T cells over time (Fig.

5 H). Thus, with our optimized protocol, we achieved a high rate of T cell transduction with retrozithromax coexpressing a CAR and mIL-15, and in the absence of exogenous cytokines, these 4G-CAR-T cells exhibit a less differentiated and inhibitory phenotype as well as enhanced expansion and viability in vitro. We next sought to evaluate the expansion of 4G- versus 2G-CAR-T cells in the presence of exogenous hIL-7/IL-15.

We observed continuous expansion of 4G- and 2G-CAR-T cells for 2 wk but at a significantly higher rate for the 4G-CAR-T cells (Fig. 6 A). Viability was similarly high for both over a 10-d period (Fig.

6 B). Notably, 4G-CAR-T cells cultured in hIL-2 demonstrated enhanced expansion at days 5 and 9 as compared with similarly cultured 2G-CAR-T cells (Fig. 6 C).

We subsequently sought to determine if increasing hIL-15 levels in the medium could augment 2G-CAR-T cell expansion. We demonstrated that 2G-CAR-T cells cultured in the presence of increasing concentrations of hIL-15 (while maintaining hIL-7 at 10 ng/ml) achieved significant increases in fold expansion, reaching or surpassing that of 4G-CAR-T cells (cultured in standard 10 ng/ml hIL-15) at day 9 after transduction in the presence of 50 ng/ml or 100 ng/ml hIL-15, respectively (Fig. 6 D and Fig.

S3 D). Notably, increasing the concentration of hIL-15 in the culture medium from 10 to 50 or 100 ng/ml significantly increased the expansion of 4G-CAR-T cells (Fig. 6 E), and the fold expansion of 4G-CAR-T cells was nearly double compared to that of 2G-CAR-T cells (cultured in equivalent increased hIL-15 concentrations) on day 9 after transduction (Fig.

6 E and Fig. S3 D). We next tested the effector capacity of 4G- as compared with 2G-CAR-T cells against target cells.

Significantly higher levels of IL-2 were produced by 4G- than 2G-CAR-T cells upon co-culture with VEGFR-2+ bEnd3 cells at 1 wk after transduction, while neither reacted against VEGFR-2− H5V cells (Fig. 6 F). We further observed mIL-15 secretion by 4G-CAR-T cells only upon co-culture with bEnd3 cells and not H5V cells (Fig.

6 G). In addition, there was significantly higher expansion of 4G- than 2G-CAR-T cells at day 4 after co-culture with bEnd3 cells, and neither expanded upon co-culture with H5V cells (Fig. 6, H and I).

The 4G-CAR-T cells also exhibited significantly higher proliferation (Fig. 6 J) and numbers of dividing CD8+ T cells compared with 2G-CAR- or control T cells at day 4 of the co-culture (Fig. 6, K and L).

The ability of 4G- and 2G-CAR-T cells to induce apoptosis of target cells was equivalent (Fig. 6 M, and N), in accordance with previous evaluation of hIL-15-CAR-T cells (Krenciute et al., 2017). We further tested the 4G- and 2G-CAR-T cells in vivo against subcutaneous B16 melanoma tumors.

Briefly, on day 8 after tumor cell injection, with tumors approaching 20–40 mm3 in volume, CD45.2+ C57BL/6 mice were lymphodepleted by sublethal total body irradiation and subsequently received two intravenous T cell injections (8–9 × 106 CD45.1+ cells at each injection. Fig. 7 A).

In mice treated with control T cells, the tumors grew rapidly, while modest tumor control was observed in mice that received 2G-CAR-T cells, similar to previous reports for this tumor vasculature targeting CAR (Chinnasamy et al., 2010, 2012). Mice treated with 4G-CAR-T cells, however, had significantly attenuated tumor growth (Fig. 7 B).

Ex vivo analysis of transferred CD45.1+ T cells in the blood, spleen, and tumor on day 11 after ACT revealed significantly higher engraftment of 4G- than 2G-CAR-T cells and control T cells (Fig. 7, C–E). In addition, CAR expression levels were higher for 4G- than 2G-CAR-T cells in blood, spleen, and tumor (Fig.

7, C, D, and F). Notably, we observed sustained presence of the mIL-15 transgene in the spleens and tumors of mice treated with 4G-CAR-T cells (Fig. 7, D and F).

Finally, in agreement with our in vitro data, 4G-CAR-T cells expressed significantly higher levels of the antiapoptotic protein Bcl-2 in vivo (Fig. 7 G. Flow cytometry gating strategy shown in Fig.

S4). Thus, mIL-15 coexpression by CAR-T cells enhances not only expansion and in vitro effector functions but also in vivo persistence and tumor control. Finally, we sought to comprehensively evaluate the effect of mIL-15 coexpression on CAR-T cells in vivo and to determine if endogenous immune cells are also impacted.

Following the same ACT strategy as demonstrated above (Fig. 8 A), we observed that 4G-CAR-T cells in the spleen (Fig. 8, B and C) and tumor-draining lymph nodes (Fig.

S5, A and B) exhibited a higher frequency of Ki67 (cellular marker for proliferation) than 2G-CAR-T cells. In the tumor, despite that Ki67 expression levels were similar for both 4G- and 2G-CAR-T cells (Fig. 8, D and E), the 4G-CAR-T cells displayed significantly lower levels of PD-1 (Fig.

8, F and G). Analysis of endogenous immune infiltrate revealed significantly higher coexpression of CD69 and Ki67 by natural killer (NK) cells in 4G- as compared with 2G-CAR-T cell–treated tumors (Fig. 8, H and I).

In addition, in 4G-CAR-T cell–treated mice there were lower levels of tumor-residing M2 (F4/80+ CD206+) macrophages, which are often associated with immunosuppression in the TME (Fig. 8 J, K). Both the activation of NK cells and lower levels of M2 macrophages may contribute to tumor control in the context of 4G-CAR-T cell transfer.

Tumor-residing B cells (CD19+ MHC II+) were not detected (Fig. S5, C and D), and there were no differences in splenic B cell frequency in any of the treated mice (Fig. S5, E and F).

Finally, similar frequencies of tumor-residing dendritic cells (DCs. CD11b− CD11c+) were observed among the control and CAR-T cell–treated mice (Fig. S5, G and H).

The flow cytometry gating strategy for the ex vivo characterization of the different immune cell populations is shown in Fig. S4. Thus, 4G-CAR-T cells coexpressing mIL-15, in addition to conferring enhanced tumor control as compared with 2G-CAR-T cells, also reprogram the TME in favor of protective endogenous immunity.

CAR-T cell therapy has yielded unprecedented clinical responses against some hematological malignancies, but not against epithelial-derived solid tumors (Irving et al., 2017). Rational combinatorial treatments and innovative CAR-T cell coengineering strategies (Lanitis et al., 2020) offer solutions for overcoming obstacles in the solid TME, but these are best evaluated in immunocompetent mice to enable the interplay of the endogenous immune system. In this study, we have presented optimized conditions for murine T cell activation, retroviral transduction, and expansion that allowed us to achieve consistently high and stable transgene expression levels, as well as robust expansion of both 2G- and 4G-CAR-T cells having a predominantly TCM cell phenotype, which is favored for ACT (Melchionda et al., 2005.

Gattinoni et al., 2005. Zhou et al., 2005). We have also elucidated the beneficial impact of mIL-15 coexpression by murine CAR-T cells both in vitro and in vivo.

Retroviral vectors, most commonly derived from the murine stem cell zithromax (MSCV), a derivative of the Moloney murine leukemia zithromax, have proven to be the most effective approach for stably introducing genes into murine T cells (Kerkar et al., 2011). Lentizithromax, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al., 2004. Tsurutani et al., 2007).

Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al., 2017. Zhang et al., 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al., 2009). Retrozithromax-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al., 2010.

Davila et al., 2013. Fu et al., 2013). Moreover, the long-term stability of CAR expression by murine T cells has not previously been thoroughly evaluated (Kusabuka et al., 2016.

Kochenderfer et al., 2010). Despite that it is common procedure to concentrate lentizithromax via ultracentrifugation, this is usually not performed for CAR-encoding retrozithromaxes. In this study, we demonstrated that retrozithromax can be efficiently concentrated, leading to significantly improved CAR transduction efficiencies.

We further observed a correlation between CD8+ T cell activation levels (the highest level was achieved by αCD3/CD28 bead stimulation) and transduction efficiency. Previous studies have presented CAR expression early after transduction (2–3 d. Tran et al., 2013.

Kusabuka et al., 2016. Kochenderfer et al., 2010) and thus cannot distinguish from pseudo-transduction (Case et al., 1999. Costello et al., 2000).

In addition, some studies have applied antibiotic selection for enrichment of CAR-T cells (Kusabuka et al., 2016) or have measured GFP (or other markers) that can overestimate transduction efficiency. Here, we have demonstrated robust, long-term CAR expression in murine T cells by staining with recombinant target antigen and in the absence of any selection/enrichment method. In this study, we have also shown the utility of the common γ-chain cytokines hIL-7/IL-15 for enhanced CAR-T cell expansion and survival, as well as for promoting a TCM cell phenotype and ameliorating effector function.

Others have reported superior tumor control by IL-7/IL-15 than IL-2–expanded T cells (Cha et al., 2010. Gattinoni et al., 2005. Mueller et al., 2008).

It has also been previously demonstrated that exposure of murine T cells to IL-2 can potentiate apoptosis by suppressing the inhibitor of Fas signaling, FLIP (FLICE-inhibitory protein), and enhancing the expression of the proapoptotic molecule Fas ligand (Lenardo, 1991. Refaeli et al., 1998). In contrast, IL-7 and IL-15 inhibit activation-induced cell death, support the proliferation and survival of T cells (Waldmann, 2015.

Jiang et al., 2004. Cha et al., 2010), promote a TCM cell phenotype characterized by longer telomeres, and elevate T cell persistence and antitumor efficacy (Melchionda et al., 2005. Gattinoni et al., 2005.

Zhou et al., 2005. Klebanoff et al., 2004. Le et al., 2009).

Similarly, it has been shown that IL-7 and IL-15 enable enhanced human CAR-T cell effector function upon antigen recognition (Xu et al., 2014. Zhou et al., 2019) and that exogenous IL-15 can expand anti-CD19 CAR-T cells in treated patients by up to 180-fold (Ramanayake et al., 2015). Contradictory reports of lower murine T cell function in vitro following culture in IL-7/IL-15 versus IL-2 alone are presumably due to the method of T cell stimulation used, differences in the concentration of IL-2 used, and the duration of expansion (Cha et al., 2010.

Gattinoni et al., 2005. Mueller et al., 2008). We further showed that our methodologies enable the efficient coexpression of mIL-15 and a CAR (encoded by a bicistronic vector) in murine T cells.

Human CAR-T cells coexpressing hIL-15 as a fusion protein tethered to the cell surface, or in a secreted form, have previously demonstrated enhanced expansion and persistence upon antigen stimulation (both in vitro and in vivo), as well as increased tumor control (Hoyos et al., 2010. Markley and Sadelain, 2010). As such, there are high expectations for clinical efficacy of IL-15–CAR-T cells.

In nonactivated murine 4G-CAR-T cells, we observed very low levels of mIL-15 in the culture supernatant, but upon antigenic stimulation, significantly higher amounts were detected, in line with reports for hIL-15 CAR-T cells (Krenciute et al., 2017. Hoyos et al., 2010). Elevated levels of pSTAT5 in the 4G- versus 2G-CAR-T cells indicated active signaling by cytokine/receptor engagement.

The functional integrity of the coexpressed mIL-15 was further supported by enhanced 4G-CAR-T cell proliferation and survival, possibly due to up-regulation of the antiapoptotic molecule Bcl-2 (Wu et al., 2002. Shenoy et al., 2014). In addition, mIL-15 coexpression promoted a TCM cell phenotype, limited PD-1 up-regulation, and conferred superior effector function upon antigenic challenge.

The culture methods presented herein comprising hIL-7/hIL-15 in the medium permitted efficient murine CAR-T cell expansion, which was significantly reinforced upon mIL-15 coexpression by CAR-T cells. This enabled us to further investigate the efficacy of 4G-CAR-T cells in vivo against B16 melanoma tumors. We observed higher tumor control and persistence of 4G- as compared with the 2G-CAR-T cells and sustained expression of the mIL-15 transgene.

Moreover 4G-CAR-T cells exhibited higher Bcl-2 levels, in line with our in vitro data, suggesting that mIL-15 can render CAR-T cells more resistant to apoptosis in vivo. The coexpression of mIL-15 was also associated with significantly lower up-regulation of PD-1, an inhibitory receptor that can impair T cell function in the TME (Ahmadzadeh et al., 2009). Finally, evaluation of endogenous tumor immune infiltrate revealed a significantly higher frequency of activated (CD69+ Ki67+) NK cells and fewer M2 (F4/80+ CD206+) macrophages upon 4G- versus 2G-CAR-T cell transfer.

As NK cells are associated with delayed melanoma tumor growth (Nath et al., 2019), and M2 macrophages have been shown to contribute to tumor progression and metastasis (Poh and Ernst, 2018), the observed TME remodeling upon 4G-CAR-T cell transfer is favorable for tumor control. Our findings are consistent with prior studies. For example, coadministration of IL-15 with tumor-directed monoclonal antibodies enhanced Ab-dependent cellular cytotoxicity by augmenting both NK cell and macrophage activation (Zhang et al., 2018).

In another study, it was shown that the absence of IL-15 in immunocompetent mice promotes the formation of M2 macrophages (Gillgrass et al., 2014). In summary, we have presented comprehensive and highly reproducible methods for efficient retroviral transduction and robust expansion of murine CAR-T cells endowed with favorable properties for ACT studies in immunocompetent mice. We further demonstrated that coexpression of mIL-15 directly promotes CAR-T cell fitness and function and remodels the TME to favor tumor control.

As it is becoming apparent that endogenous immunity can play a critical role in either suppressing or supporting CAR-T cell function in the TME (Kuhn et al., 2019), comprehensive studies in immunocompetent mice are critical for accelerating the translation of effective CAR therapies to the clinic. The murine brain endothelioma cell line bEnd3, the murine immortalized heart endothelial cell line H5V, and the murine leukemia cell line C1498 were cultured in DMEM-GlutaMAX comprising 4,500 mg/liter glucose and 110 mg/liter sodium pyruvate and supplemented with 10% heat-inactivated FBS (Gibco, Thermo Fisher Scientific), 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. The melanoma cell line B16-F10 was grown as a monolayer in DMEM-GlutaMAX supplemented with 10% FBS, 100 U/ml of penicillin, and 100 µg/ml streptomycin sulfate.

Cells were passaged twice weekly to maintain them under exponential growth conditions and were routinely tested for mycoplasma contamination. The Phoenix Eco retroviral ecotropic packaging cell line, derived from immortalized normal human embryonic kidney cells, was maintained in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin sulfate. Primary murine T cells were cultured in RPMI 1640-Glutamax medium supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin sulfate, 1 mM sodium pyruvate, 50 µM β-mercaptoethanol, and 10 mM nonessential amino acids (referred to as murine T cell culture medium).

Murine T cell culture medium was further supplemented with human cytokines as described in the method for T cell expansion. The retroviral vector pMSGV (murine stem cell zithromax [MSCV]–based splice-gag vector) comprising the MSCV LTR was used as the backbone for all CAR constructs. A 2G-CAR consisting of the anti-VEGFR-2 scFv, DC101, the CD8α hinge (H), and TM region, followed by the EDs of CD28 and CD3ζ (DC101-28-z), was kindly provided by Dr.

Steven A. Rosenberg (National Cancer Institute, Bethesda, MD. Chinnasamy et al., 2010).

The DC101-28-z CAR was built by PCR amplification of a 362-bp fragment from the 2G construct with the primers. 5′-ACG​CGC​GGC​CGC​AAC​TAC​TAC​CAA​GC-3′ and 5′-ACG​CGT​CGA​CGG​GGC​GGT​ACG​CTG​CAA​AGT​CTC-3′ followed by NotI and SalI digestion of both the PCR product and the parental 2G vector, gel purification, and ligation. To generate the 4G-CAR construct encoding both mIL-15 and the VEGFR-2–directed CAR (mIL-15-T2A-DC101-28-z), a gene-string encoding the murine Igκ leader sequence followed by codon-optimized mIL-15 and T2A, flanked by XhoI and EcoRI restriction sites at the 5′ and 3′ ends, respectively, was synthesized.

The DC101-28-z construct and fragment were then digested (XhoI and EcoRI), gel purified, and ligated together. All genes strings were synthesized by GeneArt AG, and all constructs were fully sequenced by Microsynth AG. High-titer, replication-defective retrozithromax was produced and concentrated as depicted in Fig.

1. Briefly, Phoenix Eco cells were seeded at 107 per T-150 tissue culture flask in 35 ml culture medium (Fig. 1 A, 1) 24 h before transfection with 14.4 µg pCL-Eco Retrozithromax Packaging Vector and 21.4 µg pMSGV transfer plasmid using Turbofect (Thermo Fisher Scientific.

Fig. 1 A, 2). All plasmids were purified using HiPure Plasmid Filter Maxiprep Kit (Invitrogen, Thermo Fisher Scientific).

For the transfection mixture, a 3:1 ratio of Turbofect/plasmid was prepared in 2 ml Opti-MEM and incubated for 30 min at room temperature (RT. Fig. 1 A, 2).

Medium was then removed from T-150 flasks bearing 80–90% confluent Phoenix Eco cells and the transfection mixture was applied and incubated for 1 min, followed by addition of 31 ml fresh medium (Fig. 1 A, 2). The viral supernatant was discarded 20–24 h after transfection and replaced with 33 ml fresh medium (Fig.

1 A, 5) after transfection, the supernatant was harvested, and viral particles were concentrated by ultracentrifugation for 2 h at 24,000 g at 4°C with a Beckman JS-24 rotor (Beckman Coulter) and resuspended in 0.4 ml murine T cell medium. The retrozithromax was then used immediately, or aliquoted, frozen on dry ice, and stored at −80°C. As depicted in Fig.

1 B, murine T cells were isolated from single-cell suspensions of dissociated spleens from CD45.1+ congenic C57BL/6 mice bred in-house at the animal facility of the University of Lausanne (UNIL. Epalinges, Switzerland) using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies. Fig.

1 B, 1.1). T cells were plated at 106/ml in 24- or 48-well plates in T cell medium (described above) and stimulated with αCD3/CD28 Ab-coated beads (Invitrogen) at a bead to cell ratio of 2:1 and 50 IU/ml hIL-2 (Glaxo. Fig.

1 B, 1.1). Non–treated cell-culture grade 48- or 24-well plates (Corning Falcon) were precoated with 0.25 ml or 0.5 ml, respectively, of recombinant RetroNectin (Takara Bio) at a final concentration of 20 μg/ml, overnight (O/N) at 4°C (Fig. 1 B, 1.2).

1 d after T cell activation, the retronectin-precoated plates were washed with PBS, blocked with 2% BSA in PBS for 30 min at RT (Fig. 1 B, 2.1). Subsequently, plates were washed once, retrozithromax was added at the MOI indicated in the figures, and plates were then spun at 2,000 g for 1.5 h at 32°C (Fig.

1 B, 2.2). The supernatants were then aspirated, and 0.5 to 106 of 24 h activated T cells were transferred to each coated well (48- or 24-well plates. Fig.

1 B, 2.3). The plates were centrifuged for 10 min at 300 g and incubated O/N (Fig. 1 B, 2.3).

In some experiments the transduction procedure was performed at 48 h, or at both 24 and 48 h after activation. The cultures were maintained at a cell density of 0.5 to 106 cells/ml and replenished with fresh T cell medium (supplemented with hIL-2 alone or hIL-2 followed by hIL-7/IL-15 on day 2 after transduction) every other day (Fig. 1 B, 3).

At day 7, CAR surface expression was assessed by flow cytometric analysis (as described below), and the rested engineered T cells were adjusted for equal expression before functional in vitro and in vivo assays (Fig. 1 B, 4). Murine C1498 leukemia cells were transduced as described above for primary murine T cells, except that they were not activated and were maintained afterwards in DMEM-GlutaMAX complete medium at a cell density of 3 × 105 viable cells/ml.

For flow cytometric analysis, cells were surface stained using antibodies against CD3ε (145-2C11), CD4 (GK1.5, RM4-5), CD8α (53–6.7), CD25 (PC61), CD44 (IM7), CD45.1 (A20), CD45 (30F/11), CD62L (MEL-14), CD69 (H1-2F3), IL-15-Rα (6B4C88), PD-1 (29F.1A12), Ly-6G (1A8), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD206 (C068C2), NK-1.1 (PK136), CD19 (6D5), and MHC class II (M5/114.15.2). Abs were purchased from eBioscience and BioLegend or produced in-house from hybridomas by the flow cytometry platform. DC101-CAR expression by retrovirally transduced T cells was detected by incubation with soluble mouse VEGFR-2–hIgG-Fc fusion protein (R&D Systems) followed by staining with labeled goat anti-hIgG Fc (clone HP6017.

Biolegend). Thy1.1-T cells were stained in parallel as a negative control. VEGFR-2 expression by mouse endothelial cell lines was evaluated by cell-surface staining with rat anti-VEGFR-2 Ab (clone Avas12.

BioLegend) and matched isotype control (Rat IgG2a κ isotype. Clone RTK2758. BioLegend).

For detection of phosphorylated STAT5, cells were fixed with BD Cytofix Fixation Buffer at 4°C for 15 min and permeabilized with BD Phosflow Perm Buffer III for 30 min at 4°C. Intracellular phospho-staining was performed for 1 h at RT in the dark with Ab against phospho-STAT5 (Tyr694. D47E7 XP Rabbit mAb 4322.

Cell Signaling). For intracellular staining of mIL-15 (clone AIO.3. EBioscience), Bcl-2 (clone 10C4.

EBioscience), and Ki67 (clone SolA15. EBioscience), T cells were fixed and then permeabilized using the FoxP3 transcription factor staining buffer set (eBioscience) according to the manufacturer’s recommendations. For the detection of mIL-15, the cells were further washed and incubated for 30 min with anti-rat IgG2a.

To discriminate dead cells, 7-AAD (BioLegend) staining was performed. Live/dead fixable Aqua Dead cell staining was used to exclude dead cells in the ex vivo analysis of immune cells derived from the spleens, tumors, and tumor-draining lymph nodes according to the manufacturer’s instructions (Molecular Probes, Life Technologies). Data were acquired with a BD flow cytometer and analyzed using FlowJo software (Tree Star).

Cells extracted from dissociated tumors were lysed using TRIzol reagent (Invitrogen, Thermo Fisher Scientific). Total RNA was isolated using the RNeasy Mini Kit (Qiagen). After treatment with RNase-free DNase I (Qiagen), 400 ng of total RNA was reverse transcribed using PrimeScript First Strand cDNA Synthesis Kit (Takara Bio), as indicated by the manufacturer.

Quantitative real-time PCR was performed according to the commercial protocol using SYBR Green Fast PCR Master Mix (Thermo Fisher Scientific) and the 7500 Fast Real-Time PCR System (Applied Biosystems). Primers to specifically amplify regions of the DC101 scFv of the CAR cassette, or the mIL-15 transgene, were designed using the GenScript website and are as follows. DC101 forward, 5′-GCA​ACC​CAA​ACT​CCT​CAT​CT-3′.

DC101 reverse, 5′-TAT​CAT​CAG​CCT​CCA​CAG​GA-3′. IL-15 forward, 5′-CCA​GGA​TCT​ACA​GGC​GAC​AA-3′. IL-15 reverse, 5′-ATG​CTC​TGG​ATC​AGG​CTC​TC-3′.

PCR amplification of the housekeeping gene GAPDH was performed as a control, and to allow normalization of samples. The following primers were used for GAPDH. GAPDH forward, 5′-AGG​TCG​GTG​TGA​ACG​GAT​TTG-3′.

GAPDH reverse, 5′-TGT​AGA​CCA​TGT​AGT​TGA​GGT​CA-3′. Each sample was run in triplicate, and each experiment included three nontemplate control wells. The relative mRNA levels (fold change) of each transgene among the different samples were quantified using the comparative 2−ΔΔCt method.

We wish to thank members of the Flow Cytometry Platform and the Animal Care Facility of UNIL for their excellent support. We also kindly thank Dr. Steven A.

Rosenberg (National Cancer Institute, Bethesda, MD) for sharing a second generation anti-VEGFR-2 CAR construct comprising the scFv DC101. This work was generously supported by Ludwig Cancer Research, the European Research Council (advanced grant 1400206AdG-322875 to G. Coukos), and the Biltema Foundation.

P. Romero is supported in part by Oncosuisse (grant KFS-4404-02-2018). Author contributions.

Lanitis conceived, designed, developed, and supervised the study and wrote the manuscript. E. Lanitis, G.

Spill conducted experiments and acquired and analyzed data. A. Spill supported the in vivo and ex vivo studies.

Romero and D. Dangaj reviewed the data and manuscript and provided suggestions. All authors read and approved the manuscript.Christopher Mapperley Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing - original draft, Writing - review &.

Editing 1Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK2Laboratory of Haematopoietic Stem Cell and Leukaemia Biology, Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for other works by this author on:.

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€‚For the podcast associated with this article, please visit https://academic.oup.com/eurheartj/pages/Podcasts.I would like to begin here by wishing you websites and your loved zithromax street price ones a wonderful New Year. The past year has been difficult for all of us. buy antibiotics has caused illness and mortality on a global scale, has forced us to rethink our zithromax street price habits, has dealt a huge blow to our economies, and has cast a shadow on future plans. Unfortunately, human history is studded with wars, zithromaxs, and famines, frequently in deadly combination.

Yet, it is in difficult times that zithromax street price humankind shows extraordinary resources and indomitable resilience. The buy antibiotics zithromax is no exception. The incredible progress of our knowledge in a very zithromax street price short period of time leading to innovative forms of treatment will hopefully allow us to overcome this difficult moment in the near future. We should not, however, forget the many lessons learned in this difficult period, including the devastating effects of air pollution on buy antibiotics spread and lethality,1 in addition to the well-known devastating effects on cardiovascular health.2This is a Focus Issue on epidemiology and prevention.

Exercise recommendations and eligibility criteria for sports participation in competitive athletes with cardiovascular disease (CVD) were originally published by the Sports Cardiology Section of the European Society of Cardiology in 2005,3 and some aspects were subsequently updated in 2019.4 The overarching aim of zithromax street price these recommendations was to minimize the risk of adverse events in highly trained athletes. It is important to recognize, however, that most of the exercising population engages in leisure sport and solo recreational exercise and, unlike elite athletes, these individuals have a higher prevalence of risk factors for atherosclerosis and established CVD.5 The first contribution in this issue is the ‘2020 ESC Guidelines on Sports Cardiology and Exercise in Patients with Cardiovascular Disease’6 by Antonio Pelliccia from the Institute of Sport Medicine and Science in Rome, Italy, and his colleagues of the ESC Scientific Document Group. The authors note that sports cardiology is a relatively novel and emerging specialty area, therefore the evidence base for the natural history of disease progression zithromax street price or risk of death during intensive exercise and competitive sport among individuals with CVD is relatively sparse. This is reflected by the fact that a disproportionately large number of recommendations are reliant on the wisdom and vast experience of the consensus group rather than on large prospective studies.

The authors acknowledge the inherent difficulties in formulating recommendations for all scenarios in a heterogeneous population with a diverse spectrum of zithromax street price CVDs in light of the limited availability of evidence. Therefore, these recommendations should not be considered as legally binding and should not discourage individual physicians from practising outside the remit of this document, based on their clinical experience in sports cardiology. In addition, in line with good zithromax street price clinical practice, the present document encourages shared decision-making with the athlete patient and respects the autonomy of the individual after provision of detailed information about the impact of sports and the potential risks of complications and/or adverse events. The current Guidelines also provide recommendations on the investigation, risk assessment, and management of patients with CVDs to aid physicians when prescribing exercise programmes or providing advice for participation in sports.While deep vein thrombosis of the leg following airplane travel, the so-called economy class syndrome, received much attention years ago, now a report on internal jugular vein thrombosis in astronauts in space has startled the space medical community.7 In a Current Opinion article entitled ‘The thrombotic risk of spaceflight.

Has a serious problem been overlooked for more zithromax street price than half of a century?. €™, Ulrich Limper from the German Aerospace Center (DLR) in Cologne, Germany, and colleagues discuss this topic.8 Small cell, animal, and human studies performed in ground-based models and in actual weightlessness have revealed an influence of weightlessness and gravity on the blood coagulation system. However, human zithromax street price study populations were small and limited to carefully selected participants. Evidence in individuals with medical conditions and in older persons is lacking.

Evidence for thrombotic risk in spaceflight zithromax street price is unsatisfactory. This topic deserves rapid study in heterogeneous populations to guarantee safe governmental and touristic human spaceflight.CVD and cancer remain the leading causes of death. Although the epidemiology, pathobiology, and treatment of each of these diseases have been the focus of intensive study for decades, the intersection has only recently gained broader zithromax street price interest. There is increasing recognition that common shared risk factors predispose patients to both CVD and cancer.

In addition, cancer and traditional cancer therapies are associated with CVD zithromax street price. Conversely, recent intriguing data suggest that CVD (e.g. Heart failure) may stimulate tumour growth. Novel targeted therapies and their association with hypertension, arterial events, metabolic syndrome, and myocarditis all add complexity to the relationship between cancer and CVD.9 In a clinical research manuscript entitled ‘Long-term cardiovascular disease mortality zithromax street price among 160 834 five-year survivors of adolescent and young adult cancer.

An American population-based cohort study’, Lai Wang and colleagues assessed the risk of CVD mortality in US 5-year survivors of adolescent and young adult (AYA) cancer compared with that of the general population and contemporaneous 5-year survivors of childhood cancer.10 A total of 160 834 five-year AYA cancer survivors (aged 15–39 years at diagnosis) were included, representing 2 239 390 person-years of follow-up. Overall, 2910 CVD deaths occurred, which was zithromax street price 1.4-fold more that expected in the general population, corresponding to 3.6 excess CVD deaths per 10 000 person-years (Figure 1). The highest risk of cardiac mortality was experienced after Hodgkin’s lymphoma, and the highest risk of cerebrovascular mortality was observed with central nervous system (CNS) tumours. Even in survivors in their sixth and seventh decades of life, the risk of CVD mortality remained markedly zithromax street price higher than that of the matched general population.

Competing risk analysis showed that the cumulative mortality of CVD was elevated among AYA cancer survivors compared with childhood cancer survivors during the whole study period. Figure 1Cumulative mortality of heart disease among 5-year survivors of adolescent and young adult cancer and childhood cancer according to time since diagnosis by (A) sex, (B) ethnicity, and (C) lymphoma subtypes (from Lai Wang, Fengjiao Wang, Lianyu Chen, Yawen Geng, Shulin Yu, and Zhen Chen, Long-term cardiovascular disease mortality among 160 834 5-year survivors of adolescent and young zithromax street price adult cancer. An American population-based cohort study. See pages 101–109).Figure zithromax street price 1Cumulative mortality of heart disease among 5-year survivors of adolescent and young adult cancer and childhood cancer according to time since diagnosis by (A) sex, (B) ethnicity, and (C) lymphoma subtypes (from Lai Wang, Fengjiao Wang, Lianyu Chen, Yawen Geng, Shulin Yu, and Zhen Chen, Long-term cardiovascular disease mortality among 160 834 5-year survivors of adolescent and young adult cancer.

An American population-based cohort study. See pages 101–109).The authors conclude that long-term AYA cancer survivors have a greater risk of CVD mortality zithromax street price than the US general population and childhood cancer survivors. Vulnerable subgroups, especially survivors of Hodgkin lymphoma and CNS tumours, require continued close follow-up care for cardiovascular conditions throughout survivorship. The manuscript is accompanied by an Editorial by Patrizio Lancellotti from the University Hospital of Liège in Belgium and colleagues.11 The authors note that despite the many unknowns, the present study represents a valuable contribution to the identification of at-risk patient groups requiring close follow-up care, as well as to the understanding of a major health issue.Systemic vascular inflammation plays multiple zithromax street price maladaptive roles which contribute to the progression and destabilization of atherosclerotic cardiovascular disease (ASCVD).12,13 In a state of the art review entitled ‘Targeting cardiovascular inflammation.

Next steps in clinical translation’, Patrick R. Lawler from the University of Toronto in Canada, and colleagues note that these zithromax street price roles include. (i) driving atheroprogression in the clinically stable phase of disease. (ii) inciting atheroma destabilization zithromax street price and precipitating acute coronary syndromes (ACS).

And (iii) responding to cardiomyocyte necrosis in myocardial infarction (MI).14 Despite an evolving understanding of these biological processes, successful clinical translation into effective therapies has proven challenging. Realizing the zithromax street price promise of targeting inflammation in the prevention and treatment of ASCVD will be likely to require more individualized approaches, as the degree of inflammation differs among cardiovascular patients. A large body of evidence has accumulated supporting the use of high-sensitivity C-reactive protein (hsCRP) as a clinical measure of inflammation. Appreciating the mechanistic diversity of ACS triggers and the zithromax street price kinetics of hsCRP in MI may resolve purported inconsistencies from prior observational studies.

Future clinical trial designs incorporating hsCRP may hold promise to enable individualized approaches. The aim of this Clinical Review is to summarize the current understanding of how inflammation contributes to ASCVD progression, destabilization, and adverse clinical zithromax street price outcomes. The authors offer a forward-looking perspective on what next steps may enable successful clinical translation into effective therapeutic approaches—enabling targeting the right patients with the right therapy at the right time—on the road to more individualized ASCVD care (Figure 2). Figure 2Key contemporary residual risk pathways in zithromax street price secondary prevention.

*In addition to standard evidence-based therapies, more aggressive blood pressure targets may be considered. (from Patrick R. Lawler, Deepak zithromax street price L. Bhatt, Lucas C.

Godoy, Thomas F zithromax street price. Lüscher, Robert O. Bonow, Subodh zithromax street price Verma, and Paul M Ridker, Targeting cardiovascular inflammation. Next steps in clinical translation.

See pages 113–131.)Figure 2Key zithromax street price contemporary residual risk pathways in secondary prevention. *In addition to standard evidence-based therapies, more aggressive blood pressure targets may be considered. (from Patrick R zithromax street price. Lawler, Deepak L.

Bhatt, Lucas C zithromax street price. Godoy, Thomas F. Lüscher, Robert O zithromax street price. Bonow, Subodh Verma, and Paul M Ridker, Targeting cardiovascular inflammation.

Next steps in clinical zithromax street price translation. See pages 113–131.)The issue is also complemented by Discussion Forum contributions. In a contribution entitled ‘Time for clinicians to zithromax street price revisit their perspectives on C-statistic’, Arya Aminorroaya from the Tehran University of Medical Sciences in Iran and colleagues comment on the recent publication ‘Feasibility of using deep learning to detect coronary artery disease based on facial photo’ by Shen Lin from the Peking Union Medical College in China, and colleagues.15,16 Lin et al. Respond in a separate comment.17The editors hope that readers of this issue of the European Heart Journal will find it of interest.With thanks to Amelia Meier-Batschelet, Johanna Huggler, and Martin Meyer for help with compilation of this article.

References1Copat C, zithromax street price Cristaldi A, Fiore M, Grasso A, Zuccarello P, Santo Signorelli S, Conti GO, Ferrante M. The role of air pollution (PM and NO2) in buy antibiotics spread and lethality. A systematic zithromax street price review. Environ Res 2020;191:110129.2Münzel T, Sørensen M, Gori T, Schmidt FP, Rao X, Brook J, Chen LC, Brook RD, Rajagopalan S.

Environmental stressors and cardio-metabolic zithromax street price disease. Part I—epidemiologic evidence supporting a role for noise and air pollution and effects of mitigation strategies. Eur Heart J 2017;38:550–556.3Pelliccia A, Fagard R, Bjørnstad HH, Anastassakis A, Arbustini E, Assanelli http://robertroyer.com/2011/12/14/agenda-21-beware/ D, Biffi A, Borjesson M, Carrè F, Corrado D. Recommendations for zithromax street price competitive sports participation in athletes with cardiovascular disease.

A consensus document from the Study Group of Sports Cardiology of the Working Group of Cardiac Rehabilitation and Exercise Physiology and the Working Group of Myocardial and Pericardial Diseases of the European Society of Cardiology. Eur Heart zithromax street price J 2005;26:1422–1445.4Pelliccia A, Solberg EE, Papadakis M, Adami PE, Biffi A, Caselli S, La Gerche A, Niebauer J, Pressler A, Schmied CM. Recommendations for participation in competitive and leisure time sport in athletes with cardiomyopathies, myocarditis, and pericarditis. Position statement zithromax street price of the Sport Cardiology Section of the European Association of Preventive Cardiology (EAPC).

Eur Heart J 2019;40:19–33.5Gasperetti A, James CA, Cerrone M, Delmar M, Calkins H, Duru F. Arrhythmias right ventricular zithromax street price cardiomyopathy and sports activity. From molecular pathways in diseased hearts to new insights into the athletic heart mimicry. Eur Heart J 2020;doi:10.1093/eurheartj/ehaa821.6Pelliccia A, Sharma S, Gati S, Bäck M, Börjesson M, Caselli S, Collet J-P, Corrado zithromax street price D, Drezner JA, Halle M.

2020 ESC Guidelines on sports cardiology and exercise in patients with cardiovascular disease. The Task Force on sports cardiology and exercise in patients zithromax street price with cardiovascular disease of the European Society of Cardiology (ESC). Eur Heart J 2021;42:5–15.7Auñón-Chancellor SM, Pattarini JM, Moll S, Sargsyan A. Venous thrombosis during zithromax street price spaceflight.

N Engl J Med 2020;382:89–90.8Limper U, Tank J, Ahnert T, Maegele M, Grottke O, Hein M, Jordan J. The thrombotic risk of zithromax street price spaceflight. Has a serious problem been overlooked for more than half of a century?. Eur Heart J zithromax street price 2021;42:97–100.9Kondapalli L, Moslehi J, Bonaca MP.

Inflammation begets inflammation. Cancer and acute MI zithromax street price. Eur Heart J 2020;41:2194–2196.10Wang L, Wang F, Chen L, Geng Y, Yu S, Chen Z. Long-term cardiovascular disease mortality among 160 834 five-year survivors of adolescent and young adult cancer zithromax street price.

An American population-based cohort study. Eur Heart J 2021;42:101–109.11Lancellotti P, Nguyen Trung M-L, Oury C, Moonen M zithromax street price. Cancer and cardiovascular mortality risk. Is the zithromax street price die cast?.

Eur Heart J 2021;42:110–112.12Liberale L, Montecucco F, Tardif J-C, Libby P, Camici GG. Inflamm-ageing. The role of inflammation in age-dependent cardiovascular disease zithromax street price. Eur Heart J 2020;41:2974–2982.13Stojanović SD, Fiedler J, Bauersachs J, Thum T, Sedding DG.

Senescence-induced inflammation zithromax street price. An important player and key therapeutic target in atherosclerosis. Eur Heart J 2020;41:2983–2996.14Lawler zithromax street price PR, Bhatt DL, Godoy LC, Lüscher TF, Bonow RO, Verma S, Ridker PM. Targeting cardiovascular inflammation.

Next steps in clinical zithromax street price translation. Eur Heart J 2021;42:113–131.15Aminorroaya A, Tajdini M, Masoudkabir F. Time for clinicians to revisit their perspectives on C-statistic zithromax street price. Eur Heart J 2021;42:132–133.16Lin S, Li Z, Fu B, Chen S, Li X, Wang Y, Wang X, Lv B, Xu B, Song X.

Feasibility of using deep learning zithromax street price to detect coronary artery disease based on facial photo. Eur Heart J 2020;41:4400–4411.17Lin S, Chen S, Zhe Z. Model assessment zithromax street price. New measures should be known and traditional measures should be accurately interpreted.

Eur Heart J zithromax street price 2021;42:134–135. Published on behalf of the European Society of Cardiology. All rights zithromax street price reserved. © The Author(s) 2021.

For permissions, please email zithromax street price. Journals.permissions@oup.com.The results of “Effect of Finerenone on Chronic Kidney Disease Outcomes in Type 2 Diabetes” have been published in the New England Journal of Medicine (DOI. 10.1056/NEJMoa2025845)Key pointsFinerenone in Reducing Kidney Failure and Disease Progression in Diabetic Kidney Disease (FIDELIO-DKD), an industry-promoted phase 3, randomized, double-blind, placebo-controlled, multicentre trial investigated the long-term effects on renal and cardiovascular (CV) outcomes of finerenone, a non-steroidal, selective mineralocorticoid receptor antagonist (MRA) in patients with type 2 diabetes (T2D) and chronic kidney disease (CKD).The overall population included 5734 eligible patients with a urinary albumin-to-creatinine ratio (UAC) between 30 and 300 mg/g, an estimated glomerular filtration rate (eGFR) of 25 to <60 mL/min/1.73 m2 of body surface area and diabetic retinopathy, or—in the presence of UAC of 300 to 5000 mg/g—an eGFR of 25 to <75 mL/min/1.73 m2.When added to standard treatment (including a max dose of a renin-angiotensin system blocker), finerenone (10 mg or 20 mg according to renal function) was shown to be superior zithromax street price to placebo with respect to the primary composite outcome, assessed in a time-to-event analysis, of kidney failure, a sustained decrease of at least 40% in the eGFR from baseline, or death from renal causes [hazard ratio (HR) 0.82, 95% confidence interval (CI) 0.73–0.93. P = 0.001) during a median follow-up of 2.6 years.

Finerenone also reduced the incidence of the key secondary composite outcome of death from CV causes, non-fatal myocardial infarction (MI), non-fatal stroke, or hospitalization for heart failure (HF) (HR 0.86, zithromax street price 95% CI 0.75–0.99. P = 0.003).The incidence of serious adverse events did not differ significantly between finerenone and placebo. However, overall hyperkalaemia-related adverse events were twice as frequent with finerenone as with placebo (18.3% and 9.0%, respectively) and the frequency of hyperkalaemia leading to discontinuation was also higher with finerenone than placebo (2.3% vs. 0.9%).

CommentThe rationale for the FIDELIO-DKD trial1 relies on the observation that CKD is often associated with mild hyperaldosteronism which, through mineralocorticoid receptors distributed in the distal tubule and other structures of the kidney, exerts pro-inflammatory and pro-fibrotic actions and contributes to the progression of renal damage. However, in order to translate the positive and promising findings of FIDELIO-CKD into clinical practice, a more detailed analysis of the impact of finerenone on individual outcomes, as well as of the persisting and potentially harmful side-effects of MRA reported in this study, are needed.First, while finerenone was superior compared to placebo in reducing the primary composite outcome, when the individual components of the endpoint were analysed separately, the incidence of kidney failure was not significantly different in the finerenone and placebo groups (HR 0.87, 95% CI 0.72–1.05) and the impact on the composite endpoint was largely driven by a sustained decrease of ≥40% in eGFR from baseline (HR 0.81, 95% CI 0.72–0.92).Secondly, with regard to the individual CV components of the key secondary composite outcome, finerenone had only statistically uncertain effects on death from CV causes (HR 0.86, 95% CI 0.68–1.08), non-fatal MI (HR 0.80, 95% CI 0.58–1.09), non-fatal stroke (HR 1.03, 95% CI 0.76–1.38), hospitalization for HF (HR 0.86, 95% CI 0.68–1.08), death from any cause (HR 0.90, 95% CI 0.75–1.07), and hospitalization for any cause (HR 0.95, 95% CI 0.88–1.02).Finally, the higher incidence of hyperkalaemia and of withdrawals and hospitalizations due to hyperkalaemia observed with finerenone compared to placebo continues to be an issue of particular concern, mostly in patients with CKD and may represent an important barrier to its clinical use.Another relevant contemporary issue is when and in which patients to consider finerenone. When compared to the results of the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial2 with the sodium-glucose cotransporter 2 inhibitor (SGLT2i), canagliflozin, the magnitude of the benefits achieved with finerenone in terms of CKD progression (−18%) was less impressive than in CREDENCE (−30%). Differences in the populations of these trials may have contributed to a different effect size of the intervention since CREDENCE excluded patients who received MRA and those with eGFR <30 mL/min/1.73 m2, whereas FIDELIO-CKD enrolled patients treated SGLT2i (about 7%) and those with a worse renal function (>25 mL/min/1.73 m2), but did not include those affected by HF with reduced ejection fraction.It is possible that a subpopulation of patients with T2D and CKD may benefit more from finerenone than suggested by the overall effect size.

Although it was previously demonstrated that aldosterone levels are inversely proportional to eGFR in patients with CKD, the study was clearly not powered to reliably assess the benefits of finerenone in relation to baseline renal function.Additional information on the efficacy and safety of finerenone in patients with T2D and less advanced CKD will be provided by the Finerenone in Reducing Cardiovascular Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial.3 Supplementary materialSupplementary material is available at European Heart Journal online.Conflict of interest. M.V. Reports personal fees for speaker bureau and/or consulting in Advisory Board from Amgen, Astra Zeneca, Daiichi-Sankyo, Menarini Int, MSD, Novartis Pharma, Novo Nordisk outside the submitted work. C.P.

Reports personal fees from Acticor Biotech, personal fees from Amgen, personal fees from Bayer, personal fees from GlaxoSmithKline, personal fees from Tremeau, personal fees from Zambon, grants from AIFA (Italian Drug Agency), grants from European Commission, other from Scientific Advisory Board of the International Aspirin Foundation, outside the submitted work.The results of ‘Effect of Finerenone on Chronic Kidney Disease Outcomes in Type 2 Diabetes’ have been published in the New England Journal of Medicine (DOI. 10.1056/NEJMoa2025845) References1Bakris GL, Agarwal R, Anker SD, Pitt B, Ruilope LM, Rossing P, Kolkhof P, Nowack C, Schloemer P, Joseph A, Filippatos G. For the FIDELIO-DKD Investigatorset al for the FIDELIO-DKD Investigators. Effect of finerenone on chronic kidney disease outcomes in type 2 diabetes.

N Engl J Med 2020;383:2219–2229.2Perkovic V, Jardine MJ, Neal B, Bompoint S, Heerspink HJL, Charytan DM, Edwards R, Agarwal R, Bakris G, Bull S, Cannon CP, Capuano G, Chu PL, de Zeeuw D, Greene T, Levin A, Pollock C, Wheeler DC, Yavin Y, Zhang H, Zinman B, Meininger G, Brenner BM, Mahaffey KW. CREDENCE Trial Investigators. Canagliflozin and renal outcomes in type 2 diabetes and nephropathy. N Engl J Med 2019;380:2295–2306.3Ruilope LM, Agarwal R, Anker SD, Bakris GL, Filippatos G, Nowack C, Kolkhof P, Joseph A, Mentenich N, Pitt B.

FIGARO-DKD Study Investigators. Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial. Am J Nephrol 2019;50:345–356. Published on behalf of the European Society of Cardiology.

All rights reserved. © The Author(s) 2020. For permissions, please email. Journals.permissions@oup.com..

€‚For the podcast associated with this buy zithromax online australia article, please visit https://academic.oup.com/eurheartj/pages/Podcasts.I would like to begin here by wishing you buy zithromax in usa and your loved ones a wonderful New Year. The past year has been difficult for all of us. buy antibiotics has caused illness and mortality on a global scale, has forced us to rethink our habits, has dealt a huge blow to our economies, and has cast buy zithromax in usa a shadow on future plans. Unfortunately, human history is studded with wars, zithromaxs, and famines, frequently in deadly combination.

Yet, it is in difficult times that humankind shows extraordinary buy zithromax in usa resources and indomitable resilience. The buy antibiotics zithromax is no exception. The incredible progress of our knowledge in a very short period of time leading buy zithromax in usa to innovative forms of treatment will hopefully allow us to overcome this difficult moment in the near future. We should not, however, forget the many lessons learned in this difficult period, including the devastating effects of air pollution on buy antibiotics spread and lethality,1 in addition to the well-known devastating effects on cardiovascular health.2This is a Focus Issue on epidemiology and prevention.

Exercise recommendations and eligibility criteria for sports participation in competitive athletes with cardiovascular disease (CVD) were originally published by the Sports Cardiology Section of the European Society of Cardiology in 2005,3 and some aspects were subsequently updated in 2019.4 The buy zithromax in usa overarching aim of these recommendations was to minimize the risk of adverse events in highly trained athletes. It is important to recognize, however, that most of the exercising population engages in leisure sport and solo recreational exercise and, unlike elite athletes, these individuals have a higher prevalence of risk factors for atherosclerosis and established CVD.5 The first contribution in this issue is the ‘2020 ESC Guidelines on Sports Cardiology and Exercise in Patients with Cardiovascular Disease’6 by Antonio Pelliccia from the Institute of Sport Medicine and Science in Rome, Italy, and his colleagues of the ESC Scientific Document Group. The authors note that sports cardiology is a relatively novel and emerging specialty area, therefore the evidence base for the natural history of disease progression or risk of death during intensive exercise and competitive sport among individuals with buy zithromax in usa CVD is relatively sparse. This is reflected by the fact that a disproportionately large number of recommendations are reliant on the wisdom and vast experience of the consensus group rather than on large prospective studies.

The authors acknowledge the inherent difficulties in formulating recommendations for all scenarios in a heterogeneous population with a diverse spectrum of CVDs in light of buy zithromax in usa the limited availability of evidence. Therefore, these recommendations should not be considered as legally binding and should not discourage individual physicians from practising outside the remit of this document, based on their clinical experience in sports cardiology. In addition, in line with good clinical practice, the present document encourages shared decision-making with the athlete patient and respects the autonomy of the individual after provision of detailed information about the impact of sports and the buy zithromax in usa potential risks of complications and/or adverse events. The current Guidelines also provide recommendations on the investigation, risk assessment, and management of patients with CVDs to aid physicians when prescribing exercise programmes or providing advice for participation in sports.While deep vein thrombosis of the leg following airplane travel, the so-called economy class syndrome, received much attention years ago, now a report on internal jugular vein thrombosis in astronauts in space has startled the space medical community.7 In a Current Opinion article entitled ‘The thrombotic risk of spaceflight.

Has a buy zithromax in usa serious problem been overlooked for more than half of a century?. €™, Ulrich Limper from the German Aerospace Center (DLR) in Cologne, Germany, and colleagues discuss this topic.8 Small cell, animal, and human studies performed in ground-based models and in actual weightlessness have revealed an influence of weightlessness and gravity on the blood coagulation system. However, human study buy zithromax in usa populations were small and limited to carefully selected participants. Evidence in individuals with medical conditions and in older persons is lacking.

Evidence for thrombotic risk buy zithromax in usa in spaceflight is unsatisfactory. This topic deserves rapid study in heterogeneous populations to guarantee safe governmental and touristic human spaceflight.CVD and cancer remain the leading causes of death. Although the epidemiology, pathobiology, and treatment of each of buy zithromax in usa these diseases have been the focus of intensive study for decades, the intersection has only recently gained broader interest. There is increasing recognition that common shared risk factors predispose patients to both CVD and cancer.

In addition, cancer and traditional cancer therapies are buy zithromax in usa associated with CVD. Conversely, recent intriguing data suggest that CVD (e.g. Heart failure) may stimulate tumour growth. Novel targeted therapies and their association with hypertension, arterial events, metabolic syndrome, and myocarditis all add complexity to the relationship between cancer and CVD.9 In a clinical research manuscript entitled ‘Long-term cardiovascular disease mortality among 160 834 five-year survivors of adolescent and buy zithromax in usa young adult cancer.

An American population-based cohort study’, Lai Wang and colleagues assessed the risk of CVD mortality in US 5-year survivors of adolescent and young adult (AYA) cancer compared with that of the general population and contemporaneous 5-year survivors of childhood cancer.10 A total of 160 834 five-year AYA cancer survivors (aged 15–39 years at diagnosis) were included, representing 2 239 390 person-years of follow-up. Overall, 2910 CVD deaths occurred, which was 1.4-fold more that buy zithromax in usa expected in the general population, corresponding to 3.6 excess CVD deaths per 10 000 person-years (Figure 1). The highest risk of cardiac mortality was experienced after Hodgkin’s lymphoma, and the highest risk of cerebrovascular mortality was observed with central nervous system (CNS) tumours. Even in survivors in their buy zithromax in usa sixth and seventh decades of life, the risk of CVD mortality remained markedly higher than that of the matched general population.

Competing risk analysis showed that the cumulative mortality of CVD was elevated among AYA cancer survivors compared with childhood cancer survivors during the whole study period. Figure 1Cumulative mortality of heart disease among 5-year survivors of adolescent and young adult cancer and childhood cancer according to time since diagnosis by (A) sex, (B) ethnicity, and (C) lymphoma subtypes (from Lai Wang, Fengjiao Wang, Lianyu Chen, Yawen Geng, Shulin Yu, and Zhen Chen, Long-term cardiovascular disease mortality among 160 834 5-year buy zithromax in usa survivors of adolescent and young adult cancer. An American population-based cohort study. See pages 101–109).Figure 1Cumulative mortality of heart disease among 5-year survivors of adolescent and young adult cancer and childhood cancer according to time since diagnosis by (A) sex, (B) ethnicity, and (C) lymphoma subtypes (from Lai Wang, Fengjiao Wang, Lianyu Chen, Yawen Geng, Shulin Yu, and Zhen Chen, Long-term cardiovascular disease mortality among 160 834 5-year survivors of adolescent and buy zithromax in usa young adult cancer.

An American population-based cohort study. See pages 101–109).The authors conclude that long-term AYA cancer survivors have a greater risk of CVD mortality than the US general population and buy zithromax in usa childhood cancer survivors. Vulnerable subgroups, especially survivors of Hodgkin lymphoma and CNS tumours, require continued close follow-up care for cardiovascular conditions throughout survivorship. The manuscript is accompanied by an Editorial by Patrizio Lancellotti from the University Hospital of Liège in Belgium and colleagues.11 The authors note that despite the many unknowns, the present study represents a valuable contribution to the identification of at-risk patient groups requiring close follow-up care, as well as to the understanding of a major health issue.Systemic vascular buy zithromax in usa inflammation plays multiple maladaptive roles which contribute to the progression and destabilization of atherosclerotic cardiovascular disease (ASCVD).12,13 In a state of the art review entitled ‘Targeting cardiovascular inflammation.

Next steps in clinical translation’, Patrick R. Lawler from the buy zithromax in usa University of Toronto in Canada, and colleagues note that these roles include. (i) driving atheroprogression in the clinically stable phase of disease. (ii) inciting atheroma destabilization buy zithromax in usa and precipitating acute coronary syndromes (ACS).

And (iii) responding to cardiomyocyte necrosis in myocardial infarction (MI).14 Despite an evolving understanding of these biological processes, successful clinical translation into effective therapies has proven challenging. Realizing the promise of targeting inflammation in the prevention and treatment of ASCVD will be likely to require more individualized approaches, buy zithromax in usa as the degree of inflammation differs among cardiovascular patients. A large body of evidence has accumulated supporting the use of high-sensitivity C-reactive protein (hsCRP) as a clinical measure of inflammation. Appreciating the mechanistic diversity of ACS triggers and the kinetics of hsCRP in MI may resolve purported inconsistencies from prior observational studies buy zithromax in usa.

Future clinical trial designs incorporating hsCRP may hold promise to enable individualized approaches. The aim of this Clinical Review is to summarize the current understanding of how inflammation buy zithromax in usa contributes to ASCVD progression, destabilization, and adverse clinical outcomes. The authors offer a forward-looking perspective on what next steps may enable successful clinical translation into effective therapeutic approaches—enabling targeting the right patients with the right therapy at the right time—on the road to more individualized ASCVD care (Figure 2). Figure 2Key contemporary residual risk buy zithromax in usa pathways in secondary prevention.

*In addition to standard evidence-based therapies, more aggressive blood pressure targets may be considered. (from Patrick R. Lawler, Deepak L buy zithromax in usa. Bhatt, Lucas C.

Godoy, Thomas F buy zithromax in usa. Lüscher, Robert O. Bonow, Subodh Verma, and Paul buy zithromax in usa M Ridker, Targeting cardiovascular inflammation. Next steps in clinical translation.

See pages 113–131.)Figure 2Key buy zithromax in usa contemporary residual risk pathways in secondary prevention. *In addition to standard evidence-based therapies, more aggressive blood pressure targets may be considered. (from Patrick R buy zithromax in usa. Lawler, Deepak L.

Bhatt, Lucas buy zithromax in usa C. Godoy, Thomas F. Lüscher, Robert buy zithromax in usa O. Bonow, Subodh Verma, and Paul M Ridker, Targeting cardiovascular inflammation.

Next steps buy zithromax in usa in clinical translation. See pages 113–131.)The issue is also complemented by Discussion Forum contributions. In a contribution entitled ‘Time for clinicians to revisit their perspectives on C-statistic’, Arya Aminorroaya from the Tehran University of Medical Sciences in Iran and colleagues comment on the recent publication ‘Feasibility of using deep learning to detect coronary artery disease based on facial photo’ by Shen Lin from buy zithromax in usa the Peking Union Medical College in China, and colleagues.15,16 Lin et al. Respond in a separate comment.17The editors hope that readers of this issue of the European Heart Journal will find it of interest.With thanks to Amelia Meier-Batschelet, Johanna Huggler, and Martin Meyer for help with compilation of this article.

References1Copat C, Cristaldi A, buy zithromax in usa Fiore M, Grasso A, Zuccarello P, Santo Signorelli S, Conti GO, Ferrante M. The role of air pollution (PM and NO2) in buy antibiotics spread and lethality. A systematic review buy zithromax in usa. Environ Res 2020;191:110129.2Münzel T, Sørensen M, Gori T, Schmidt FP, Rao X, Brook J, Chen LC, Brook RD, Rajagopalan S.

Environmental stressors and buy zithromax in usa cardio-metabolic disease. Part I—epidemiologic evidence supporting a role for noise and air pollution and effects of mitigation strategies. Eur Heart J 2017;38:550–556.3Pelliccia A, Fagard R, Bjørnstad HH, Anastassakis A, Arbustini E, Assanelli D, Biffi A, Borjesson M, Carrè F, Corrado D. Recommendations for competitive sports participation in buy zithromax in usa athletes with cardiovascular disease.

A consensus document from the Study Group of Sports Cardiology of the Working Group of Cardiac Rehabilitation and Exercise Physiology and the Working Group of Myocardial and Pericardial Diseases of the European Society of Cardiology. Eur Heart J 2005;26:1422–1445.4Pelliccia A, Solberg EE, Papadakis M, Adami PE, Biffi A, Caselli S, buy zithromax in usa La Gerche A, Niebauer J, Pressler A, Schmied CM. Recommendations for participation in competitive and leisure time sport in athletes with cardiomyopathies, myocarditis, and pericarditis. Position statement of the Sport Cardiology Section of the European Association buy zithromax in usa of Preventive Cardiology (EAPC).

Eur Heart J 2019;40:19–33.5Gasperetti A, James CA, Cerrone M, Delmar M, Calkins H, Duru F. Arrhythmias right buy zithromax in usa ventricular cardiomyopathy and sports activity. From molecular pathways in diseased hearts to new insights into the athletic heart mimicry. Eur Heart J 2020;doi:10.1093/eurheartj/ehaa821.6Pelliccia A, Sharma S, Gati S, Bäck M, Börjesson M, Caselli S, Collet J-P, Corrado D, Drezner JA, buy zithromax in usa Halle M.

2020 ESC Guidelines on sports cardiology and exercise in patients with cardiovascular disease. The Task buy zithromax in usa Force on sports cardiology and exercise in patients with cardiovascular disease of the European Society of Cardiology (ESC). Eur Heart J 2021;42:5–15.7Auñón-Chancellor SM, Pattarini JM, Moll S, Sargsyan A. Venous thrombosis buy zithromax in usa during spaceflight.

N Engl J Med 2020;382:89–90.8Limper U, Tank J, Ahnert T, Maegele M, Grottke O, Hein M, Jordan J. The thrombotic risk buy zithromax in usa of spaceflight. Has a serious problem been overlooked for more than half of a century?. Eur buy zithromax in usa Heart J 2021;42:97–100.9Kondapalli L, Moslehi J, Bonaca MP.

Inflammation begets inflammation. Cancer and buy zithromax in usa acute MI. Eur Heart J 2020;41:2194–2196.10Wang L, Wang F, Chen L, Geng Y, Yu S, Chen Z. Long-term cardiovascular disease mortality among 160 834 five-year survivors of adolescent and young adult cancer buy zithromax in usa.

An American population-based cohort study. Eur Heart J 2021;42:101–109.11Lancellotti P, Nguyen buy zithromax in usa Trung M-L, Oury C, Moonen M. Cancer and cardiovascular mortality risk. Is the buy zithromax in usa die cast?.

Eur Heart J 2021;42:110–112.12Liberale L, Montecucco F, Tardif J-C, Libby P, Camici GG. Inflamm-ageing. The role of inflammation in age-dependent buy zithromax in usa cardiovascular disease. Eur Heart J 2020;41:2974–2982.13Stojanović SD, Fiedler J, Bauersachs J, Thum T, Sedding DG.

Senescence-induced inflammation buy zithromax in usa. An important player and key therapeutic target in atherosclerosis. Eur Heart J 2020;41:2983–2996.14Lawler buy zithromax in usa PR, Bhatt DL, Godoy LC, Lüscher TF, Bonow RO, Verma S, Ridker PM. Targeting cardiovascular inflammation.

Next steps in clinical buy zithromax in usa translation. Eur Heart J 2021;42:113–131.15Aminorroaya A, Tajdini M, Masoudkabir F. Time for clinicians buy zithromax in usa to revisit their perspectives on C-statistic. Eur Heart J 2021;42:132–133.16Lin S, Li Z, Fu B, Chen S, Li X, Wang Y, Wang X, Lv B, Xu B, Song X.

Feasibility of using deep learning to detect coronary artery disease based on facial buy zithromax in usa photo. Eur Heart J 2020;41:4400–4411.17Lin S, Chen S, Zhe Z. Model assessment buy zithromax in usa. New measures should be known and traditional measures should be accurately interpreted.

Eur Heart J buy zithromax in usa 2021;42:134–135. Published on behalf of the European Society of Cardiology. All rights buy zithromax in usa reserved. © The Author(s) 2021.

For permissions, buy zithromax in usa please email. Journals.permissions@oup.com.The results of “Effect of Finerenone on Chronic Kidney Disease Outcomes in Type 2 Diabetes” have been published in the New England Journal of Medicine (DOI. 10.1056/NEJMoa2025845)Key pointsFinerenone in Reducing Kidney Failure and Disease Progression in Diabetic Kidney Disease (FIDELIO-DKD), an industry-promoted phase 3, randomized, double-blind, placebo-controlled, multicentre trial investigated the long-term effects on renal and cardiovascular (CV) outcomes of finerenone, a non-steroidal, selective mineralocorticoid receptor antagonist (MRA) in patients with type 2 diabetes (T2D) and chronic kidney disease (CKD).The overall population included 5734 eligible patients with a urinary albumin-to-creatinine ratio (UAC) between 30 and 300 mg/g, an estimated glomerular filtration rate (eGFR) of 25 to <60 mL/min/1.73 m2 of body surface area and diabetic retinopathy, or—in the presence of UAC of 300 to 5000 mg/g—an eGFR of 25 to <75 mL/min/1.73 m2.When added to standard treatment (including a max dose of a renin-angiotensin system blocker), finerenone (10 mg or 20 mg according to renal function) was shown buy zithromax in usa to be superior to placebo with respect to the primary composite outcome, assessed in a time-to-event analysis, of kidney failure, a sustained decrease of at least 40% in the eGFR from baseline, or death from renal causes [hazard ratio (HR) 0.82, 95% confidence interval (CI) 0.73–0.93. P = 0.001) during a median follow-up of 2.6 years.

Finerenone also reduced the incidence of the key secondary composite outcome of death from CV causes, non-fatal myocardial buy zithromax in usa infarction (MI), non-fatal stroke, or hospitalization for heart failure (HF) (HR 0.86, 95% CI 0.75–0.99. P = 0.003).The incidence of serious adverse events did not differ significantly between finerenone and placebo. However, overall hyperkalaemia-related adverse events were twice as frequent with finerenone as with placebo (18.3% and 9.0%, respectively) and the frequency of hyperkalaemia leading to discontinuation was also higher with finerenone than placebo (2.3% vs. 0.9%).

CommentThe rationale for the FIDELIO-DKD trial1 relies on the observation that CKD is often associated with mild hyperaldosteronism which, through mineralocorticoid receptors distributed in the distal tubule and other structures of the kidney, exerts pro-inflammatory and pro-fibrotic actions and contributes to the progression of renal damage. However, in order to translate the positive and promising findings of FIDELIO-CKD into clinical practice, a more detailed analysis of the impact of finerenone on individual outcomes, as well as of the persisting and potentially harmful side-effects of MRA reported in this study, are needed.First, while finerenone was superior compared to placebo in reducing the primary composite outcome, when the individual components of the endpoint were analysed separately, the incidence of kidney failure was not significantly different in the finerenone and placebo groups (HR 0.87, 95% CI 0.72–1.05) and the impact on the composite endpoint was largely driven by a sustained decrease of ≥40% in eGFR from baseline (HR 0.81, 95% CI 0.72–0.92).Secondly, with regard to the individual CV components of the key secondary composite outcome, finerenone had only statistically uncertain effects on death from CV causes (HR 0.86, 95% CI 0.68–1.08), non-fatal MI (HR 0.80, 95% CI 0.58–1.09), non-fatal stroke (HR 1.03, 95% CI 0.76–1.38), hospitalization for HF (HR 0.86, 95% CI 0.68–1.08), death from any cause (HR 0.90, 95% CI 0.75–1.07), and hospitalization for any cause (HR 0.95, 95% CI 0.88–1.02).Finally, the higher incidence of hyperkalaemia and of withdrawals and hospitalizations due to hyperkalaemia observed with finerenone compared to placebo continues to be an issue of particular concern, mostly in patients with CKD and may represent an important barrier to its clinical use.Another relevant contemporary issue is when and in which patients to consider finerenone. When compared to the results of the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial2 with the sodium-glucose cotransporter 2 inhibitor (SGLT2i), canagliflozin, the magnitude of the benefits achieved with finerenone in terms of CKD progression (−18%) was less impressive than in CREDENCE (−30%). Differences in the populations of these trials may have contributed to a different effect size of the intervention since CREDENCE excluded patients who received MRA and those with eGFR <30 mL/min/1.73 m2, whereas FIDELIO-CKD enrolled patients treated SGLT2i (about 7%) and those with a worse renal function (>25 mL/min/1.73 m2), but did not include those affected by HF with reduced ejection fraction.It is possible that a subpopulation of patients with T2D and CKD may benefit more from finerenone than suggested by the overall effect size.

Although it was previously demonstrated that aldosterone levels are inversely proportional to eGFR in patients with CKD, the study was clearly not powered to reliably assess the benefits of finerenone in relation to baseline renal function.Additional information on the efficacy and safety of finerenone in patients with T2D and less advanced CKD will be provided by the Finerenone in Reducing Cardiovascular Mortality and Morbidity in Diabetic Kidney Disease (FIGARO-DKD) trial.3 Supplementary materialSupplementary material is available at European Heart Journal online.Conflict of interest. M.V. Reports personal fees for speaker bureau and/or consulting in Advisory Board from Amgen, Astra Zeneca, Daiichi-Sankyo, Menarini Int, MSD, Novartis Pharma, Novo Nordisk outside the submitted work. C.P.

Reports personal fees from Acticor Biotech, personal fees from Amgen, personal fees from Bayer, personal fees from GlaxoSmithKline, personal fees from Tremeau, personal fees from Zambon, grants from AIFA (Italian Drug Agency), grants from European Commission, other from Scientific Advisory Board of the International Aspirin Foundation, outside the submitted work.The results of ‘Effect of Finerenone on Chronic Kidney Disease Outcomes in Type 2 Diabetes’ have been published in the New England Journal of Medicine (DOI. 10.1056/NEJMoa2025845) References1Bakris GL, Agarwal R, Anker SD, Pitt B, Ruilope LM, Rossing P, Kolkhof P, Nowack C, Schloemer P, Joseph A, Filippatos G. For the FIDELIO-DKD Investigatorset al for the FIDELIO-DKD Investigators. Effect of finerenone on chronic kidney disease outcomes in type 2 diabetes.

N Engl J Med 2020;383:2219–2229.2Perkovic V, Jardine MJ, Neal B, Bompoint S, Heerspink HJL, Charytan DM, Edwards R, Agarwal R, Bakris G, Bull S, Cannon CP, Capuano G, Chu PL, de Zeeuw D, Greene T, Levin A, Pollock C, Wheeler DC, Yavin Y, Zhang H, Zinman B, Meininger G, Brenner BM, Mahaffey KW. CREDENCE Trial Investigators. Canagliflozin and renal outcomes in type 2 diabetes and nephropathy. N Engl J Med 2019;380:2295–2306.3Ruilope LM, Agarwal R, Anker SD, Bakris GL, Filippatos G, Nowack C, Kolkhof P, Joseph A, Mentenich N, Pitt B.

FIGARO-DKD Study Investigators. Design and baseline characteristics of the finerenone in reducing cardiovascular mortality and morbidity in diabetic kidney disease trial. Am J Nephrol 2019;50:345–356. Published on behalf of the European Society of Cardiology.

All rights reserved. © The Author(s) 2020. For permissions, please email. Journals.permissions@oup.com..