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Archive for the ‘Stem Cell Complications’ Category

Energy drinks may damage the heart, researchers warnshould the FDA get involved? – Cardiovascular Business

Sunday, February 14th, 2021

Drinking certain energy drinks may cause significant damage to the heart, according to new findings published in Food and Chemical Toxicology.

Because the consumption of these beverages is not regulated and they are widely accessible over the counter to all age groups, the potential for adverse health effects of these products is a subject of concern and needed research, lead researcher Ivan Rusyn, MD, PhD, a professor at Texas A&M University in College Station, said in a prepared statement.

Rusyn et al. assessed a total of 17 popular energy drinks, studying their chemical profiles and looking for any associations with potential cardiac complications. Energy drinks sold by Adrenaline, Shoc, Bang Star, C4, CELSIUS, HEAT, EBOOST, Game Fuel, GURU, Kill Cliff, Kickstart, Monster Energy, Red Bull, Reign, Rockstar, RUNA, UPTIME, Venom Energy and Xyience Energy were all part of the teams analysis.

Overall, the authors found that stem cell-derived cardiomyocyteshuman heart cells grown in a laboratoryshowed signs of an increased beat rate after being exposed to some energy drinks. Also, theophylline, adenine and azelate were all ingredients the team associated with potentially contributing to QT prolongation in cardiomyocytes.

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Easter Ross mum of blood cancer tot urges would-be stem cell donors to show the love this Valentine’s Day; Alness lass Adeline Davidson’s plight…

Sunday, February 14th, 2021

Adeline Davidson and dad Jordan.Picture: Callum Mackay

THE Easter Ross mum of a little girl with an extremely rare form of blood cancer has urged life-saving donors to step up to help others.

Steph Davidson made the appeal as blood cancer charity DKMS asked people to show the love this Valentine's Day by signing up for a worldwide register of potential blood stem cell donors.

Her daughter Adeline (3) is awaiting a blood stem cell donation from a stranger and has endured many "false starts" and complications as a result of the coronavirus crisis.

Ms Davidson, who lives in Alness, is backing the DKMS campaign at a time when UK-wide registrations have slumped by 28 per cent.

Big-hearted Highlanders have bucked that trend with an increase in registrations during the pandemic.

With no match within her family, a blood stem cell donation from a complete stranger is Adelines best chance of survival.

The brave little girl, who has missed out on her first year at nursery and valiantly gone through painful and invasive treatment over the last year, has finally got a date for her lifesaving transplant.

With one donor pulling out at the last minute, and her transplant date pushed back several times due to the pandemic, her family have their heart set on finally having their healthy and happy little girl back home and able to play with her friends and family.

Her mum said: Shes such a sweet and friendly little girl, so confident and the best big sister to her little brother and sister. Its just felt like the world has been against us this past year with so many treatments, needles and false starts.

"We are so incredibly grateful to this stranger, who could be anywhere in the world. I want to give them the biggest hug in the world. I cant begin to imagine how awful it is for other families who have a loved one in need of a lifesaving transplant where no match has been found.

"As a parent it makes you feel so powerless being unable to protect your child. Anyone who is healthy and able to register, please, please do. Its such a small commitment for you and could give someone a second chance at life Adelines not even had a chance to start hers and we were so close to it being taken away from her.

The Valentines Day campaign by DKMS is asking people to celebrate with their loved one by taking five minutes to sign up to the stem cell donor register to potentially save the love of someone elses life.

Every 20 minutes, someone in the UK is diagnosed with blood cancer. Around 2000 people each year are dealt the shocking news that they need a blood stem cell transplant.

For these people the perfect match doesnt necessarily have a compatible Zodiac sign or share the same taste in films they need to have a genetically similar make up to give them the best shot at a second chance of life.

With two in three of those people not finding a perfect match within their family, they must turn to the worldwide donor registry and rely on a stranger to save their lives. By signing up to the register you could one day be a match for someone who needs you to help save their life.

The pandemic has had a destructive impact on the lives of people with blood cancer. Not only has it led to a huge drop in the number of people registering as donors, it has meant fewer people are visiting the GP with cancer symptoms, and resulted in hospital appointments and treatments being postponed or cancelled. Due to this, DKMS expects to see a surge in blood cancer diagnoses and increased demand for blood stem cell donors when we are back to normal, making it all the more important that people register now.

Jonathan Pearce, chief executive of DKMS UK, said: At DKMS, we are dedicated to the fight against blood cancer and are proud to have registered over 780,000 blood stem cell donors. Hearing the stories of people like Adeline shows why people registering as blood stem cell donors is so important.

"With the shocking drop in registrations over the last 10 months we are calling on Scots to save the love of someone elses life this Valentines Day. We want every worried family to get the reassuring call that a match for their loved on has been found. If youre inspired by Adelines story, please register as a stem cell donor to give the ultimate gift this Valentines Day by saving a life.

How to sign up

Signing up to save the life of someone like Adeline is easy to do. Register as a potential lifesaver online at dkms.org.uk to receive your home swab kit. It takes a few moments to swab. When you return your swab kit you go on standby to help save someones life.

Taking the first steps to register as a potential blood stem cell donor can be done within a few minutes from the comfort of your own home. If you are aged between 17-55 and in general good health you can sign up for a home swab kit online. Your swabs can then be returned with the enclosed pre-paid envelope to DKMS in order to ensure that your details are added to the UKs aligned stem cell registry.

Related: Alness parents make heartfelt plea after coronavirus-related donor blow

Waiting game as stem cell donor found in United States

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Global Induced Pluripotent Market Positive Outlook, Revenue Generation & Leading Manufacturers, Forecast 2026||CELGENE CORPORATION; Astellas…

Sunday, February 14th, 2021

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After Bone Marrow Donation Saves 9-Year-Old Boy With Cancer, Boston Mom Fights To Raise Awareness – Here And Now

Sunday, February 7th, 2021

Every year, about 10,000 people in the U.S. need a stem cell transplant but cant find a donor.

The intense medical procedure, which can help those with leukemia, lymphoma, sickle cell anemia and other blood diseases, can save lives but securing a donor can be like finding a needle in a haystack.

Be The Match is a nonprofit, national registry where people can sign up to donate their stem cells. More than 35 million people around the world have volunteered yet only a small percentage of those donors are Americans, and even the registry admits most Americans dont know it exists.

The mother of a 12-year-old boy with leukemia has set out to change that.

Mandy Goldman is a hairdresser who lives with her husband and four children outside Boston. She remembers the devastating day five years ago when doctors told her the chemotherapy they gave her son Mateo Goldman, 9 years old at the time, didnt work.

They told us that our only option of curing Mateo was a bone marrow transplant, she says, a risky procedure that often involves a host of complications. But they had no other choice, she says.

The family got to work on the monumental task finding Mateo Goldman a close enough match.

Linda Matchan first reported the Goldman familys experience for The Boston Globe. In her research, she found very, very few people had any awareness of the need for bone marrow and stem cells donors. The awareness campaign around the subject is severely lacking compared to other campaigns like the importance of donating blood, she says.

For example, there's a little boy right now in North Carolina named Thor Forte, who's 10 and has sickle cell disease. And he has been waiting for literally half his life, five years, for a donor to be available, Matchan says. He's a tough match, but they finally did find somebody. And then when the time came for the procedure, the person backed out. So two years later, the boy is still waiting.

Fortunately, quickly after finding out Mateo Goldman didnt match with anyone in his family, he was paired with a donor on the registry from Germany. Mandy Goldman says Laura Stterlin of Frankfurt was ready to go and donate, ultimately saving her son.

Mateo Goldman wrote Stterlin, whose name he did not know at the time, a thank you note reading: Dear Donor, thank you for giving me the bone marrow. You feel like youre already part of my family, he says.

And unlike usual Make-A-Wish requests, Mateo Goldman asked to meet Stterlin in person halfway across the world. The trip to Germany was planned for summer of 2020 but has since been canceled due to the pandemic.

In 2019 when she was reporting this story, Matchan had a trip planned to Germany. She ended up meeting Stterlin and hearing the story of how she became a donor. Stterlin said she was at a sporting event with her husband when she got hungry and went on the hunt for some grub.

Dear Donor, thank you for giving me the bone marrow. You feel like youre already part of my family.

Germany has a robust public service campaign to get citizens to donate bone marrow, Matchan says. So it came to no surprise to Stterlin when she came across a kiosk to sign up.

Just three months later, she got a call and an email from the registry saying that there is somebody in the United States for whom she could be a match and was asked if she would donate, Matchan says. A couple of days later, she went into the hospital and did the donation.

Stterlins stem cells then crossed the Atlantic Ocean, making their way to America during a snowstorm.

The cells started working in Mateo Goldman right away but not without some difficulties, Mandy Goldman says. He battled total body stiffness from graft-versus-host disease, a complication of the transplant.

But, you know, Matteo's an amazing kid, she says, so through it all, he was smiling and making the best of it, even though he was suffering for a lot of the time.

Two years later, in July of 2020, the cancer came back. But since Mateo Goldmans first transplant, the science had evolved greatly.

So much so that his older brother, Leo Goldman, became a candidate to donate his cells for the second stem cell transplant.

I didn't realize how I could get my brother's cells, Mateo Goldman, now 12 years old, says. Once that sank in, I felt that it would connect me and my brother more.

Right before Christmas last year, the family got extraordinary news: Mateo Goldman had zero cancer in his bone marrow, Mandy Goldman says.

Now the mom of four is on a mission to raise awareness on stem cell donations and share the story of how it saved her sons life.

The amazing feeling Leo got from being able to be the person who saved his brother's life is something he's going to carry with him forever, she says. And even Laura [Stterlin], she gave him three and a half years of his life that we get to spend with him. I just really want to educate people about how empowering it is to do something so incredible for somebody else.

When she started talking to others to raise awareness, she was shocked to discover how fearful people were in committing to be a donor.

If people could see the trauma these patients go through her son had a drain placed in his stomach, total body radiation, chemotherapy that left him head-to-toe in a skin-burning rash she says then maybe they wouldnt be scared to dedicate a small action for someone whose only cure is through a stem cell transplant.

Once people are educated about how much of a difference it makes, she says, then I feel like they would do it.

Click here to learn more about the Be The Match Registry.

Tinku Rayproduced and edited this interview for broadcast.Serena McMahonadapted it for the web.

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After Bone Marrow Donation Saves 9-Year-Old Boy With Cancer, Boston Mom Fights To Raise Awareness - Here And Now

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Leukemia in children: Symptoms, causes, treatment, outlook, and more – Medical News Today

Sunday, February 7th, 2021

Leukemia is a type of cancer that affects the blood. The two most common types in children are acute lymphoblastic leukemia and acute myelogenous leukemia.

In a person with leukemia, blood cells are released into the bloodstream before they are fully formed, so there are fewer healthy blood cells in the body.

Below, we describe the types of childhood leukemia, the symptoms, and the treatments. We then look at when to contact a doctor, what questions to ask, and where to find support.

Childhood leukemia is the most common form of cancer in children. It affects up to 3,800 children under the age of 15 in the United States each year.

Leukemia occurs when bone marrow releases new blood cells into the bloodstream before they are fully mature.

These immature blood cells do not function as they should, and eventually, the number of immature cells overtakes the number of healthy ones.

Leukemia can affect red and white blood cells and platelets.

The bone marrow produces stem cells. A blood stem cell can become a myeloid stem cell or a lymphoid stem cell.

Lymphoid stem cells become white blood cells. Myeloid stem cells can become:

Leukemia is typically acute or chronic, and chronic types are rare in children. They can include chronic myeloid leukemia or chronic lymphocytic leukemia.

Most childhood leukemias are acute, meaning that they progress quickly and need treatment as soon as possible.

Acute lymphoblastic leukemia (ALL) is the most common type in children, accounting for 75% of childhood leukemia cases.

It affects cells called lymphocytes, a type of white blood cell.

In a person with ALL, the bone marrow releases a large number of underdeveloped white blood cells called blast cells. As the number of these increases, the number of red blood cells and platelets decreases.

There are two subtypes of ALL: B-cell and T-cell.

In most childhood cases of ALL, the cancer develops in the early forms of B-cells. The other type, T-cell ALL, typically affects older children.

Research from 2020 reports that the majority of people diagnosed with ALL are under 18 and typically between 2 and 10 years old.

The American Cancer Society report that children under 5 years old have the highest risk of developing ALL and that this risk slowly declines until a person reaches their mid-20s.

The outlook for ALL depends on the subtype, the persons age, and factors specific to each person.

Myeloid leukemias account for approximately 20% of childhood leukemia cases, and most myeloid leukemias are acute.

Acute myelogenous leukemia (AML) affects white blood cells other than the lymphocytes. It may also affect red blood cells and platelets.

AML can begin in:

Juvenile myelomonocytic leukemia (JMML) accounts for approximately 12% of leukemia cases in children.

This rare type is neither acute nor chronic. JMML begins in the myeloid cells, and it typically affects children younger than 2 years.

Symptoms can include:

The symptoms of leukemia may be nonspecific similar to those of other common childhood illnesses.

A doctor will ask how long the child has been experiencing the symptoms, which can include:

Children may experience specific symptoms depending on the type of blood cell that the leukemia is affecting.

A low number of red blood cells can cause:

A low number of healthy white blood cells can cause infections or a fever with no other sign of an infection.

A low platelet count can cause:

Various factors can increase a childs risk of leukemia, and most are not preventable.

The following genetic conditions can increase the risk of leukemia:

Also, having a sibling with leukemia may increase the risk of developing it.

These can include exposure to:

If a child has symptoms that might indicate leukemia, a doctor may perform or request:

A bone marrow aspiration involves using a syringe to take a liquid sample of bone marrow cells. The doctor may give the child a drug that allows them to sleep through this test.

During the diagnostic process, a person might ask:

The doctor may recommend a variety of treatments for childhood leukemia, and the best option depends on a range of factors specific to each person.

The treatment usually consists of two phases. The first aims to kill the leukemia cells in the childs bone marrow, and the second aims to prevent the cancer from coming back.

The child may need:

Before or during treatment, a person might ask the doctor:

Questions to ask after the treatment might include:

Children who have undergone leukemia treatments require follow-up care, as the treatments often cause late effects.

These can develop in anyone who has received treatment for cancer, and they may not arise for months or years after the treatment has ended.

Treatments that can cause late effects include:

These complications may affect:

The late effects that may come can also depend on the type of treatment and the form of leukemia.

Because many leukemia symptoms can also indicate other issues, it can be hard to know when to contact a doctor.

Overall, it is best to seek medical advice if a child shows symptoms or behaviors that are not normal for them.

If a child has received a leukemia diagnosis, the effects can extend to parents, other family members, caregivers, and friends.

A person can find support and additional resources from:

The following organizations based in the United Kingdom also provide support and guidance:

Childhood leukemia can affect mental health, as well as physical health.

Learn more about mental health resources here.

According to the American Cancer Society, most children with leukemia have no known risk factors. There is no way to prevent leukemia from developing.

Because there are very few lifestyle-related or environmental causes of childhood leukemia, it is very unlikely that a caregiver can do anything to help prevent the disease.

A childs outlook depends on the type of leukemia. It is important to keep in mind that current estimates do not take into account recent advances in technology and medicine.

For example, the most recent 5-year survival rate estimates reflect the experiences of children who received their diagnoses and treatments more than 5 years ago.

The American Cancer Society report that the 5-year survival rate for children with ALL is 90%. The same rate for children with AML is 6570%.

Childhood leukemia is typically acute, which means that it develops quickly. As a result, a person should contact a doctor if they notice any of the symptoms.

The most common type of childhood leukemia is ALL, representing 3 out of 4 leukemia cases in children.

Treatment may include a combination of chemotherapy, targeted drugs, immunotherapy, stem cell transplants, surgery, and radiation.

The prognosis depends on the type of leukemia and the childs age.

This diagnosis can affect mental as well as physical health, and the effects can extend to caregivers, family members, and friends. Many different resources are available for support.

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Leukemia in children: Symptoms, causes, treatment, outlook, and more - Medical News Today

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Understanding bone marrow transplant: The guidelines and the protocols – The New Indian Express

Thursday, February 4th, 2021

The outbreak of the Covidpandemic has made many patients reluctantto undergotreatments. While their apprehension seems to overpower them, doctors need to ensure thatstrict guidelines and protocols which assure the best quality service are followed.

Among elective surgeries andtransplants, bone marrow transplant cases have increased substantially in the past few months. Adhering to guidelines for pre-transplant evaluation and the management of a common complication, graft versus host disease (GVHD)is essential.

With the diversity of practice and expertise, the following guidelines will provide a pivotal tool for learning about the rapidly updated therapy landscape in Hematopoietic stem cell transplantation (HSCT).

The guidelines intended to provide a systematic approach for transplantation and help streamline clinical practices and educate new generations of physicians-in-training. Additionally, guidelines can help to evaluate a potential transplant recipient anddetermine if the patient is an eligible candidate for the procedure.

Types and selection of transplantation:

Selection of the type of transplantation for a patient depends on factors such as the type of malignancy, availability of a suitable donor, age of the recipient, the ability to collect a tumor-free autograft, the stage, the malignancy's susceptibility to the GVM effect, and status of disease -- bone marrow involvement, the bulk of disease, chemosensitivity to conventional chemotherapy. This method is particularly applicable for Autologous or Allogeneic Transplantation where one can have a sibling donor or a matched unrelated donor. In the case of a matched unrelated donor, ensure that the collection is adequate and stem cells are available well in time especially if they are imported from countries in Europe.

A haploidentical transplant is another type of transplant that uses healthy, blood-forming cells from a half-matched donor to replace the unhealthy ones. The ideal donor in this case is a family member.

That said, for bone marrow transplant blood products are the backbone and it is important to ensure to have adequate supply before you begin with the transplant.

What are the guidelines and protocols that can be adopted in current times?

Some measures for consideration are: Minimize face-to-face visits including monitoring and consider shifting to telehealth where feasible. Some adaptive community measures like the hospital in the home services, community practices for blood collection, imaging, and support services. For radiation oncology treatment, consider reducing fractions when supported by evidence Consider alternative and less resource-intensive treatment regimes. Minimize unnecessary visitors to cancer centers, for instance, limiting to only patients and their essential caregivers based on frailty and language needs Screen for possible symptoms of COVID-19 and triage patients for admission. If necessary, the admission has to be directed to oncology/hematology departments rather than emergency departments. Immunocompromised patients are likely to have atypical presentations of COVID-19 For suspected checkpoint inhibitor-related pneumonitis prioritizes COVID-19 testing for an early decision regarding corticosteroid therapy.

These are some guidelines that you should heed during a bone marrow transplant. While it is imperative to be updated about the guidelines, timely intervention can reduce the other possible complications during the process.

(The author is the Director, Medical Oncology and Hemato Oncology, atFortis Cancer Institute, Bangalore)

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Why Cynata is hopeful its COVID treatment trial will succeed where others have failed – Business News Australia

Thursday, February 4th, 2021

Cynata Therapeutics (ASX: CYP), founded by two clever stem cell researchers and one wise Australian techpreneur, is in the process of developing a treatment for COVID-19.

Using its in-house stem cell technology Cymerus, the ASX-listed biotech hopes to treat one of the deadliest complications of COVID-19 -acute respiratory distress syndrome (ARDS).

In doing so Cynata would achieve what competitor Mesoblast (ASX: MSB) couldn't with FDA approval.

By deploying an industrialised approach to stem cell therapeutics, Cynata CEO Ross Macdonald (pictured) is confident the clinical trial process won't leave the company hamstrung.

In 1981 scientists discovered a way to derive embryonic stem cells from early mouse embryos.

The discovery thrilled scientists, and eventually led to the development of a method to do the same in lab-grown human embryos by 1998.

While there have been plenty of discussions surrounding the ethics of using of embryonic stem cells, these major scientific movements have pushed researchers to discover new and inventive ways of treating a whole raft of diseases and infections.

One such researcher, Dr Ian Dixon, saw potential for the use of mesenschymal stem cells (MSCs) - a type of stem cell that can differentiate into a variety of cell types enabling the treatment of many diseases and infections.

However there was still an obstacle to overcome: how do you mass produce enough cells needed to commercialise a treatment?

Luckily, two researchers at the University of Wisconson, Professor Igor Slukvin and Dr Maksym Vodyanik, had invented a biotechnological breakthrough called Cymerus.

The technology was able to do exactly what Dixon needed: the consistent manufacture of MSCs on an ultra-large scale; basically what Henry Ford did to the industrialisation of the auto industry, but for stem cells.

So in 2003 Dixon partnered with the two researchers to start Cynata - now an ASX-listed biotechnology company trialing a number of different treatments for a wide variety of ailments.

Most recently, Cynata's focus has been on developing a treatment for a complication of COVID-19 called acute respiratory distress syndrome (ARDS).

The complication ravages COVID-19 infected patients, destroying their organs through what is known as a cytokine storm. The complication is estimated to kill up to half of COVID-19 patients that suffer from it.

Melbourne-based Cynata is currently in the very early stages of its investigation into whether its MSCs will be able to treat the coronavirus complication overwhelming hospitals globally.

If this all sounds familiar, you might be thinking of another ASX-listed biotech called Mesoblast (ASX: MSB).

In March last year Mesoblast, also based in Melbourne, saw its shares surge after announcing plans to evaluate its stem cell treatment solutions on COVID-19 patients.

The group commenced the arduous clinical trial process to see if its remestemcel-L therapy could treat ARDS by using bone marrow aspirate from healthy donors - a similar approach the company had already taken to treat a condition many suffer from after receiving bone marrow transplants.

Mesoblast was riding high on the ASX following positive announcements surrounding the clinical traila process, especially back in April 2020 when a trial at New York City's Mt Sinai hospital found its remestemcel-L therpay achieved "remarkable" results.

Serious attention gathered around Mesoblast, with the company even securing $138 in funds from investors to continue its important research.

The company went so far as to sign a commercialisation deal for the COVID-19 treatment with Novartis, and the US Food and Drugs Administration (FDA) fast tracked the approvals process for the potential game-changing treatment.

However, in December 2020, Mesoblast hit a stumbling block.

Mesoblast's COVID-19 treatment flunked the test - its remestemcel-L therapy failed to show a lower mortality rate for patients in the prescribed 30-day timeframe of treatment.

At that point Cynata had commenced research into its own ARDS treatment. But did Mesoblast's failure unnerve Cynata CEO Ross Macdonald? Not a chance.

"I'm more confident that our trial will be successful where theirs was a failure," Macdonald said.

"If you use a process like we have developed - we don't rely on multiple different [stem cell] donations. You start with exactly the same material every time."

To explain, Macdonald used the analogy of a local caf; you normally expect a coffee from one caf to taste more or less exactly the same every time you go there - the same beans are used every time.

Whereas Macdonald said Mesoblast's process is like going to the same caf every day, but each visit they use different beans from a different supplier which leads to inconsistency in taste and flavour.

Cynata's approach with its MSCs is in line with the first example - what you get the first time from them will be replicated in each and every dose of the drug - while MSB's is like the latter.

"Yes, you still got the coffee, but the experience of the taste is totally different than it was yesterday," he said.

"The FDA said to Mesoblast, well you've got a manufacturing problem that is reliant upon multiple donors prepared to donate bone marrow and that is flawed.

"So with that in mind it's perhaps not surprising that they had a pretty disappointing result in the clinical trials."

Additionally, Macdonald said the initial investor reactions to MSB's early COVID-19 trail results were overblown.

"The initial data from their trial that got everybody excited was, in my view, quite flawed, because they said "look at how many patients are dying in intensive care units with COVID compared the patients that we treated," he said.

"But the reality of the situation was quite different. The control group at that time - the death rate was way, way higher than you would typically see for ARDS, whether its COVID or anything else. And it was simply because of the chaos that existed in intensive care units in New York in the first wave.

"So we think that the initial enthusiasm was perhaps a little misguided."

When asked why Mesoblast is receiving so much attention compared to Cynata, especially considering the above, Macdonald said it was simply because MSB is bigger and has been around for longer. For context, MSB has a market capitalisation of $1.46 billion, whereas Cynata's is just $94.56 million.

"I'd love to know why there is less attention, and how we can get our market cap above a billion dollars," joked Macdonald.

"I think the answer though is that they've been around for a lot longer than we have, they have spent a hell of a lot more money than we've spent - their monthly spend is more than we've spent for pretty much our entire existence.

"But I think the fundamental reason why is that data drives value in biotech, so the more clinical data you generate that shows your product works, the more attention you attract from investors."

That's not to say Cynata is being totally ignored in favour of the larger Mesoblast.

The company secured a $15 million placement led by $10 million from healthcare investor BioScience Managers in December.

The funds will be used to expand Cynata's clinical development pipeline and scale their operations in Australia.

As such, the company is preparing to expand its clinical development pipeline to include idiopathic pulmonary fibrosis, renal transplantation, and diabetic foot ulcers.

"So we're starting to garner that attention now that says two things - one, cell therapies are definitely a medical revolution and two, Cynata is part of that new generation of companies," Macdonald said.

As for the company's pipeline, in addition to the COVID treatment trials, Cynata is planning on launching three new clinical candidates that will get under way this year.

There's also Cynata's osteoarthritis trial, which Macdonald describes as significant for the biotech company; with 2 million patients in Australia and 30 million in the United States the company is hoping to tap into an $11 billion plus addressable market.

"It will ultimately show whether MSCs are useful in that particularly devastating condition," he said.

"It doesn't just affect people who want to go and play golf or tennis, it affects, particularly manual labourers who can no longer work.

"So the cost to the economy of osteoarthritis is quite significant, which is of course one of the reasons why the Australian Government is funding this trial."

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Why Cynata is hopeful its COVID treatment trial will succeed where others have failed - Business News Australia

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Mobilize family caregivers to speed the rollout of Covid-19 vaccines – STAT

Thursday, February 4th, 2021

Five years ago, I sat in a hospital lounge as a nurse laid out everything I as a caregiver would need to feed my husband intravenously: a bag of fluid called total parenteral nutrition, a vitamin solution, syringe, pump, a new 9-volt battery, a quarter for opening the battery chamber, and tiny alcohol wipes. She demonstrated the fussy, time-consuming process of hooking up the whole thing to my husbands central line.

Then she handed me a 20-page pamphlet detailing the procedure and I was on my own.

At the time, my husband, Brad, had been hospitalized for more than four months after a stem cell transplant that was plagued by complications for relapsed, aggressive lymphoma. He was unable to eat much by mouth, so he relied on IV nutrition, night after night, to survive at home.

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Over the years of Brads medical ordeal, I have administered IV antibiotics, flushed and sterilized port lines, checked blood sugar, given shots of Neulasta (a booster shot for immunity after chemotherapy), and much more. My shock and anger at suddenly being expected to be a bedside nurse faded, and handling needles and central lines became routine.

Brad no longer needs this level of care, but his immune system is still suppressed and he is chronically ill. Were now mired in the long wait for Covid-19 vaccines which, in my state, California, could take several confusing, frustrating months.

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The Covid-19 vaccines are miracles of fast-moving science, but Covid-19 vaccination has so far been a slow grind. With daily deaths in the U.S. continuing to number in the thousands and the country barreling toward the devastating figure of half a million deaths, we need all hands on deck to turn vaccines into vaccinations.

The gigantic systems that must figure out how to get shots into arms U.S. health care and the federal government are better known for ponderous bureaucracy than nimble pivots and stopgap solutions. As Ive watched the delays, Ive thought often of the times I was deputized to give my husband care during the medical ordeal that left him chronically ill.

I could give him the shot, I keep thinking.

Every day, family caregivers like me, with no medical experience, volunteer or are voluntold to provide care that a generation or two ago would have been administered only by a nurse. According to a 2020 AARP survey, there are more than 50 million family caregivers in the U.S. We represent a resource that, if deployed, could help get vaccines into arms, the well-known last mile problem.

In some ways, caregivers have been sidelined during the pandemic. Because of justified concerns about infection, hospitals have had to ban family caregivers from hospital visits. When we went on lockdown, I worried about my husbands immune suppression, but briefly consoled myself with the idea that if he did get sick, I would know how to advocate and care for him, given my prior experience. Then I soon realized I wouldnt even get in the door.

Of course, the slow pace of vaccination has many causes: dose shortages, the negligence of the Trump administration, the ethical questions surrounding who to prioritize, and personnel shortages.

What if the valuable experience of each and every caregiver could be applied to the most critical effort of this moment, vaccination?

Caregivers, many of us already trained in basic at-home care and used to cutting through health-care bureaucracy, could pitch in to speed up vaccinations. Deputizing caregivers to give shots at home could ease the enormous, yearlong strain on the medical and public health systems while also providing a safer, more accessible vaccination method for vulnerable seniors, those with limited mobility, and immune-compromised people like my husband, who shouldnt be waiting for hours in mass clinics.

The Biden administrations strategic plan to combat the pandemic promises federal leadership and faster action on vaccination nationwide, bringing much-needed hope in a dark time. Reports indicate the administration will deploy the National Guard and the Federal Emergency Management Agency to aid the effort.

I hope it will also explore tapping ordinary citizens. In addition to finding ways to use caregivers, we should consider mass volunteer efforts for both direct care and support. The United Kingdom, for instance, started a volunteer corps in which not only retired doctors but also teachers and other laypeople are giving shots. In Connecticut and Alberta, veterinarians have been deputized. Its time to extend such efforts more broadly.

Vaccination campaigns in regions beset by childhood diseases have long relied heavily on volunteers for last-mile delivery. My sister-in-law recently told me that, at age 17, she went on a volunteer summer program to vaccinate kids in Ecuador. She and her fellow teens practiced, first on oranges and then on each other, with syringes filled with saline. Im not proposing we send out needle-wielding minors, even in a crisis as deep as the present one, but the moment cries out for innovative solutions.

Both the individual family care I am expected to provide for my husband and the broad tragedies of the pandemic reflect the profound strains on American health care. But shifting higher-level care tasks to family caregivers, hard though it is on individuals, represents innovation a workaround to save lives despite systemic challenges and has given millions of Americans experience that could help meet the moment.

With the availability of Covid-19 vaccines, the end of the pandemic is tantalizingly close. Lets use every resource we have to get there, including an often-overlooked one: caregivers.

Kate Washington, a journalist based in Sacramento, Calif., is the author of Already Toast: Caregiving and Burnout in America (Beacon Press, March 2021).

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People With Cancer Should Receive COVID-19 Vaccine, Experts Say – Cancer Health Treatment News

Thursday, February 4th, 2021

People living with cancerincluding those undergoing treatmentshould receive COVID-19 vaccines as soon as they are available, according to new guidelines from the National Comprehensive Cancer Network (NCCN).

While people with some types of cancer and those receiving certain cancer treatments may not respond quite as well, the vaccines should still provide partial protection, which is especially important because some cancer patients are at higher risk for COVID-19 complications.

Right now, there is urgent need and limited data, said committee co-leader Steve Pergam, MD, MPH,of the Seattle Cancer Care Alliance and the Fred Hutchinson Cancer Research Center, headquarters of the National Institutes of Healths COVID-19 Prevention Network. Our number one goal is helping to get the vaccine to as many people as we can. That means following existing national and regional directions for prioritizing people who are more likely to face death or severe illness from COVID-19. The evidence we have shows that people receiving active cancer treatment are at greater risk for worse outcomes from COVID-19, particularly if they are older and have additional comorbidities, like immunosuppression.

Much remains to be learned about COVID-19 in people with cancer. Studies have shown that people with blood cancers like leukemia or lymphoma and lung cancer are at greater risk for severe COVID-19 and death, but those with other types, such as breast or lung cancer, do not appear to be at higher risk. Patients with active or advanced cancer are likely to fare worse. Although studies of the effects of cancer treatment on COVID-19 outcomes have yielded conflicting results, therapies that cause immune suppression seem to lead to poorer outcomes.

Two mRNA vaccines from Pfizer/BioNTech and Moderna were authorized by the Food and Drug Administration in December. These vaccines were 95% and 94% effective for preventing symptomatic COVID-19 in Phase III clinical trials. Vaccine candidates from AstraZeneca, Johnson and Johnson and Novavax are also effective, especially for preventing severe disease, and are likely to receive emergency use authorization in the coming months. All the vaccines were shown to be safe.

A Centers for Disease Control and Prevention (CDC) advisory committee developed a vaccine prioritization plan that puthealth care workers and residents of long-term care facilitiesfirst in line, followed bypeople overage 75 and certain frontline essential workers. The CDC later expanded eligibility to include everyone over 65 and people with underlying health conditionsincluding cancerthat put them at risk for more severe COVID-19. But current supplies are nowhere near adequate to vaccinate everyone whos eligible.

The NCCNs COVID-19 Vaccine Committee, which includes top hematology and oncology experts in the areas of infectious diseases, vaccine development and delivery, medical ethics and health information technology, recommends that all people with cancer should get a vaccine. The committee also advises that caregivers and people living in the same household with cancer patients should also get vaccinated when they are eligible.

While clinical trials have shown that the vaccines are highly effective at reducing the risk of becoming ill with COVID-19, it is still not clear how well they prevent asymptomatic infection and transmission, so the committee emphasizes the importance of continuing to follow precautions such as wearing masks and social distancing.

Although people on cancer treatment were excluded from the COVID-19 vaccine trials, experts say theres no reason to think the vaccines wont be safe for this group. The currently authorized vaccines do not contain live virus and therefore cannot cause disease, even in immunocompromised people.

The data we have on these vaccines shows theyre remarkably safe in the general population based on the trials. Admittedly, very few patients with active cancer or in active therapy were included in the trials. But having gone through all the documentation for both of these vaccines, it looks remarkably safe, Gary Lyman, MD, of Fred Hutch, who helped start the COVID-19 and Cancer Consortium, told the Fred Hutch News Service. I have no real concerns that there will be big surprises when it comes to safety for the cancer patient population. The risk to these patients from COVID is high and the risks from the vaccines appear very low.

While the vaccines appear safe for people with cancer, some patients may not respond as well, particularly those whose cancer or treatment causes immune suppression. Some blood cancers affect B cells, the white blood cells that produce antibodiesa key player in vaccine response. Chemotherapy and radiation can deplete white blood cells, and people undergoing stem cell transplants or receiving CAR-T therapy have their own immune cells killed off with chemo or radiation to make room for the new cells.

The NCCN committee recommends that people receiving intensive chemotherapy for leukemia should wait to be vaccinated until their white blood cell count recovers. Stem cell transplant and CAR-T recipients should delay vaccination until three months after the procedure to improved the chances that the vaccine will produce a good immune response. People undergoing major surgery should wait at least a few days. But everyone elseincluding patients receiving chemotherapy for solid tumors, targeted therapy, immunotherapy or radiation therapyshould get a vaccine as soon as they can.

If it is necessary to prioritize among people with cancer, the committee recommends moving those on active treatment (except those taking only hormone therapy), those who plan to start treatment soon and those who have recently finished treatment to the front of the line. Cancer patients with other risk factors, including older age and additional health conditions, should also be prioritized.

Finally, the guidance acknowledges the disparities and social inequities related to COVID-19Black and Latino people are more likely to be exposed to the coronavirus and more likely to develop severe disease and die from it, but are less likely to get vaccinated.

One of our primary goals is reducing morbidity and mortality, saidSirisha Narayana, MD,chair of the University of California at San Francisco Ethics Committee. We also have to take social determinants of health into account and make special efforts for people in high-risk communities.

The medical community is rising to one of the biggest challenges we have ever faced, addedNCCN CEO Robert Carlson, MD. The COVID-19 vaccines exemplify the heights of scientific achievement. Now we have to distribute them quickly, equitably, safely and efficiently, using clearly defined and transparent principles.

Given their higher risk for COVID-19, the NCCN, the American Society of Clinical Oncology and other advocates are asking that people with cancer be given priority for vaccination.

People with metastatic and active cancers die at a rate similar to people over age 75; if we die at the rate of 75 year olds we should be vaccinated with the 75 year olds, Kelly Shanahan, an advocate living with metastatic breast cancer, told Cancer Health. Those of us with active and metastatic cancers dont have the luxury of just staying home. We must get our treatments and scans and see our oncologists. Keep us out of the hospitalsand morguesby prioritizing us for the COVID19 vaccinations!

Click here to read the full NCCN COVID-19 vaccine guidance.

Click here for more news about COVID-19.For more, visit our sister site, COVID Health.com.

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Evotec and Medical Center Hamburg-Eppendorf Enter Partnership to Develop iPSC-Based Tissue Therapy f – PharmiWeb.com

Thursday, February 4th, 2021

DGAP-News: Evotec SE / Key word(s): Miscellaneous04.02.2021 / 07:30 The issuer is solely responsible for the content of this announcement.

Hamburg, Germany, 04 February, 2021:Evotec SE (Frankfurt Stock Exchange: EVT, MDAX/TecDAX, ISIN: DE0005664809) today announced that the Company has entered into a multi-year partnership with the Medical Center Hamburg-Eppendorf ("UKE") for the development of a highly innovative first-in-class cell therapy approach for the treatment of heart failure.

Under the terms of the partnership, Evotec and UKE will leverage their complementary strengths for the development of a new cell therapy approach using Engineered Heart Tissue for the treatment of heart failure. Heart failure is frequently associated with ischemic heart disease and often comes with a poor prognosis. Mortality is comparable to that of the most common cancers, with <50% 4-year survival. Treatment of patients suffering from heart failure is expected to deliver significant patient benefit through improved heart function, ultimately leading to an improved prognosis.

Evotec leverages its industry-leading human induced pluripotent stem cells ("hiPSCs") platform to establish GMP-compatible process development and upscaling for large-scale generation of clinical-grade heart muscle cells known as cardiomyocytes. Evotec will also contribute genetically modified GMP iPSC lines, which contain alterations preventing rejection of the cardiomyocyte-containing product by patient immune systems ("cloaking"), and include additional safety mechanisms to control unwanted proliferation of graft cells. By using these GMP-grade iPSC lines, the project will deliver off-the-shelf products, which can be implanted in broad patient populations with little to no immunosuppression. UKE applies its proprietary Giga Patch Method for the generation of fully functional heart tissue suitable for cardiac transplantation. Further in vivo validation and development activities will be shared jointly between the partners. Evotec will be responsible for GMP and pre-clinical activities as well as for any subsequent partnering of the programme.

Dr Cord Dohrmann, Chief Scientific Officer of Evotec, commented: "We are very excited about this collaboration with the UKE. Both Evotec and UKE have developed and refined their respective technology platforms over a number of years and have now decided to jointly drive this cardiac cell therapy programme towards clinical development. We are confident that this partnership will deliver a new therapeutic option for patients who suffer from heart failure."

Prof. Dr Thomas Eschenhagen, Director of the Institute of Experimental Pharmacology and Toxicology at UKE, added: "We are excited about the new opportunities the partnership with Evotec will create. After having worked on means to repair injured heart by 3-dimensional heart muscle patches for over two decades, joining forces with Evotec and its industrialized hiPSC platform and new cell lines, will bring this development to a new stage. We are aiming at the most efficient and safest therapy in the field."

"We are very happy to see a scientific success story advance to a feat of technology transfer. Translation of scientific insights into therapeutic options is a key mission of our University Medical Center", says Prof. Dr Blanche Schwappach-Pignataro, the Dean of Faculty of Medicine of the UKE.

No financial terms of the agreement were disclosed.

About heart failureHeart failure is a severe global health burden with more than 26 million people suffering with the condition worldwide, disproportionately affecting elderly people. While there are options to treat heart failure both medicinally and with devices, there is currently no treatment that targets the cause of the disease or significantly slows down its progression.

About Evotec and iPSCInduced pluripotent stem cells (also known as iPS cells or iPSCs) are a type of pluripotent stem cell that can be generated directly from adult cells. Pluripotent stem cells hold great promise in the field of regenerative medicine. Because they can propagate indefinitely, as well as give rise to every other cell type in the body (such as neurons, heart, pancreatic and liver cells), they represent a single source of cells that could be used to replace those lost to damage or disease.

Evotec has built an industrialised iPSC infrastructure that represents one of the largest and most sophisticated iPSC platforms in the industry. Evotec's iPSC platform has been developed over the last years with the goal to industrialise iPSC-based drug screening in terms of throughput, reproducibility and robustness to reach the highest industrial standards, and to use iPSC-based cells in cell therapy approaches via the Company's proprietary EVOcells platform.

ABOUT THE MEDICAL CENTER HAMBURG-EPPENDORF (UKE)Since its foundation in 1889, the Medical Center Hamburg-Eppendorf (UKE) has been one of the leading clinics in Europe. With about 13,600 employees, the UKE is one of the largest employers in Hamburg. Each year, the UKE treats around 511,000 patients, 106,000 of whom are inpatients and 405,000 outpatients. The emphasis in UKE's research are the neurosciences, cardiovascular research, care research, oncology, as well as infections and inflammations. Other potential areas of the UKE are molecular imaging and skeletal biology research. The UKE trains about 3,400 medical specialists and dentists.Knowledge, Research, Healing through Shared Competence: The UKE | http://www.uke.de

ABOUT EVOTEC SEEvotec is a drug discovery alliance and development partnership company focused on rapidly progressing innovative product approaches with leading pharmaceutical and biotechnology companies, academics, patient advocacy groups and venture capitalists. We operate worldwide and our more than 3,500 employees provide the highest quality stand-alone and integrated drug discovery and development solutions. We cover all activities from target-to-clinic to meet the industry's need for innovation and efficiency in drug discovery and development (EVT Execute). The Company has established a unique position by assembling top-class scientific experts and integrating state-of-the-art technologies as well as substantial experience and expertise in key therapeutic areas including neuronal diseases, diabetes and complications of diabetes, pain and inflammation, oncology, infectious diseases, respiratory diseases, fibrosis, rare diseases and women's health. On this basis, Evotec has built a broad and deep pipeline of more than 100 co-owned product opportunities at clinical, pre-clinical and discovery stages (EVT Innovate). Evotec has established multiple long-term alliances with partners including Bayer, Boehringer Ingelheim, Bristol Myers Squibb, CHDI, Novartis, Novo Nordisk, Pfizer, Sanofi, Takeda, UCB and others. For additional information please go to http://www.evotec.com and follow us on Twitter @Evotec.

FORWARD-LOOKING STATEMENTSInformation set forth in this press release contains forward-looking statements, which involve a number of risks and uncertainties. The forward-looking statements contained herein represent the judgement of Evotec as of the date of this press release. Such forward-looking statements are neither promises nor guarantees, but are subject to a variety of risks and uncertainties, many of which are beyond our control, and which could cause actual results to differ materially from those contemplated in these forward-looking statements. We expressly disclaim any obligation or undertaking to release publicly any updates or revisions to any such statements to reflect any change in our expectations or any change in events, conditions or circumstances on which any such statement is based.

Media Contact Evotec SE:Gabriele Hansen, SVP Head of Global Corporate Communications & Marketing, Phone: +49.(0)40.56081-255, gabriele.hansen@evotec.com

IR Contact Evotec SE:Volker Braun, SVP Head of Global Investor Relations & ESG, Phone: +49.(0)40.56081-775, volker.braun@evotec.com

04.02.2021 Dissemination of a Corporate News, transmitted by DGAP - a service of EQS Group AG.The issuer is solely responsible for the content of this announcement.

The DGAP Distribution Services include Regulatory Announcements, Financial/Corporate News and Press Releases. Archive at http://www.dgap.de

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APOE Tied to Increased Susceptibility to SARS-CoV-2 | ALZFORUM – Alzforum

Thursday, February 4th, 2021

29 Jan 2021

Part 2 of a two-part series. Click here for Part 1.It turns out a persons risk associated with ApoE4 goes beyond Alzheimers and vascular disease. This apolipoprotein allele can also worsen infections caused by certain viruses. One of those appears to be SARS-CoV-2, the cause of COVID-19, according to two epidemiological studies. In the January 11 Gerontology, researchers led by Miguel Calero, Queen Sofia Foundation Alzheimer Research Center, Madrid, Spain, reported that aged ApoE4 carriers are more likely to show COVID-19 symptoms than ApoE3 carriers. This supports a paper in the September 16, 2020, Journals of Gerontology. Researchers led by David Melzer, University of Exeter, England, U.K., reported that older ApoE4 carriers are more likely to test positive for the virus and more likely to die from COVID-19 than are ApoE3s carriers. Why might this be?

In the January 4 Cell Stem Cell, researchers led by Yanhong Shi, Beckman Research Institute, Duarte, California, and Vaithilingaraja Arumugaswami, University of California, Los Angeles, reported that the new coronavirus infected more ApoE4 neurons and astrocytes than their ApoE3 counterparts in cell culture. Astrocytes stoked the fire, upping the number of infected cells in co-cultures and in astrocyte-containing brain organoids. Infected neurons degenerated, while astrocytes swelled and their nuclei broke apart. Though this may not explain why ApoE4 carriers are at higher risk of COVID-19, it suggests that they may be more prone to long-term neurological symptoms of the disease (see Part 1 of this series).

To probe how various factors, including ApoE genotype, affect the severity of a persons COVID-19, Calero and colleagues phoned people ages 75-94 who, as part of in the Vallecas Project in Madrid, had been genotyped for ApoE. From 20112013, this observational cohort study tracked markers that might predict future dementia (Olarzaran et al., 2015).In April 2020, first author Teodoro del Ser Claero asked Vallecas participants if they had any COVID-19 symptoms, and learned that ApoE4 carriers were 2.4 times as likely to reply in the affirmative, and to have had a COVID-19 diagnosis.

This aligns with Melzer and colleagues data. They sifted through the U.K. Biobank database, which now includes COVID-19 infection data. First author Chia-Ling Kuo stratified participants based on APOE genotype. ApoE4/4 carriers were 2.2 times as likely to have tested positive or have had severe disease, and 4.3 times as likely to have died from COVID-19 than were ApoE3/3s. These differences remained even after the researchers corrected for comorbidities known to worsen COVID-19, such as dementia, hypertension, and Type 2 diabetes.

Even so, Caleb Finch, University of Southern California, Los Angeles, and Alexander Kulminski, Duke University, Durham, North Carolina, think the comorbidities may explain the association. ApoE cluster haplotypes associate with the same morbidities from cardiovascular disease and obesity that increase vulnerability to COVID-19, they note in a review in the same journal. The ApoE locus was first recognized as a genetic determinant of cardiovascular disease in the 1980s, through its effect on blood lipid and cholesterol levels (Sing et al., 1985). Some research even indicated ApoE4 protected people from lipophilic pathogens (Martin, 1999). But at least for COVID, the latest data suggest the opposite. The ApoE trail, like a Moebius strip, takes us back to where we started from, four decades ago, with another view, wrote Finch and Kulminski. To understand how ApoE4 may increase COVID-19 infectivity and mortality, we have returned to the original associations of ApoE variants with blood lipids, vascular disease, and cognition.

Zooming in to the cellular level may provide insight on how ApoE4 renders cells more susceptible to viruses. For example, HIV more easily penetrates human cells if they are ApoE4/4 (Burt et al., 2008). In mice, herpes simplex virus (HSV) exploits the lipoprotein to enter brain cells, leading to a higher viral burden in ApoE4 than ApoE3 transgenic mice (Burgos et al., 2006). What about SARS-CoV-2?

ApoE4 Worsens SARS-CoV-2 Damage. The virus infected neurons and astrocytes in cell culture and brain organoids. ApoE4 cells fared worse, remdesivir protected them. [Courtesy of Wang et al., Cell Stem Cell, 2021.]

Shi wondered if ApoE4 could explain the neurological effects of SARS-CoV-2 in some people. To begin with, co-first authors Cheng Wang, Mingzi Zhang, and colleagues confirmed the virus penchant for certain brain cells (see Part 1 of this series). They differentiated human induced pluripotent stem cells (hiPSCs) into neuronal progenitors (NPCs), neurons, astrocytes, oligodendrocyte progenitor, or brain endothelial cells, then infected them with SARS-CoV-2. Immunostaining detected the viral spike protein in less than 5 percent of NPCs, neurons, and astrocytes, which Shi called a low-grade infection. The virus also infected 60-day-old brain organoids comprising NPCs and neurons.

Astrocytes are known to spread neurotropic viruses in the CNS, including Japanese encephalitis, West Nile, and Zika viruses (Soung et al., 2018; Potokar et al., 2019). Could astrocytes stoke the COVID fire in neurons and organoids? Indeed, more neurons tested positive for SARS-CoV-2 spike protein in neuron-astrocyte co-cultures than in monoculture. The scientists also saw higher viral RNA loads in neurons from organoids that had incorporated astrocytes than in those that did not.

Wang, Zhang, and colleagues then homed in on APOE genotype. They used CRISPR/Cas9 to create isogenic cell lines from iPSCs taken from ApoE3/3 and ApoE4/4 donors. They differentiated the cells into neurons and co-cultured them with ApoE3 astrocytes for three weeks. Then they added SARS-CoV-2, using one viral particle per cell. Immunostaining revealed spike protein in all neurons within 24 hours. After 72 hours, the viral protein content in all neurons had grown, but E4 neurons had 1.5 times more than E3 neurons (see image below).

Viral Invasion. After SARS-Cov-2 infects neurons (purple, left panels), its spike protein (green) popped up within 24 hours (middle) and accumulated over 72 hours (right panel). Infected ApoE4 neurons (bottom) had more viral protein than isogenic ApoE3 lines (top). [Courtesy of Wang et al., Cell Stem Cell, 2021.]

At that point, infected cells formed fewer neurites than did uninfected cells, and the neurites were short. Infected ApoE4 cells had even fewer neurites than ApoE3 cells, and they were shorter still. Staining with Syn 1 revealed fewer synapses in both infected neurons.

What about astrocytes? More iPSC-derived ApoE4/4 astrocytes were infected than iPSC-derived ApoE3/3 cells. The former had fatter somas, longer processes, and nuclei that were more fragmented compared to infected ApoE3 cells (see image below). Taken together, these findings hint at an ApoE-dependent reaction to viral infection, with ApoE4 neurons and astrocytes more severely damaged.

Angrier Astrocytes. SARS-CoV-2-infected (red) E4 astrocyte soma (right four panels) had fragmented nuclei (blue) and grew fatter (green) than E3 astrocyte soma (left four panels) whose nuclei remained intact. [Courtesy of Wang et al., Cell Stem Cell, 2021.]

Why were E4 astrocytes worse off than the E3 cells? Jessica Young, University of Washington, Seattle, thinks it may have to do with endosomes. Of 40 genes previously identified as crucial for SARS-CoV-2 infection, two, the endosomal entry receptor ACTR2 and the ATP6AP2 ATPase, are involved in endosome function (Daniloski et al., 2021). Both are more highly expressed in ApoE4 astrocytes, which have larger early endosomes than do E3 cells (Oct 2020 news). Proteins involved in endosomal entry and transport are more abundant in APOE4 cells, which may facilitate the cellular infectivity of the virus, Young told Alzforum (full comment below). G. William Rebeck, Georgetown University, Washington, D.C., agreed the endosome might be involved. The speculation that these ApoE effects may be due to differentially expressed genes related to endosomal trafficking builds on a model that has been developed across several labs over the past two decades. (Full comment below.)

The FDA-approved drug remdesivir quelled SARS-CoV-2 infection in cultured neurons and astrocytes. When Wang, Zhang, and colleagues pretreated cells with 10 M remdesivir two hours before adding the virus, fewer neurons and astrocytes became infected. The drug also bumped up the number and length of neurites and reduced the number of fragmented nuclei in infected astrocytes compared to vehicle treatment.

Whether this remdesivir is relevant in the clinic remain to be seen. Remdesivir is thought to poorly enter the brain, Rik van der Kant, Vrije Universiteit Amsterdam, and Diederik van de Beek, Amsterdam UMC, wrote in a joint comment (below). David Clifford, Washington University, St. Louis, cautioned against overinterpreting these results based on what he hears from fellow clinicians who are treating COVID-19 patients. Clinically, remdesivir appears minimally effective and is increasingly considered unimportant in COVID-19 patient care, he wrote. Treating the CNS is always more challenging than treating peripheral infections.Chelsea Weidman Burke

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Transforming Outcomes in Advanced CSCC with Immunotherapy – LWW Journals

Thursday, February 4th, 2021

Are there updated data for LIBTAYO in advanced CSCC? What do they show?

Longer-term data from EMPOWER-CSCC-1 were presented at the 2020 American Society of Clinical Oncology (ASCO) virtual meeting. These results showed an ORR of 46% (95% CI: 39%-53%) following treatment with LIBTAYO, with a median time to response of 2 months (interquartile range: 2-4 months) across the three treatment groups, which were metastatic CSCC and locally advanced CSCC dosed at 3mg/kg every 2 weeks and metastatic CSCC dosed at 350mg every 3 weeks. The median time to CR was 11 months (interquartile range: 7.4-14.8months) among those who achieved a CR in any group. The median DoR hadyet to be reached for any treatment group (range for groups combined: 1.9-34.3 months).4,10

Updated response rates arein the table below.4,10

Safety was generally consistent with previous data. The most common adverse reactions reported were fatigue (35%), diarrhea (28%) and nausea (24%). The most common Grade 3 or higher adverse reactions were pneumonitis (3%), autoimmune hepatitis (2%), anemia, colitis and diarrhea (each 1%).

Warnings and Precautions

Severe and Fatal Immune-Mediated Adverse Reactions

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue at any time after starting treatment. While immune-mediated adverse reactions usually occur during treatment, they can also occur after discontinuation. Immune-mediated adverse reactions affecting more than one body system can occur simultaneously. Early identification and management are essential to ensuring safe use of PD-1/PD-L1 blocking antibodies. The definition of immune-mediated adverse reactions included the required use of systemic corticosteroids or other immunosuppressants and the absence of a clear alternate etiology. Monitor closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

No dose reduction for LIBTAYO is recommended. In general, withhold LIBTAYO for severe (Grade 3) immune-mediated adverse reactions. Permanently discontinue LIBTAYO for life-threatening (Grade 4) immune-mediated adverse reactions, recurrent severe (Grade 3) immune-mediated adverse reactions that require systemic immunosuppressive treatment, or an inability to reduce corticosteroid dose to 10 mg or less of prednisone equivalent per day within 12 weeks of initiating steroids.

Withhold or permanently discontinue LIBTAYO depending on severity. In general, if LIBTAYO requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose immune-mediated adverse reactions are not controlled with corticosteroids.

Immune-mediated pneumonitis:LIBTAYO can cause immune-mediated pneumonitis. In patients treated with other PD-1/PD-L1 blocking antibodies, the incidence of pneumonitis is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.7% (22/591) of patients receiving LIBTAYO, including fatal (0.3%), Grade 4 (0.3%), Grade 3 (1.0%), and Grade 2 (1.9%). Pneumonitis led to permanent discontinuation in 1.9% of patients and withholding of LIBTAYO in 1.9% of patients. Systemic corticosteroids were required in all patients with pneumonitis. Pneumonitis resolved in 59% of the 22 patients. Of the 11 patients in whom LIBTAYO was withheld, 7 reinitiated after symptom improvement; of these 1/7 (14%) had recurrence of pneumonitis. Withhold LIBTAYO for Grade 2, and permanently discontinue for Grade 3 or 4. Resume in patients with complete or partial resolution (Grade 0 to 1) after corticosteroid taper. Permanently discontinue if no complete or partial resolution within 12 weeks of initiating steroids or inability to reduce prednisone to less than 10 mg per day (or equivalent) within 12 weeks of initiating steroids.

Immune-mediated colitis: LIBTAYO can cause immune-mediated colitis. The primary component of immune-mediated colitis was diarrhea. Cytomegalovirus (CMV) infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis treated with PD-1/PD-L1 blocking antibodies. In cases of corticosteroid-refractory immune-mediated colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.2% (7/591) of patients receiving LIBTAYO, including Grade 3 (0.3%) and Grade 2 (0.7%). Colitis led to permanent discontinuation in 0.2% of patients and withholding of LIBTAYO in 0.7% of patients. Systemic corticosteroids were required in all patients with colitis. Colitis resolved in 71% of the 7 patients. Of the 4 patients in whom LIBTAYO was withheld, none reinitiated LIBTAYO. Withhold LIBTAYO for Grade 2 or 3, and permanently discontinue for Grade 4. Resume in patients with complete or partial resolution (Grade 0 to 1) after corticosteroid taper. Permanently discontinue if no complete or partial resolution within 12 weeks of initiating steroids or inability to reduce prednisone to less than 10 mg per day (or equivalent) within 12 weeks of initiating steroids.

Immune-mediated hepatitis:LIBTAYO can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 1.9% (11/591) of patients receiving LIBTAYO, including fatal (0.2%), Grade 4 (0.2%), and Grade 3 (1.5%). Hepatitis led to permanent discontinuation of LIBTAYO in 0.8% of patients and withholding of LIBTAYO in 0.8% of patients. Systemic corticosteroids were required in all patients with hepatitis. Additional immunosuppression with mycophenolate was required in 9% (1/11) of these patients. Hepatitis resolved in 64% of the 11 patients. Of the 5 patients in whom LIBTAYO was withheld, none reinitiated LIBTAYO.

For hepatitis with no tumor involvement of the liver: Withhold LIBTAYO if AST or ALT increases to more than 3 and up to 8 times the upper limit of normal (ULN) or if total bilirubin increases to more than 1.5 and up to 3 times the ULN. Permanently discontinue LIBTAYO if AST or ALT increases to more than 8 times the ULN or total bilirubin increases to more than 3 times the ULN.

For hepatitis with tumor involvement of the liver: Withhold LIBTAYO if baseline AST or ALT is more than 1 and up to 3 times ULN and increases to more than 5 and up to 10 times ULN. Also, withhold LIBTAYO if baseline AST or ALT is more than 3 and up to 5 times ULN and increases to more than 8 and up to 10 times ULN. Permanently discontinue LIBTAYO if AST or ALT increases to more than 10 times ULN or if total bilirubin increases to more than 3 times ULN. If AST and ALT are less than or equal to ULN at baseline, withhold or permanently discontinue LIBTAYO based on recommendations for hepatitis with no liver involvement.

Resume in patients with complete or partial resolution (Grade 0 to 1) after corticosteroid taper. Permanently discontinue if no complete or partial resolution within 12 weeks of initiating steroids or inability to reduce prednisone to less than 10 mg per day (or equivalent) within 12 weeks of initiating steroids.

Immune-mediated endocrinopathies: For Grade 3 or 4 endocrinopathies, withhold until clinically stable or permanently discontinue depending on severity.

Immune-mediated nephritis with renal dysfunction: LIBTAYO can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.5% (3/591) of patients receiving LIBTAYO, including Grade 3 (0.3%) and Grade 2 (0.2%). Nephritis led to permanent discontinuation in 0.2% of patients and withholding of LIBTAYO in 0.3% of patients. Systemic corticosteroids were required in all patients with nephritis. Nephritis resolved in all 3 patients. Of the 2 patients in whom LIBTAYO was withheld, none reinitiated LIBTAYO. Withhold LIBTAYO for Grade 2 or 3 increased blood creatinine, and permanently discontinue for Grade 4 increased blood creatinine. Resume in patients with complete or partial resolution (Grade 0 to 1) after corticosteroid taper. Permanently discontinue if no complete or partial resolution within 12 weeks of initiating steroids or inability to reduce prednisone to less than 10 mg per day (or equivalent) within 12 weeks of initiating steroids.

Immune-mediated dermatologic adverse reactions: LIBTAYO can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson Syndrome (SJS), toxic epidermal necrolysis (TEN), and Drug Rash with Eosinophilia and Systemic Symptoms (DRESS) has occurred with PD-1/PD-L1 blocking antibodies. Immune-mediated dermatologic adverse reactions occurred in 2.0% (12/591) of patients receiving LIBTAYO, including Grade 3 (1.0%) and Grade 2 (0.8%). Immune-mediated dermatologic adverse reactions led to permanent discontinuation in 0.3% of patients and withholding of LIBTAYO in 1.4% of patients. Systemic corticosteroids were required in all patients with immune-mediated dermatologic adverse reactions. Immune-mediated dermatologic adverse reactions resolved in 42% of the 12 patients. Of the 8 patients in whom LIBTAYO was withheld for dermatologic adverse reaction, 5 reinitiated LIBTAYO after symptom improvement; of these 60% (3/5) had recurrence of the dermatologic adverse reaction. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate non-exfoliative rashes. Withhold LIBTAYO for suspected SJS, TEN, or DRESS. Permanently discontinue LIBTAYO for confirmed SJS, TEN, or DRESS. Resume in patients with complete or partial resolution (Grade 0 to 1) after corticosteroid taper. Permanently discontinue if no complete or partial resolution within 12 weeks of initiating steroids or inability to reduce prednisone to less than 10 mg per day (or equivalent) within 12 weeks of initiating steroids.

Other immune-mediated adverse reactions: The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% in 591 patients who received LIBTAYO or were reported with the use of other PD-1/PD-L1 blocking antibodies. Severe or fatal cases have been reported for some of these adverse reactions.

Infusion-related reactions

Severe infusion-related reactions (Grade 3) occurred in 0.2% of patients receiving LIBTAYO. Monitor patients for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or 2, and permanently discontinue for Grade 3 or 4.

Complications of Allogeneic HSCT

Fatal and other serious complications can occur in patients who receive allogeneic hematopoietic stem cell transplantation (HSCT) before or after being treated with a PD-1/PD-L1 blocking antibody. Transplant-related complications include hyperacute graft-versus-host-disease (GVHD), acute GVHD, chronic GVHD, hepatic veno-occlusive disease (VOD) after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between PD-1/PD-L1 blockade and allogeneic HSCT. Follow patients closely for evidence of transplant-related complications and intervene promptly. Consider the benefit versus risks of treatment with a PD-1/PD-L1 blocking antibody prior to or after an allogeneic HSCT.

Embryo-fetal toxicity

LIBTAYO can cause fetal harm when administered to a pregnant woman due to an increased risk of immune-mediated rejection of the developing fetus resulting in fetal death. Advise women of the potential risk to a fetus. Advise females of reproductive potential to use effective contraception during treatment with LIBTAYO and for at least 4 months after the last dose.

Adverse reactions

Use in specific populations

Please click here for full Prescribing Information.

INDICATIONAND USAGE

LIBTAYO is indicated for the treatment of patients with metastatic cutaneous squamous cell carcinoma (mCSCC) or locally advanced CSCC (laCSCC) who are not candidates for curative surgery or curative radiation.

++++

References:

1. LIBTAYO (cemiplimab-rwlc) injection full U.S. prescribing information. Regeneron Pharmaceuticals, Inc., and sanofi-aventis U.S. LLC. Available at: https://www.regeneron.com/sites/default/files/Libtayo_FPI.pdf

2. Mansouri B, Housewright C. The treatment of actinic keratosesthe rule rather than the exception. J Am Acad Dermatol 2017; 153(11):1200. doi:10.1001/jamadermatol.2017.3395.

3.Schmults CD, et al. High-Risk Cutaneous Squamous Cell Carcinoma A Practical Guide for Patient Management. Springer. ISBN 978-3-662-47081-7 (eBook).DOI 10.1007/978-3-662-47081-7.

4. Data on File. Regeneron Pharmaceuticals Inc. 2020.

5. Data on File. Regeneron Pharmaceuticals Inc. 2018.

6. Migden M, Rischin D, Schmults C, Guminski A, Hauschild A, Lewis K et al. PD-1 Blockade with Cemiplimab in Advanced Cutaneous Squamous-Cell Carcinoma. New England Journal of Medicine. 2018;379(4):341-351.

7. NCCNClinical Practice Guidelines in Oncology (NCCN Guidelines) forSquamous Cell Skin Cancer V.2.2020. National Comprehensive CancerNetwork, Inc. 2020.

8. Califano JA, Lydiatt WM, Nehal KS, et al. Cutaneous squamous cell carcinoma of the head and neck. In: Amin MB, Edge SB, Greene FL, et al, eds. AJCC Cancer Staging Manual. 8th ed. Springer; 2017:171-181.

9. Jennings L, Schmults CD. Management of high-risk cutaneous squamous cell carcinoma. J Clin Aesthet Dermatol. 2010;3(4):39-48.

10. RischinD, Khushalani NI, Schmults CD, et al. Phase 2 study of cemiplimab in patients with advanced cutaneous squamous cell carcinoma (CSCC): longer follow-up. Poster presented at: American Society of Clinical Oncology (ASCO) 2020 Virtual Scientific Program; May 29-31, 2020.

LIB.20.04.0063 1/21

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Transforming Outcomes in Advanced CSCC with Immunotherapy - LWW Journals

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Ashley Cain is living his worst nightmare as his baby daughter battles leukaemia in hospital – The Sun

Thursday, February 4th, 2021

ASHLEY Cain praised his sick daughter for 'smiling everyday' as she battles leukaemia.

The former footballer's baby daughter Azaylia underwent a stem cell transplant 10 days ago after her leukaemia had returned.

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He told The Sun that four-month old is at high risk of complications and that the transplant has likely a lower success outcome after her leukaemia returned.

But he has since opened up about how the tot is doing after the transplant, admitting Azaylia was in a lot of pain.

Taking to Instagram to share some adorable pictures of his smiling daughter, Ashley let his fans know how was she was going.

He captioned the series of pics: "It has now been 10 days since Azaylias Stem Cell transplant and she still gives Daddy bright eyes and big smiles everyday despite her pain to help me through my worries. "

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"She is going through quite some pain at the moment and has ran into a few side effects over the last 10 days.

Ashley added: "But, she is doing well and her love for life seems to outshine any trouble she is going through. She is a remarkable little girl and her strength fuels my soul every single day!

"LETS GO CHAMP! I BELIVE IN YOU! "

His fans rushed to send words of encouragement to him, Azaylia and his partner, Safiyya Vorajee.

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"Keep going champs," wrote model, Brandon Myers.

MMA fighter Kane Mousah wrote: "Shes Amazing Brother Shes Has Got This LETS GO CHAMP !!! "

Another Instagram user wrote: "What an inspirational little baby girl you have ! Strongest human Ive known yet ... just an inspiring family all in all."

Azaylia had undergone a second round of chemotherapy to clear the cancer ahead of the stem cell transplant.

However doctors informed the couple that despite their best efforts, the leukaemia had returned quickly.

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Azaylia, who is battling the rarest form of the disease, will undergo the transplant tomorrow at Birmingham Childrens Hospital.

Speaking to The Sun, Ashley, 30, explained: All we wanted for Christmas was a match for our daughter. We have been told that we have found a match for our daughter. It is amazing. We have had our routes of ups and downs and this is the next stage."

Ashley and Safiyya have been staying at in theHoliday Inn at Snow Hill, next to the hospital, to be close to Azaylia at all times.

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Speaking ahead of the transplant, Ashley said the news Azaylias leukaemia had returned was hard to comprehend.

He said: It came as a massive blow to us and brought us to our knees. It means she will have to go into transplant with Leukaemia which is not ideal at all.

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We were told that because of her age and the aggressiveness of her leukaemia she is in the high risk category for transplant complications and the poor risk category for transplant success.

This news was incredibly hard to hear and has made every day even more difficult to face since.

Ashley said: Were remaining strong and positive for our daughter, she has got past the first stage of treatment against the odds and we truly believe that she will do the same this time around.

She is a fighter, she is a warrior and she will do this and we will be by her side every step of the way!

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Ashley Cain is living his worst nightmare as his baby daughter battles leukaemia in hospital - The Sun

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Canada’s blood supply has a diversity problem and people are dying because of it – CBC.ca

Monday, February 1st, 2021

Lauren Sanostill wonders,if things were different, whether her father's life couldhave been saved.

"You always wonder if there was someone in the registry who was a bettermatch would have resulted in better outcomes and less transplant complications."

Mark Sanowas a 52-year-old Toronto father of three. He worked in the financial industry as a marketing manager and in his spare time was an avid sportsman who loved tennis, hockey and especially skiing.

In November of 2019 he was diagnosed with a rare form of leukemia. The only thing that couldsave his life, doctors told his family,was a stem cell transplant;a critical treatment for blood cancers and dozens of other diseases.

According to Canadian Blood Services (CBS), stem cells are the body's basic building blocks the raw material from which all cells are made. In blood, stem cells can become red, white blood cells or even blood platelets.

"Without stem cells, the body cannot make the blood cells needed for the immune system to function," CBS says, which runs the national blood bank.

It says a patient must find a match with a donor, and that is usually a person who shares the same ethnic background.

CBS says right now, donors to Canada's blood stem cell registry are more than two-thirds Caucasian, with the other third fracturedin uneven splintersacross race and ethnicity.

It means an Asian patient like Sano, according to the Canadian Blood Services stem cell registry, would have anywhere from seven to less than one per cent chanceof finding a match, depending on hisparticular genetic background.

So when the Sano family sought a match, they found a lack of minority donors who were aclose enough. Sano's daughter Lauren was the closest they could find and even then, she was far from ideal.

"I ended up being a half-match for him and was his donor.It was the most fulfilling and grounding experience."

As fulfilling as it was, it wasn't enough.Sano died at Princess Margaret Hospital in October 2020, 18 months after he was first diagnosed.

Lauren still wonders, whether her dad's life could have been saved, had they found the right donor from a more racially diverse pool of donors.

"I feel very lucky I was able to give him the gift of life.I was at least grateful that I was able to do this for my dad."

The dearth of diversity in Canada's stem cell registry is a problem Canadian Blood Services is familiar with, according toHeidi Elmoazzen, the agency's director of stem cells. Shehas been actively working on increasing the pool of minority donors to give minority patientsa better shot at getting better.

"We find that people tend to find matches within the same ethnic or racial background as them, which is why we're trying to build a registry that reflects the unique diversity we have here in Canada."

Some groups are more diverse than others when it comes to the make up of their stem cells, according to Elmoazzen. For example, she saysBlack people tend to be the most diverse.

A Black person whose ancestors are from the Caribbean might not have the same markers as someone from say, eastern Africa, which makes finding a match challenging.

Adding to the complication is that to harvest stem cells, you literally need young blood.Only young people, between the ages of 17 and 35 can apply.

The ongoing COVID-19 pandemic has also disrupted recruitment efforts, Elmoazzen says.Canadian Blood Services finds 60 to 70 per cent of its potential stem cell donors during its community clinics and with everyone staying home, the number of people visiting is down.

"It's had a heavy impact on our ability to recruit donors this year," she adds.

Still, virtual drives are underway. People interested in donating can still sign up through the Canadian Blood Services website.

There are also volunteers like Lauren Sano, who along with a number of Western University students will be pushing for donations in a virtual blood stem cell drive this month in honour of Black History Month and in April.

The hope is that by reaching out to diverse communities, Sanosays her goal is to help people make donating blood a habit. She says she hopes that willnot only will boost the blood supply, but the supply of blood products, such as stem cells and platelets as well.

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Canada's blood supply has a diversity problem and people are dying because of it - CBC.ca

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Autologous Stem Cell and Non Stem Based therapies Market Share, Size 2021 Global Industry Future Trends, Growth, Strategies,, Segmentation, In-depth…

Monday, February 1st, 2021

Autologous Stem Cell and Non Stem Based therapies Market delivers a succinct analysis of industry size, regional growth and revenue forecasts for the upcoming years. The report further sheds light on significant challenges and the latest growth strategies adopted by manufacturers who are a part of the competitive spectrum of this business domain.

Autologous Stem Cell and Non Stem Based therapies Market: Global Size, Trends, Competitive, Historical & Forecast Analysis, 2021-2027. Rise in the prevalence of Cancer and Diabetes in all age groups population. Furthermore, the growing geriatric population is another key factor which drives the Autologous Stem Cell and Non Stem Based therapies Market.

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Scope Of Market Reports

Autologous Stem Cell transplantation is a process in which cells from which all blood cells develop are removed, preserved and later given to the same person after severe treatment. In autologous stem cell transplantation, the patient itself acts as stem cell donor. These cells are collected in advance while they are in remission and returned to the patient at a later stage i.e., after two months. They are used to replace stem cells which have been impaired by high doses of chemotherapy.It is important to realize that the processes required in a stem cell transplant are lengthy and complicated. A transplant involves a lot of preparation and a lot of care after procedure. Many people have a single autologous stem cell transplant while others mainly having myeloma or tumors; have two or more continuous transplants.

The initial step in an autologous stem cell transplant is gathering the stem cells. Physicians usually collect stem cells from the bloodstream (peripheral blood stem cells) in advance. A mobilization treatment is used. When the stem cells are in the bloodstream, then collection process starts.The blood is separated using an Apheresis machine. This procedure requires a few hours, and is repeated until the appropriate amount of stem cells is collected. Once the stem cells are harvested, they are frozen in our Stem Cell Processing and Cryopreservation Laboratory until its time to transplant.

Autologous Stem Cell and Non Stem Based therapies Market is segmented on the basis of Application, product, End user and Geography. Based upon ApplicationAutologous Stem Cell and Non Stem Based therapies Market is classified as Neurodegenerative Disorders,Autoimmune Diseases, cancer &Tumors, Cardiovascular Diseases and Others. Based on the ProductAutologous Stem Cell and Non Stem Based therapies Market is classified into Blood Pressure Monitoring Devices, Pulmonary Pressure Monitoring Devices and Intracranial Pressure Monitoring Devices. On the basis of End users Autologous Stem Cell and Non Stem Based therapies Market is classified into Hospitals, Ambulatory Surgical Centers and Others.

The regions covered in Autologous Stem Cell and Non Stem Based therapies Market report are North America, Europe, Asia-Pacific and Rest of the World. On the basis of country level, Global Melanoma Drug Market sub divided in to U.S., Mexico, Canada, U.K., France, Germany, Italy, China, Japan, India, South East Asia, GCC, Africa, etc.

Rising prevalence of cancer and diabetes among people across all age groups, growing geriatric population, increasing demand for autologous stem cell and non-stem cell based therapies is another factor, which is likely to create a heightened demand. Moreover, Favorable reimbursement policies across several nations are also boosting market. Risks and complications associated with the Autologous Stem Cell and Non Stem Based therapy such as diarrhea, hair loss, nausea, severe infections, vomiting, heart complications, and infertility and thehigh cost of autologous cellular therapies ranging from $500,000 to $1,000,000 restraint the market. Innovation of some newtherapies with improved efficacy, fewer side effects are expected to offer good opportunity for growth of Autologous Stem Cell and Non Stem Based therapies Market in the future.

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North America is probable to attain the largest share of the Autologous Stem Cell and Non Stem Based therapies Market in terms of revenue and expected to hold the position followed by Europe region. This is due to less risk related with the treatment. Also, the demand for these treatments is high due to their ability to cure a significant number of infectious diseases. Autologous stem cell and non-stem cell based therapies do not require an outside donor hence the treatment is less infectious and cheap. However, Asia Pacific is expected to show the high growth in the forecast period. The demand in this region will be led by countries such as China, India, Malaysia, and Vietnam. The demand is likely to grow as autologous stem cell and non-stem cell based therapies aid in the efficient management of cardiovascular diseases as well. Rising healthcare facilities as well as increasing tax and reimbursement procedures is also estimated to help in the growth of the autologous stem cell and non-stem cell based therapies market in the Asia Pacific.

Furthermore, increase in awareness of disease and government initiatives for improving health care facilities are expected to boost the regional market to a certain extent.

By Application Analysis Neurodegenerative Disorders, Autoimmune Diseases, Cancer & Tumors, Cardiovascular Diseases, Others

By Product Analysis Blood Pressure Monitoring Devices, Pulmonary Pressure Monitoring Devices, Intracranial Pressure Monitoring Devices, Others

By End User Analysis Hospitals, Ambulatory Surgical centers, Others

North America, US, Mexico, Chily, Canada, Europe, UK, France, Germany, Italy, Asia Pacific, China, South Korea, Japan, India, Southeast Asia, Latin America, Brazil, The Middle East and Africa, GCC, Africa, Rest of Middle East and Africa

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https://www.marketwatch.com/press-release/at-1971-cagr-zero-trust-security-market-size-is-projected-to-reach-9435-billion-by-2027-says-brandessence-market-research-2021-01-25?tesla=y

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Merck Receives Positive EU CHMP Opinion for Expanded Approval of KEYTRUDA (pembrolizumab) in Certain Patients With Relapsed or Refractory Classical…

Monday, February 1st, 2021

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced that the Committee for Medicinal Products for Human Use (CHMP) of the European Medicines Agency (EMA) has adopted a positive opinion recommending approval of an expanded label for KEYTRUDA, Mercks anti-PD-1 therapy. The opinion is recommending KEYTRUDA as monotherapy for the treatment of adult and pediatric patients aged 3 years and older with relapsed or refractory classical Hodgkin lymphoma (cHL) who have failed autologous stem cell transplant (ASCT) or following at least two prior therapies when ASCT is not a treatment option.

This recommendation is based on results from the pivotal Phase 3 KEYNOTE-204 trial, in which KEYTRUDA monotherapy demonstrated a significant improvement in progression-free survival (PFS) compared with brentuximab vedotin (BV), a commonly used treatment. KEYTRUDA reduced the risk of disease progression or death by 35% (HR=0.65 [95% CI, 0.48-0.88]; p=0.00271) and showed a median PFS of 13.2 months versus 8.3 months for patients treated with BV. The recommendation is also based on supportive data from an updated analysis of the KEYNOTE-087 trial, which supported the European Commissions (EC) approval of KEYTRUDA for the treatment of adult patients with relapsed or refractory cHL who have failed ASCT and BV or who are transplant ineligible and have failed BV. The CHMPs recommendation will now be reviewed by the EC for marketing authorization in the European Union (EU), and a final decision is expected in the first quarter of 2021. If approved, this will be the first pediatric indication for KEYTRUDA in the EU.

This positive opinion reinforces the importance of KEYTRUDA for certain adult and pediatric patients with relapsed or refractory classical Hodgkin lymphoma in the European Union, said Dr. Vicki Goodman, vice president, clinical research, Merck Research Laboratories. We look forward to the decision by the European Commission and will continue to expand our clinical development program in blood cancers with KEYTRUDA and our recently acquired investigational therapies to help address the unmet needs of patients.

Merck is studying KEYTRUDA across hematologic malignancies through a broad clinical program, including multiple registrational trials in cHL and primary mediastinal large B-cell lymphoma and more than 60 investigator-initiated studies across 15 tumors. In addition to KEYTRUDA, Merck is evaluating two clinical-stage assets for the treatment of patients with hematologic malignancies: MK-1026 (formerly ARQ 531), a Brutons tyrosine kinase inhibitor, and VLS-101, an antibody-drug conjugate targeting ROR1.

About KEYNOTE-204

KEYNOTE-204 (ClinicalTrials.gov, NCT02684292) is a randomized, open-label, Phase 3 trial evaluating KEYTRUDA monotherapy compared with BV for the treatment of patients with relapsed or refractory cHL. The primary endpoints are PFS and overall survival (OS), and the secondary endpoints include objective response rate (ORR), complete remission rate (CRR) and safety. The study enrolled 304 patients, aged 18 years and older, who were randomized to receive either:

About Hodgkin Lymphoma

Hodgkin lymphoma is a type of lymphoma that develops in the white blood cells called lymphocytes, which are part of the immune system. Hodgkin lymphoma can start almost anywhere most often in lymph nodes in the upper part of the body, with the most common sites being in the chest, neck or under the arms. Worldwide, there were approximately 83,000 new cases of Hodgkin lymphoma diagnosed, and more than 23,000 people died from the disease in 2020. In the EU, there were nearly 20,000 new cases of Hodgkin lymphoma diagnosed, and nearly 4,000 people died from the disease in 2020. Classical Hodgkin lymphoma accounts for more than nine in 10 cases of Hodgkin lymphoma in developed countries.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,300 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Small Cell Lung Cancer

KEYTRUDA is indicated for the treatment of patients with metastatic small cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy and at least 1 other prior line of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS 10), as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test, with disease progression after one or more prior lines of systemic therapy.

Cervical Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test. This indication is approved under accelerated approval based on progression-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the programmed death receptor-1 (PD-1) or the programmed death ligand 1 (PD-L1), blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% of these patients interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (1.1%), and Grade 2 (0.4%) reactions. Systemic corticosteroids were required in 69% (33/48); additional immunosuppressant therapy was required in 4.2% of patients. Colitis led to permanent discontinuation of KEYTRUDA in 0.5% (15) and withholding in 0.5% (13) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Colitis resolved in 85% of the 48 patients.

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.4%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 68% (13/19) of patients; additional immunosuppressant therapy was required in 11% of patients. Hepatitis led to permanent discontinuation of KEYTRUDA in 0.2% (6) and withholding in 0.3% (9) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Hepatitis resolved in 79% of the 19 patients.

KEYTRUDA with Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen, which was at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.3%) reactions. Systemic corticosteroids were required in 77% (17/22) of patients; of these, the majority remained on systemic corticosteroids. Adrenal insufficiency led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.3% (8) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Hypophysitis

KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.2%) reactions. Systemic corticosteroids were required in 94% (16/17) of patients; of these, the majority remained on systemic corticosteroids. Hypophysitis led to permanent discontinuation of KEYTRUDA in 0.1% (4) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in <0.1% (1) of patients.

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in <0.1% (2) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. Hypothyroidism occurred in 8% (237/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (6.2%). It led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.5% (14) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. The majority of patients with hypothyroidism required long-term thyroid hormone replacement. The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC, occurring in 16% of patients receiving KEYTRUDA as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. The incidence of new or worsening hypothyroidism was higher in 389 adult patients with cHL (17%) receiving KEYTRUDA as a single agent, including Grade 1 (6.2%) and Grade 2 (10.8%) hypothyroidism.

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in <0.1% (1) and withholding of KEYTRUDA in <0.1% (1). All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.1%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 89% (8/9) of patients. Nephritis led to permanent discontinuation of KEYTRUDA in 0.1% (3) and withholding in 0.1% (3) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Nephritis resolved in 56% of the 9 patients.

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received KEYTRUDA or were reported with the use of other antiPD-1/PD-L1 treatments. Severe or fatal cases have been reported for some of these adverse reactions. Cardiac/Vascular: Myocarditis, pericarditis, vasculitis; Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: Myositis/polymyositis rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica; Endocrine: Hypoparathyroidism; Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic HSCT before or after antiPD-1/PD-L1 treatment. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute and chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between antiPD-1/PD-L1 treatment and allogeneic HSCT. Follow patients closely for evidence of these complications and intervene promptly. Consider the benefit vs risks of using antiPD-1/PD-L1 treatments prior to or after an allogeneic HSCT.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with an antiPD-1/PD-L1 treatment in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

Adverse Reactions

In KEYNOTE-006, KEYTRUDA was discontinued due to adverse reactions in 9% of 555 patients with advanced melanoma; adverse reactions leading to permanent discontinuation in more than one patient were colitis (1.4%), autoimmune hepatitis (0.7%), allergic reaction (0.4%), polyneuropathy (0.4%), and cardiac failure (0.4%). The most common adverse reactions (20%) with KEYTRUDA were fatigue (28%), diarrhea (26%), rash (24%), and nausea (21%).

In KEYNOTE-054, KEYTRUDA was permanently discontinued due to adverse reactions in 14% of 509 patients; the most common (1%) were pneumonitis (1.4%), colitis (1.2%), and diarrhea (1%). Serious adverse reactions occurred in 25% of patients receiving KEYTRUDA. The most common adverse reaction (20%) with KEYTRUDA was diarrhea (28%).

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Merck Receives Positive EU CHMP Opinion for Expanded Approval of KEYTRUDA (pembrolizumab) in Certain Patients With Relapsed or Refractory Classical...

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Merck Presents Results From Head-to-Head Phase 3 KEYNOTE-598 Trial Evaluating KEYTRUDA (pembrolizumab) in Combination With Ipilimumab Versus KEYTRUDA…

Monday, February 1st, 2021

In KEYNOTE-598, the addition of ipilimumab to KEYTRUDA did not improve overall survival or progression-free survival, and patients who received the combination were more likely to experience serious side effects than those who received KEYTRUDA monotherapy, said Dr. Michael Boyer, chief clinical officer and conjoint chair of thoracic oncology, Chris OBrien Lifehouse, Camperdown, NSW, Australia. KEYTRUDA monotherapy remains a standard of care for the first-line treatment of certain patients with metastatic non-small cell lung cancer whose tumors express PD-L1.

As a leader in lung cancer, we are pursuing a broad clinical program to better understand the potential of KEYTRUDA-based combinations to improve survival outcomes for patients with this devastating disease, said Dr. Roy Baynes, senior vice president and head of global clinical development, chief medical officer, Merck Research Laboratories. KEYNOTE-598 is the first head-to-head study designed to answer the question of whether combining KEYTRUDA with ipilimumab provided additional clinical benefits beyond treatment with KEYTRUDA alone in certain patients with metastatic non-small cell lung cancer. The results are clear the combination did not add clinical benefit but did add toxicity.

These results were presented in the Presidential Symposium at the IASLC 2020 World Conference on Lung Cancer hosted by the International Association for the Study of Lung Cancer on Friday, Jan. 29 and published in the Journal of Clinical Oncology. As previously announced in Nov. 2020, the study was discontinued due to futility based on the recommendation of an independent Data Monitoring Committee (DMC), which determined the benefit/risk profile of KEYTRUDA in combination with ipilimumab did not support continuing the trial. The DMC also advised that patients in the study discontinue treatment with ipilimumab/placebo.

KEYNOTE-598 Study Design and Additional Data (Late-Breaking Abstract #PS01.09)

KEYNOTE-598 (ClinicalTrials.gov, NCT03302234) is a randomized, double-blind, Phase 3 trial designed to evaluate KEYTRUDA in combination with ipilimumab compared to KEYTRUDA monotherapy as first-line treatment for patients with metastatic NSCLC without EGFR or ALK genomic tumor aberrations and whose tumors express PD-L1 (TPS 50%). The dual primary endpoints are OS and PFS. Secondary endpoints include objective response rate (ORR), duration of response (DOR) and safety.

The study enrolled 568 patients who were randomized 1:1 to receive KEYTRUDA (200 mg intravenously [IV] on Day 1 of each three-week cycle for up to 35 cycles) in combination with ipilimumab (1 mg/kg IV on Day 1 of each six-week cycle for up to 18 cycles); or KEYTRUDA (200 mg IV on Day 1 of each three-week cycle for up to 35 cycles) as monotherapy. Non-binding futility criteria for the study were based on restricted mean survival time (RMST), an alternative outcome measure estimated as the area under the survival curve through a fixed timepoint. The pre-specified criteria were differences in RMST for KEYTRUDA in combination with ipilimumab and KEYTRUDA monotherapy of 0.2 at the maximum observation time and 0.1 at 24 months of follow-up.

As of data cut-off, the median study follow-up was 20.6 months. Findings showed the median OS was 21.4 months for patients randomized to KEYTRUDA in combination with ipilimumab (n=284) versus 21.9 months for those randomized to KEYTRUDA monotherapy (n=284) (HR=1.08 [95% CI, 0.85-1.37]; p=0.74). The differences in RMST for KEYTRUDA in combination with ipilimumab and KEYTRUDA monotherapy were -0.56 at the maximum observation time and -0.52 at 24 months, meeting the futility criteria for the trial and confirming the benefit/risk profile of the combination did not support continuing the study. Additionally, the median PFS was 8.2 months for patients randomized to KEYTRUDA in combination with ipilimumab versus 8.4 months for those randomized to KEYTRUDA monotherapy (HR=1.06 [95% CI, 0.86-1.30]; p=0.72). In both arms of the study, ORR was 45.4%; the median DOR was 16.1 months for patients randomized to KEYTRUDA in combination with ipilimumab versus 17.3 months for those randomized to KEYTRUDA monotherapy.

No new safety signals for KEYTRUDA monotherapy were observed. Treatment-related adverse events (TRAEs) occurred in 76.2% of patients treated with KEYTRUDA in combination with ipilimumab versus 68.3% of patients treated with KEYTRUDA monotherapy. Of these TRAEs, 35.1% vs. 19.6% were Grade 3-5, 27.7% vs. 13.9% were serious, 6.0% vs. 3.2% led to discontinuation of ipilimumab or placebo, 19.1% vs. 7.5% led to discontinuation of both drugs and 2.5% vs. 0.0% (no patients) led to death. Additionally, immune-mediated adverse events (AEs) and infusion reactions occurred in 44.7% of patients treated with KEYTRUDA in combination with ipilimumab versus 32.4% of patients treated with KEYTRUDA monotherapy. Of these immune-mediated AEs, 20.2% vs. 7.8% were Grade 3-5, 19.1% vs. 7.1% were serious, 1.8% vs. 1.1% led to discontinuation of ipilimumab or placebo, 12.1% vs. 4.3% led to discontinuation of both drugs and 2.1% vs. 0.0% (no patients) led to death.

About Lung Cancer

Lung cancer, which forms in the tissues of the lungs, usually within cells lining the air passages, is the leading cause of cancer death worldwide. Each year, more people die of lung cancer than die of colon and breast cancers combined. The two main types of lung cancer are non-small cell and small cell. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 85% of all cases. Small cell lung cancer (SCLC) accounts for about 10% to 15% of all lung cancers. Before 2014, the five-year survival rate for patients diagnosed in the U.S. with NSCLC and SCLC was estimated to be 5% and 6%, respectively.

About KEYTRUDA (pembrolizumab) Injection, 100 mg

KEYTRUDA is an anti-PD-1 therapy that works by increasing the ability of the bodys immune system to help detect and fight tumor cells. KEYTRUDA is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, thereby activating T lymphocytes which may affect both tumor cells and healthy cells.

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,300 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

Selected KEYTRUDA (pembrolizumab) Indications in the U.S.

Melanoma

KEYTRUDA is indicated for the treatment of patients with unresectable or metastatic melanoma.

KEYTRUDA is indicated for the adjuvant treatment of patients with melanoma with involvement of lymph node(s) following complete resection.

Non-Small Cell Lung Cancer

KEYTRUDA, in combination with pemetrexed and platinum chemotherapy, is indicated for the first-line treatment of patients with metastatic nonsquamous non-small cell lung cancer (NSCLC), with no EGFR or ALK genomic tumor aberrations.

KEYTRUDA, in combination with carboplatin and either paclitaxel or paclitaxel protein-bound, is indicated for the first-line treatment of patients with metastatic squamous NSCLC.

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with NSCLC expressing PD-L1 [tumor proportion score (TPS) 1%] as determined by an FDA-approved test, with no EGFR or ALK genomic tumor aberrations, and is stage III where patients are not candidates for surgical resection or definitive chemoradiation, or metastatic.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with metastatic NSCLC whose tumors express PD-L1 (TPS 1%) as determined by an FDA-approved test, with disease progression on or after platinum-containing chemotherapy. Patients with EGFR or ALK genomic tumor aberrations should have disease progression on FDA-approved therapy for these aberrations prior to receiving KEYTRUDA.

Small Cell Lung Cancer

KEYTRUDA is indicated for the treatment of patients with metastatic small cell lung cancer (SCLC) with disease progression on or after platinum-based chemotherapy and at least 1 other prior line of therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

Head and Neck Squamous Cell Cancer

KEYTRUDA, in combination with platinum and fluorouracil (FU), is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent head and neck squamous cell carcinoma (HNSCC).

KEYTRUDA, as a single agent, is indicated for the first-line treatment of patients with metastatic or with unresectable, recurrent HNSCC whose tumors express PD-L1 [combined positive score (CPS) 1] as determined by an FDA-approved test.

KEYTRUDA, as a single agent, is indicated for the treatment of patients with recurrent or metastatic HNSCC with disease progression on or after platinum-containing chemotherapy.

Classical Hodgkin Lymphoma

KEYTRUDA is indicated for the treatment of adult patients with relapsed or refractory classical Hodgkin lymphoma (cHL).

KEYTRUDA is indicated for the treatment of pediatric patients with refractory cHL, or cHL that has relapsed after 2 or more lines of therapy.

Primary Mediastinal Large B-Cell Lymphoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with refractory primary mediastinal large B-cell lymphoma (PMBCL), or who have relapsed after 2 or more prior lines of therapy. KEYTRUDA is not recommended for treatment of patients with PMBCL who require urgent cytoreductive therapy.

Urothelial Carcinoma

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (CPS 10), as determined by an FDA-approved test, or in patients who are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status. This indication is approved under accelerated approval based on tumor response rate and duration of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trials.

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic urothelial carcinoma (mUC) who have disease progression during or following platinum-containing chemotherapy or within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.

KEYTRUDA is indicated for the treatment of patients with Bacillus Calmette-Guerin (BCG)-unresponsive, high-risk, non-muscle invasive bladder cancer (NMIBC) with carcinoma in situ (CIS) with or without papillary tumors who are ineligible for or have elected not to undergo cystectomy.

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)

This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with MSI-H central nervous system cancers have not been established.

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

KEYTRUDA is indicated for the first-line treatment of patients with unresectable or metastatic MSI-H or dMMR colorectal cancer (CRC).

Gastric Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic gastric or gastroesophageal junction (GEJ) adenocarcinoma whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test, with disease progression on or after two or more prior lines of therapy including fluoropyrimidine- and platinum-containing chemotherapy and if appropriate, HER2/neu-targeted therapy. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent locally advanced or metastatic squamous cell carcinoma of the esophagus whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test, with disease progression after one or more prior lines of systemic therapy.

Cervical Cancer

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cervical cancer with disease progression on or after chemotherapy whose tumors express PD-L1 (CPS 1) as determined by an FDA-approved test. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Hepatocellular Carcinoma

KEYTRUDA is indicated for the treatment of patients with hepatocellular carcinoma (HCC) who have been previously treated with sorafenib. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Merkel Cell Carcinoma

KEYTRUDA is indicated for the treatment of adult and pediatric patients with recurrent locally advanced or metastatic Merkel cell carcinoma (MCC). This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Renal Cell Carcinoma

KEYTRUDA, in combination with axitinib, is indicated for the first-line treatment of patients with advanced renal cell carcinoma (RCC).

Tumor Mutational Burden-High

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic tumor mutational burden-high (TMB-H) [10 mutations/megabase] solid tumors, as determined by an FDA-approved test, that have progressed following prior treatment and who have no satisfactory alternative treatment options. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials. The safety and effectiveness of KEYTRUDA in pediatric patients with TMB-H central nervous system cancers have not been established.

Cutaneous Squamous Cell Carcinoma

KEYTRUDA is indicated for the treatment of patients with recurrent or metastatic cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation.

Triple-Negative Breast Cancer

KEYTRUDA, in combination with chemotherapy, is indicated for the treatment of patients with locally recurrent unresectable or metastatic triple-negative breast cancer (TNBC) whose tumors express PD-L1 (CPS 10) as determined by an FDA-approved test.

This indication is approved under accelerated approval based on progression-free survival. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

KEYTRUDA is a monoclonal antibody that belongs to a class of drugs that bind to either the programmed death receptor-1 (PD-1) or the programmed death ligand 1 (PD-L1), blocking the PD-1/PD-L1 pathway, thereby removing inhibition of the immune response, potentially breaking peripheral tolerance and inducing immune-mediated adverse reactions. Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue, can affect more than one body system simultaneously, and can occur at any time after starting treatment or after discontinuation of treatment. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions.

Monitor patients closely for symptoms and signs that may be clinical manifestations of underlying immune-mediated adverse reactions. Early identification and management are essential to ensure safe use of antiPD-1/PD-L1 treatments. Evaluate liver enzymes, creatinine, and thyroid function at baseline and periodically during treatment. In cases of suspected immune-mediated adverse reactions, initiate appropriate workup to exclude alternative etiologies, including infection. Institute medical management promptly, including specialty consultation as appropriate.

Withhold or permanently discontinue KEYTRUDA depending on severity of the immune-mediated adverse reaction. In general, if KEYTRUDA requires interruption or discontinuation, administer systemic corticosteroid therapy (1 to 2 mg/kg/day prednisone or equivalent) until improvement to Grade 1 or less. Upon improvement to Grade 1 or less, initiate corticosteroid taper and continue to taper over at least 1 month. Consider administration of other systemic immunosuppressants in patients whose adverse reactions are not controlled with corticosteroid therapy.

Immune-Mediated Pneumonitis

KEYTRUDA can cause immune-mediated pneumonitis. The incidence is higher in patients who have received prior thoracic radiation. Immune-mediated pneumonitis occurred in 3.4% (94/2799) of patients receiving KEYTRUDA, including fatal (0.1%), Grade 4 (0.3%), Grade 3 (0.9%), and Grade 2 (1.3%) reactions. Systemic corticosteroids were required in 67% (63/94) of patients. Pneumonitis led to permanent discontinuation of KEYTRUDA in 1.3% (36) and withholding in 0.9% (26) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Pneumonitis resolved in 59% of the 94 patients.

Pneumonitis occurred in 8% (31/389) of adult patients with cHL receiving KEYTRUDA as a single agent, including Grades 3-4 in 2.3% of patients. Patients received high-dose corticosteroids for a median duration of 10 days (range: 2 days to 53 months). Pneumonitis rates were similar in patients with and without prior thoracic radiation. Pneumonitis led to discontinuation of KEYTRUDA in 5.4% (21) of patients. Of the patients who developed pneumonitis, 42% interrupted KEYTRUDA, 68% discontinued KEYTRUDA, and 77% had resolution.

Immune-Mediated Colitis

KEYTRUDA can cause immune-mediated colitis, which may present with diarrhea. Cytomegalovirus infection/reactivation has been reported in patients with corticosteroid-refractory immune-mediated colitis. In cases of corticosteroid-refractory colitis, consider repeating infectious workup to exclude alternative etiologies. Immune-mediated colitis occurred in 1.7% (48/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (1.1%), and Grade 2 (0.4%) reactions. Systemic corticosteroids were required in 69% (33/48); additional immunosuppressant therapy was required in 4.2% of patients. Colitis led to permanent discontinuation of KEYTRUDA in 0.5% (15) and withholding in 0.5% (13) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 23% had recurrence. Colitis resolved in 85% of the 48 patients.

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

KEYTRUDA can cause immune-mediated hepatitis. Immune-mediated hepatitis occurred in 0.7% (19/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.4%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 68% (13/19) of patients; additional immunosuppressant therapy was required in 11% of patients. Hepatitis led to permanent discontinuation of KEYTRUDA in 0.2% (6) and withholding in 0.3% (9) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Hepatitis resolved in 79% of the 19 patients.

KEYTRUDA with Axitinib

KEYTRUDA in combination with axitinib can cause hepatic toxicity. Monitor liver enzymes before initiation of and periodically throughout treatment. Consider monitoring more frequently as compared to when the drugs are administered as single agents. For elevated liver enzymes, interrupt KEYTRUDA and axitinib, and consider administering corticosteroids as needed. With the combination of KEYTRUDA and axitinib, Grades 3 and 4 increased alanine aminotransferase (ALT) (20%) and increased aspartate aminotransferase (AST) (13%) were seen at a higher frequency compared to KEYTRUDA alone. Fifty-nine percent of the patients with increased ALT received systemic corticosteroids. In patients with ALT 3 times upper limit of normal (ULN) (Grades 2-4, n=116), ALT resolved to Grades 0-1 in 94%. Among the 92 patients who were rechallenged with either KEYTRUDA (n=3) or axitinib (n=34) administered as a single agent or with both (n=55), recurrence of ALT 3 times ULN was observed in 1 patient receiving KEYTRUDA, 16 patients receiving axitinib, and 24 patients receiving both. All patients with a recurrence of ALT 3 ULN subsequently recovered from the event.

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

KEYTRUDA can cause primary or secondary adrenal insufficiency. For Grade 2 or higher, initiate symptomatic treatment, including hormone replacement as clinically indicated. Withhold KEYTRUDA depending on severity. Adrenal insufficiency occurred in 0.8% (22/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.3%) reactions. Systemic corticosteroids were required in 77% (17/22) of patients; of these, the majority remained on systemic corticosteroids. Adrenal insufficiency led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.3% (8) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Hypophysitis

KEYTRUDA can cause immune-mediated hypophysitis. Hypophysitis can present with acute symptoms associated with mass effect such as headache, photophobia, or visual field defects. Hypophysitis can cause hypopituitarism. Initiate hormone replacement as indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Hypophysitis occurred in 0.6% (17/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.3%), and Grade 2 (0.2%) reactions. Systemic corticosteroids were required in 94% (16/17) of patients; of these, the majority remained on systemic corticosteroids. Hypophysitis led to permanent discontinuation of KEYTRUDA in 0.1% (4) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Thyroid Disorders

KEYTRUDA can cause immune-mediated thyroid disorders. Thyroiditis can present with or without endocrinopathy. Hypothyroidism can follow hyperthyroidism. Initiate hormone replacement for hypothyroidism or institute medical management of hyperthyroidism as clinically indicated. Withhold or permanently discontinue KEYTRUDA depending on severity. Thyroiditis occurred in 0.6% (16/2799) of patients receiving KEYTRUDA, including Grade 2 (0.3%). None discontinued, but KEYTRUDA was withheld in <0.1% (1) of patients.

Hyperthyroidism occurred in 3.4% (96/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (0.8%). It led to permanent discontinuation of KEYTRUDA in <0.1% (2) and withholding in 0.3% (7) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. Hypothyroidism occurred in 8% (237/2799) of patients receiving KEYTRUDA, including Grade 3 (0.1%) and Grade 2 (6.2%). It led to permanent discontinuation of KEYTRUDA in <0.1% (1) and withholding in 0.5% (14) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement. The majority of patients with hypothyroidism required long-term thyroid hormone replacement. The incidence of new or worsening hypothyroidism was higher in 1185 patients with HNSCC, occurring in 16% of patients receiving KEYTRUDA as a single agent or in combination with platinum and FU, including Grade 3 (0.3%) hypothyroidism. The incidence of new or worsening hypothyroidism was higher in 389 adult patients with cHL (17%) receiving KEYTRUDA as a single agent, including Grade 1 (6.2%) and Grade 2 (10.8%) hypothyroidism.

Type 1 Diabetes Mellitus (DM), Which Can Present With Diabetic Ketoacidosis

Monitor patients for hyperglycemia or other signs and symptoms of diabetes. Initiate treatment with insulin as clinically indicated. Withhold KEYTRUDA depending on severity. Type 1 DM occurred in 0.2% (6/2799) of patients receiving KEYTRUDA. It led to permanent discontinuation in <0.1% (1) and withholding of KEYTRUDA in <0.1% (1). All patients who were withheld reinitiated KEYTRUDA after symptom improvement.

Immune-Mediated Nephritis With Renal Dysfunction

KEYTRUDA can cause immune-mediated nephritis. Immune-mediated nephritis occurred in 0.3% (9/2799) of patients receiving KEYTRUDA, including Grade 4 (<0.1%), Grade 3 (0.1%), and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 89% (8/9) of patients. Nephritis led to permanent discontinuation of KEYTRUDA in 0.1% (3) and withholding in 0.1% (3) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, none had recurrence. Nephritis resolved in 56% of the 9 patients.

Immune-Mediated Dermatologic Adverse Reactions

KEYTRUDA can cause immune-mediated rash or dermatitis. Exfoliative dermatitis, including Stevens-Johnson syndrome, drug rash with eosinophilia and systemic symptoms, and toxic epidermal necrolysis, has occurred with antiPD-1/PD-L1 treatments. Topical emollients and/or topical corticosteroids may be adequate to treat mild to moderate nonexfoliative rashes. Withhold or permanently discontinue KEYTRUDA depending on severity. Immune-mediated dermatologic adverse reactions occurred in 1.4% (38/2799) of patients receiving KEYTRUDA, including Grade 3 (1%) and Grade 2 (0.1%) reactions. Systemic corticosteroids were required in 40% (15/38) of patients. These reactions led to permanent discontinuation in 0.1% (2) and withholding of KEYTRUDA in 0.6% (16) of patients. All patients who were withheld reinitiated KEYTRUDA after symptom improvement; of these, 6% had recurrence. The reactions resolved in 79% of the 38 patients.

Other Immune-Mediated Adverse Reactions

The following clinically significant immune-mediated adverse reactions occurred at an incidence of <1% (unless otherwise noted) in patients who received KEYTRUDA or were reported with the use of other antiPD-1/PD-L1 treatments. Severe or fatal cases have been reported for some of these adverse reactions. Cardiac/Vascular: Myocarditis, pericarditis, vasculitis; Nervous System: Meningitis, encephalitis, myelitis and demyelination, myasthenic syndrome/myasthenia gravis (including exacerbation), Guillain-Barr syndrome, nerve paresis, autoimmune neuropathy; Ocular: Uveitis, iritis and other ocular inflammatory toxicities can occur. Some cases can be associated with retinal detachment. Various grades of visual impairment, including blindness, can occur. If uveitis occurs in combination with other immune-mediated adverse reactions, consider a Vogt-Koyanagi-Harada-like syndrome, as this may require treatment with systemic steroids to reduce the risk of permanent vision loss; Gastrointestinal: Pancreatitis, to include increases in serum amylase and lipase levels, gastritis, duodenitis; Musculoskeletal and Connective Tissue: Myositis/polymyositis rhabdomyolysis (and associated sequelae, including renal failure), arthritis (1.5%), polymyalgia rheumatica; Endocrine: Hypoparathyroidism; Hematologic/Immune: Hemolytic anemia, aplastic anemia, hemophagocytic lymphohistiocytosis, systemic inflammatory response syndrome, histiocytic necrotizing lymphadenitis (Kikuchi lymphadenitis), sarcoidosis, immune thrombocytopenic purpura, solid organ transplant rejection.

Infusion-Related Reactions

KEYTRUDA can cause severe or life-threatening infusion-related reactions, including hypersensitivity and anaphylaxis, which have been reported in 0.2% of 2799 patients receiving KEYTRUDA. Monitor for signs and symptoms of infusion-related reactions. Interrupt or slow the rate of infusion for Grade 1 or Grade 2 reactions. For Grade 3 or Grade 4 reactions, stop infusion and permanently discontinue KEYTRUDA.

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

Fatal and other serious complications can occur in patients who receive allogeneic HSCT before or after antiPD-1/PD-L1 treatments. Transplant-related complications include hyperacute graft-versus-host disease (GVHD), acute and chronic GVHD, hepatic veno-occlusive disease after reduced intensity conditioning, and steroid-requiring febrile syndrome (without an identified infectious cause). These complications may occur despite intervening therapy between antiPD-1/PD-L1 treatments and allogeneic HSCT. Follow patients closely for evidence of these complications and intervene promptly. Consider the benefit vs risks of using antiPD-1/PD-L1 treatments prior to or after an allogeneic HSCT.

Increased Mortality in Patients With Multiple Myeloma

In trials in patients with multiple myeloma, the addition of KEYTRUDA to a thalidomide analogue plus dexamethasone resulted in increased mortality. Treatment of these patients with an antiPD-1/PD-L1 treatment in this combination is not recommended outside of controlled trials.

Embryofetal Toxicity

Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. Advise women of this potential risk. In females of reproductive potential, verify pregnancy status prior to initiating KEYTRUDA and advise them to use effective contraception during treatment and for 4 months after the last dose.

See more here:
Merck Presents Results From Head-to-Head Phase 3 KEYNOTE-598 Trial Evaluating KEYTRUDA (pembrolizumab) in Combination With Ipilimumab Versus KEYTRUDA...

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Disabled People Are Waiting, Anxiously, For Lifesaving Covid-19 Vaccinations – Forbes

Monday, February 1st, 2021

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On January 26, 2021, Governor Gavin Newsom announced that California would alter its previous plan to start offering vaccines to high risk adults under 65 in the next vaccination phase. Instead, future eligibility levels in the state will be determined solely by age.

The United States Centers for Disease Controls current non-binding recommendation is to offer vaccines to disabled and chronically ill people aged 1664 years with underlying medical conditions which increase the risk of serious, life-threatening complications from COVID-19 in Phase 1c. In many states that would mean eligibility in a month or two, once the current first phases are completed. That was the plan for California, too, until this past week.

This move in California deeply disappointed the disabled community, and intensified growing concern among disabled and chronically ill people nationwide.

In a January 28 press conference, Andy Imparato, Executive Director of Disability Rights California, explained that based on current rates of vaccine production, going strictly by age will mean disabled and chronically ill people wont have access until June.

Another speaker at the virtual press conference, San Francisco disabled activist Alice Wong, has been both profoundly affected by the Covid-19 pandemic, and active in drawing attention to the unique risks and hardships the virus poses to people with disabilities and chronic illnesses. Californias change in vaccination priorities spurred her to further action:

"When I found out that Governor Newsom was eliminating prioritization for groups under Phase 1C in the state's vaccination plan I felt a surge of rage and fear at the injustice of it all. In response, I tweeted with the hashtag #HighRiskCA as a way for people from multiple communities disproportionately impacted by the pandemic to share their stories.

This is a localized variation on a hashtag thats been active since the pandemic started in March 2020, #HighRiskCovid19. Another important hashtag that has since the beginning expressed the feelings of high risk populations is #NoBodyIsDisposable.

Other disabled people also spoke out at the January 28 press conference.

Elena Escalera, Ph.D. of St. Mary's College and the #NoBodyIsDisposable Coalition said that the prospect of being included in the next phase of vaccinations is encouraging to people with disabilities and chronic illnesses. But when those priorities were changed in California to leave out people with disabilities under 65, ... there went the glimmer of hope of survival.

Anesthesiologist and bioethicist Dr. Alyssa Burgart highlighted the deep disability bias at the core of these decisions.

The bias against people with disabilities is pervasive. It is pervasive in health care because many of these folks are largely invisible. As you can see, many of these speakers have been confined to their homes because of the pandemic, and how much this has truly limited their ability to be engaged.

And Claudia Center, Legal Director of the Disability Rights Education and Defense Fund, noted the multiple layers of disability and chronic illness discrimination that disabled and chronically ill people have faced throughout the pandemic, and which also intersect with racial and other biases. These issues have included not just the latest setbacks in vaccine prioritization, but also denial of Covid-19 treatment through crisis standards of care, disabled people not being allowed to bring essential support staff with them to the hospital, and lack of data collection on how the pandemic affects disabled people.

It seems like California is making this change in priorities so it can avoid complicated and subtle decision-making, and instead go by more easily confirmed age. If so, it will achieve this simplicity by throwing some of its highest risk populations under the proverbial bus. Whatever the reasons for this change, and whether or how long its current priority system stands, it is adding to an already tense undercurrent of feeling among people with disabilities all over the country. There is a growing fear and conviction that disabled and chronically ill people, and our very specific kinds of risk from COVID-19, are once again being misunderstood and overlooked.

Obviously, everyone who isnt a vaccine or Covid skeptic is anxious to get vaccinated for the virus. And we all face the same fundamental barriers to vaccination, such as lack of sufficient supply and clumsy distribution systems. Its also important to recognize that putting any group higher on a priority list by itself doesnt do much. You can be at the top of the list, but if you cant figure out how to get a shot, or if your local provider runs out of doses, you are out of luck.

However, disabled and chronically ill people generally have more reason than most to be anxious and impatient. Some specific disabilities and conditions dont put people at that much more of a risk from Covid-19 infection, serious illness, or death, but a great many do. This is not mere speculation or paranoia. It is a documented medical fact recognized by most medical and epidemiological authorities.

Plus, being disabled exposes us to other, less direct hardships from the pandemic. For one thing, disabled people are more likely to be institutionalized in congregate care like nursing homes, assisted living, and group homes making it impossible for us to isolate ourselves. Many others of us require home care, which is less risky than nursing homes, but still exposes us to vectors of infection that we cant really do much on our own to avoid.

In a Los Angeles Times Op-Ed, Tim Jin writes:

Many people with disabilities are dealing with comorbidities of health that make us more vulnerable if we get the virus, while routine contact with multiple caregivers and other people who support us increases our risk of being exposed to COVID-19 As a person with cerebral palsy who lives on my own with support, I am more at risk because I rely on my staff to help me. I am exposed to multiple support people who come and go each day.

And Its not just people with conditions conventionally seen as disabilities who face higher risk from Covid-19. In an article for CNN, organ transplant recipient Kendall Ciesemier underscores the risk to people with chronic illnesses and other specific medical conditions:

The ones with cancer, with HIV, who have recovered from a bone marrow, stem cell, or solid organ transplant are increasingly becoming deprioritized across the country, sent to the back of the vaccine line.

She adds that these more recent setbacks only add to the sense of hopeless invisibility disabled, chronically ill, and other marginalized people have experienced throughout the pandemic:

To me and many like me, living in this pandemic has provided a daily reminder that our needs are unseen to those around us, that our lives hold little value to those who refuse to wear masks, who gather in groups or fly to a vacation destination. This is especially true for immunocompromised Black and brown people, who are among the most marginalized.

As disabled and chronically ill people we arent saying we have to be the very highest priority. We also recognize that other groups, particularly the elderly, have also at various points during the pandemic been ill-served, forgotten, or written off as acceptable losses. Most of us agree that prioritizing elderly people and healthcare workers makes sense. But we are dismayed to see disabled people who dont fit these categories seemingly forgotten.

Prioritizing everyone over 65 or 75 certainly puts some disabled people at the front of the line. But while many elderly people are also disabled, most disabled people are not elderly. According to the U.S. Census, about 34% of Americans over 65 have some kind of disability, a substantially higher disability rate than for the overall population. But only about 27% of Americans with disabilities are over 65. Disability and age overlap, but only partly.

Likewise, prioritizing health care and congregate care employees and residents is important to the disabled community, but only addresses some of us, not the vast majority who dont live in these facilities. Everyone knows about the tragedy of infection and death in nursing homes. Fewer people realize the same risk to developmentally disabled people in large institutions and smaller group homes. Meanwhile, people with disabilities who live on their own, or at home with home care, are virtually forgotten.

As a result, while we are nominally recognized to be high risk, most states vaccine priorities fail to recognize people with disabilities and chronic health conditions as a priority. Despite CDC recommendations, only 6 states currently offer vaccines to high risk adults who arent either elderly or health care / long term care workers. Many of us face the real possibility of our high-risk conditions not being recognized at all, resulting in more unnecessary illness, death, and long-term suffering.

Given the present scarcity of vaccine doses though, what is the fairer answer? This question is often presented as a false choice between deciding when to vaccinate disabled people based on science, and giving priority to the disability community for social or political reasons. In fact, it should be a combination of the two.

Scientists may know better which specific chronic illnesses and disabilities are and arent higher risk for Covid-19. But they arent always good at knowing and remembering the other ways Covid-19 disproportionately affects and endangers disabled and chronically ill people. One reason why a lot of disabled people are getting not just anxious but angry is that so many of us know from experience that without our own deliberate advocacy, its entirely possible for disabled and chronically ill people to be simply overlooked.

Deciding in a more targeted way who should have earlier access to Covid-19 vaccines is hard. Nobody is saying its not. But simply going by age, or focusing on a few specific environments and professions, isnt the answer. Its not logical, scientific, or humane.

On the other hand, the new Biden Administration appears to be a little bit ahead of the game in recognizing disabled and chronically ill peoples higher risk, and making them a higher priority. Its initial Covid-19 proposals include:

Implementation is always difficult, but a few more basic recommendations arent hard to think of. For example:

Among the many fears generated by the Covid-19 pandemic, one affecting the disabled community from the start is that our fellow Americans and portions of our government just dont care as much if we die. This idea has its roots in over a hundred years of on and off enthusiasm for eugenics the idea that society is better off without disabled people, and that disabled people themselves are, in a sense, better off dead.

A more specific fear and a profound sense of insult took hold in the early days of the pandemic when the fact that elderly and disabled people were at much higher risk of death was reported as a way to reassure other Americans that at least they werent in danger. This also turned out to be untrue, but even if it had been, it was not a proud moment in the history of American bravery or solidarity. Things only looked worse when states and localities proposed rationing policies that would deny care to people with certain kinds of disabilities who got Covid-19 explicitly and in policy writing them off.

Now its already looking like the vaccine rollout might sacrifice or simply overlook disabled people. Its probably still an exaggeration to say, as many disabled people are saying now, on social media and elsewhere, They want us dead. But the slightly less dramatic assertion that They dont care about us honestly doesnt seem far fetched these days. And even if we have our fellow Americans and governments intentions all wrong, their actions have not been promising.

Theres still time for a turnaround, but that time is running out fast.

See the article here:
Disabled People Are Waiting, Anxiously, For Lifesaving Covid-19 Vaccinations - Forbes

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Family of Belfast woman Eimear Gooderham (25) share memories and dealing with grief in special UTV programme – Belfast Telegraph

Monday, February 1st, 2021

The story of Belfast woman Eimear Gooderham (nee Smyth), who passed away after a brave battle with cancer and sparked awareness of the stem cell register in Northern Ireland, will be told in a UTV programme this week.

imear was diagnosed with Hodgkins Lymphoma, a type of blood cancer, in 2016 aged 22 and underwent a dozen rounds of chemotherapy.

She manage to beat the cancer in the spring of 2017 and was given the all-clear by doctors, only for the disease to return again a few weeks later.

The disease went into remission following an autologous stem cell transplant, which involved using her own cells and high-dose chemotherapy.

In 2018, however, the Hodgkins Lymphoma returned once again and doctors said Eimear required another stem cell transplant, but from an anonymous donor.

This prompted her father Sean to launch a campaign, alongside UTV, to get people to sign the stem cell register and eventually a match was found.

Eimear had surgery, but sadly she passed away in hospital of organ failure on June 27, 2019, after suffering complications.

She had been due to marry her fianc Phillip Gooderham in October 2019, however with her condition worsening the wedding was organised to take place in hospital before she passed away.

UTV presenter Sarah Clarke followed Eimears story from the summer of 2018 and now that story will be told in a special programme, Eimears Wish, airing this Thursday at 10.45pm.

The programme will feature extracts from her video diary and dad Sean and sister Seainin, share memories of Eimear and talk about the positive ways they have been dealing with their grief since she passed away.

Sean Smyth said he hopes the programme will highlight the need for more people in Northern Ireland to join the stem cell donor register, especially men aged between 16 and 30.

There is also a lack of age-appropriate care for teenagers and young adults with life threatening illnesses such as blood cancer, he said.

The current facilities and the environment in which our teenagers and young adults receive their treatment and care is very poor. There also needs to be better facilities for the childrens carers.

Sarah Clarke added: It was Eimears dying wish to raise awareness of stem cell donation and to help further research into the treatment to help others. And although this programme is an entirely different one from the one we set out to make, I hope that it will in some way help to do that.

Belfast Telegraph

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Family of Belfast woman Eimear Gooderham (25) share memories and dealing with grief in special UTV programme - Belfast Telegraph

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Single-cell molecular profiling of all three components of the HPA axis reveals adrenal ABCB1 as a regulator of stress adaptation – Science Advances

Monday, February 1st, 2021

Abstract

Chronic activation and dysregulation of the neuroendocrine stress response have severe physiological and psychological consequences, including the development of metabolic and stress-related psychiatric disorders. We provide the first unbiased, cell typespecific, molecular characterization of all three components of the hypothalamic-pituitary-adrenal axis, under baseline and chronic stress conditions. Among others, we identified a previously unreported subpopulation of Abcb1b+ cells involved in stress adaptation in the adrenal gland. We validated our findings in a mouse stress model, adrenal tissues from patients with Cushings syndrome, adrenocortical cell lines, and peripheral cortisol and genotyping data from depressed patients. This extensive dataset provides a valuable resource for researchers and clinicians interested in the organisms nervous and endocrine responses to stress and the interplay between these tissues. Our findings raise the possibility that modulating ABCB1 function may be important in the development of treatment strategies for patients suffering from metabolic and stress-related psychiatric disorders.

The hypothalamic-pituitary-adrenal (HPA) axis is pivotal for the maintenance of homeostasis in the presence of real or perceived challenges (1, 2). This process requires numerous adaptive responses involving those of the neuroendocrine and central nervous systems (3). When a situation is perceived as stressful, the paraventricular nucleus (PVN) of the hypothalamus releases corticotropin-releasing factor (CRF) to the hypophyseal portal system, connecting the hypothalamus with the anterior pituitary gland, where it stimulates the secretion of adrenocorticotropic hormone (ACTH) into the peripheral bloodstream. In turn, upon binding to the melanocortin 2 receptor, ACTH stimulates the production and secretion of glucocorticoids (GCs) from the adrenal cortex that bind to corticosteroid receptors (4). These act as transcriptional regulators providing the necessary energy resources and behavioral (emotional and cognitive) adaptations to cope with the stressful challenge and also to exert the main negative feedback at different levels of the HPA axis. While necessary for immediate response, prolonged GC exposure can increase morbidity and mortality (5, 6). Dysregulation of the neuroendocrine stress response can have severe psychological and physiological consequences, and chronic activation of the HPA axis has been linked to stress-related disorders such as anxiety disorders, major depression, posttraumatic stress disorder, and metabolic syndrome (7). As exemplified in Cushings syndrome, endogenous overproduction of GCs has detrimental effects such as impaired glucose metabolism; infectious, musculoskeletal, and cardiovascular complications; and neuropsychiatric comorbidities (8). However, despite decades of research, the molecular underpinnings of HPA dysfunction after prolonged exposure to stress are still not fully understood. Furthermore, most of the work in the field has largely focused on investigating chronic stress effects in the brain, yet much less is known about how chronic stress exposure affects the peripheral components of the HPA axis at the molecular level (9). Recent advances in the field of genomics now allow us to obtain genome-wide data on an individual cell level. Single-cell transcriptomics thereby provide powerful insight into the complexity of different tissues by enabling the identification and characterization of molecular signatures at extraordinary resolution, which can ultimately reveal previously unidentified dimensions of cell identities and their relationships with disease (10).

In this study, using single-cell RNA sequencing (scRNA-seq), we comprehensively cataloged transcriptional changes associated with chronic stress exposure in all three components of the HPA axis. We analyzed 21,723 single cells from the PVN, the pituitary, and the adrenal gland from 10 mice across two conditions (controls, n = 5; stress, n = 5). We found cell typespecific transcriptional signatures of chronic stress adaptation across the HPA axis. We identified a novel subpopulation of stress-responsive adrenocortical cells, which play an important role in the plasticity and adaptation process associated with chronic stress exposure in the adrenal cortex. We validated our findings using mouse tissues, human adrenal samples from patients with ACTH-dependent Cushings syndrome, in vitro adrenal cell models, and peripheral cortisol and genotyping data from treatment-nave, depressed patients. Our study provides the first unbiased and systematic characterization of cell typespecific signatures of the HPA axis under baseline (unstressed) and chronic stress conditions. Furthermore, our results allow a deeper understanding of HPA axis activity and its association with stress-related and metabolic disorders. Ultimately, these findings could lead to more accurate, and more reliable, molecular signatures to monitor disease progression and efficacy of treatment.

To induce chronic activation of the HPA axis, we used the chronic social defeat stress (CSDS) model, a validated, commonly used paradigm to induce long-lasting, depression- and anxiety-like endophenotypes in mice (Fig. 1A) (11). Stress exposure resulted in hallmark features of chronically stressed mice, including reduced social interaction, as demonstrated by the social avoidance test (SAT), a significant increase in basal corticosterone (CORT) levels, enhanced adrenal weight, and reduced fur quality, which is a measure associated with decreased grooming behavior (Fig. 1, B to F) (12). Body weight was not significantly different across groups after CSDS (Fig. 1G). Notably, the natural variability shown by control (unstressed) mice in the SAT did not correlate or was indicative of any of the hallmark features of chronically stressed mice (fig. S1). Five mice from each group (controls versus stressed) were selected for molecular characterization. The PVN, pituitary, and adrenal gland from these mice were used for scRNA-seq experiments (Fig. 1H).

(A) Experimental timeline of chronic social defeat stress (CSDS) paradigm for control (n = 15) and stressed (n = 15) mice. (B) CSDS reduced interaction ratios in stressed mice during the social avoidance test. Bigger dots represent the five mice from each group selected for molecular characterization (P = 0.0084, unpaired t test, two-tailed). (C and D) Twenty-one days of social defeat exposure significantly increased (a.m.) basal corticosterone (CORT) levels (P < 0.0001, unpaired t test, two-tailed) and enhanced adrenal weight (P < 0.0001, unpaired t test, two-tailed). (E) Representative adrenal glands from control and stressed mice. Scale bars, 0.5 mm. (F) CSDS significantly reduced fur quality in stressed mice [two-way analysis of variance (ANOVA), P < 0.0001]. Coat state score: (0) no wounds, well-groomed and bright coat, and clean eyes; (1) no wounds, less groomed and shiny coat OR unclean eyes; (2) small wounds, AND/OR dull and dirty coat and not clear eyes; (3) extensive wounds, OR broad piloerection, alopecia, or crusted eyes. (G) Body weight was not significantly affected by chronic stress (two-way ANOVA, P > 0.05). (H) Experimental design for scRNA-seq experiment. Individual cell suspensions were prepared from the PVN, pituitary gland (PG), and adrenal gland (AG) from selected control (n = 5) and stressed mice (n = 5). **P < 0.01, ****P < 0.0001.

To characterize inter-and intratissue heterogeneity of the HPA axis, we sequenced the transcriptome of 21,723 single cells from the PVN, pituitary, and adrenal, obtained from both unstressed (n = 5) and chronically stressed (n = 5) mice. We systematically cataloged cell identities using Scanpy, a scalable toolkit for analyzing single-cell gene expression data (13) following best practices. Graph-based clustering was performed to group cells according to their unique gene expression profiles, and dimension reduction (UMAP, Uniform Manifold Approximation and Projection) plots were used for visualization (Fig. 2) (14). In the PVN, unsupervised cluster analysis revealed a total of 18 cell clusters with distinct gene expression signatures (Fig. 2A). We determined the identity of each cluster based on the expression of established cell typespecific markers from the literature (1520). Expression of these markers across all PVN clusters can be found in fig. S2 (A to D). The 18 clusters from the PVN were further subdivided into eight major cell types as neurons, oligodendrocytes, astrocytes, microglia, endothelial, ependymal, tanycytes, and vascular cells (Fig. 2B). In the pituitary, we identified 22 unique cell clusters across 12 populations, which were grouped into somatotropes, lactotropes, corticotropes, melanotropes, gonadotropes, thyrotropes, stem cells, Pou1f1-expressing mixed cells, macrophages, endothelial cells, vascular cells, and posterior pituitary cells (Fig. 2, C and D, and fig. S3, A and B). Last, in the adrenal gland, we identified 16 unique clusters grouped into eight major groups of cells from the zona glomerulosa, zona fasciculata, a transition zone of cortical cells, medullar cells, capsular and vascular cells, macrophages, endothelial cells, and a small cluster of unknown cells (Fig. 2, E and F, and fig. S4, A and B). Expression of the top 100 genes defining the individual clusters in each of the three tissues can be found in tables S1 to S3.

(A) Dimensionality reduction Uniform Manifold Approximation and Projection (UMAP) plot depicting 6966 single cells from the PVN of the hypothalamus. Colors represent each of the 18 Louvain groups of individual cell types labeled with an abbreviation as follows: glutamatergic neurons (nGLUT1 and nGLUT2), GABAergic neurons (nGABA1 and nGABA2), mixed neurons (nMixed), vasopressin neurons (nAVP), neuropeptides (nNeuP), oligodendrocytes (Oligo1 and Oligo2), committed oligodendrocyte progenitor cells (COPs), oligodendrocyte progenitor cells (OPCs), astrocytes, endothelial, microglia, macrophages, ependymal, tanycytes, and vascular cells. (B) Distribution and percentage of eight major cell types in the PVN (purple). (C) UMAP plot depicting 9879 single cells from the pituitary. Colors represent each of the 22 Louvain groups representing individual cell types labeled with an abbreviation as follows: somatotropes (Somato1 to Somato8), lactotropes (Lacto1 and Lacto2), corticotropes (Cortico1 and Cortico2), melanotropes, gonadotropes (Gonado1 and Gonado2), thyrotropes (Thyro), endothelial, macrophages, vascular cells, stem cells, Pou1f1 mixed cells (Pou1g1 MCs), and posterior pituitary cells (PPCs). (D) Distribution and percentage of 12 major cell types in the pituitary (green). (E) UMAP plot depicting 4878 single cells from the adrenal. Colors represent each of the 16 Louvain groups representing individual cell types labeled with an abbreviation as follows: zona fasciculata (zFasc1 to zFasc5), zona glomerulosa (zGlom1 and zGlom2), transition zone of mixed fasciculata and glomerulosa cells (tZone1 and tZone2), cycling adrenocortical cells (cACCs), macrophages 1 and 2, endothelial, medullar cells, capsular and vascular cells, and unknown cells. (F) Distribution and percentage of eight major cell types in the adrenal (blue).

Next, we performed inter- and intratissue analyses to characterize cell typespecific molecular signatures of chronic stress in all three tissues of the HPA axis. First, we assessed the distribution of cell numbers for each cluster by comparing the total number of cells from the stressed group to controls (Fig. 3, A to C, and fig. S5, A to C). In the PVN, we observed a significant decrease in the number of cells from the Glut2 (32%) and neuropeptide (25%) neuronal clusters (Fig. 3A). In the pituitary, we found a significant increase in two subclusters of somatotropes (Somato6 and Somato8, 67 and 69%, respectively) (Fig. 3B). Last, in the adrenal gland, we identified the largest and most significant changes in cell distribution between the two groups. Specifically, we observed a significant increase in the number of zona fasciculata 1 cells (82%) and macrophages 2 (70%), as well as a significant decrease in the number of zona glomerulosa 1 cells (40%) (Fig. 3C).

(A to C) Distribution of cell numbers by cluster in each condition (control versus stress). Bars represent the percentage of cells from the control and stressed group per cluster (0 to 100%). All controls (gray), PVN (purple), pituitary (green), and adrenal (blue). Fishers exact test *P < 0.05, **P < 0.01, ***P < 0.001. (D) Sixty-six DEGs in 10 clusters of the PVN. Dark purple represents neurons, purple represents glial cells, and light purple represents vascular cells. (E) Six hundred ninety-two DEGs in 17 clusters of the pituitary. Dark green represents endocrine cells, green represents support cells, and light green represents stem/progenitor cells. (F) Nine hundred twenty-two DEGs in 10 clusters of the adrenal gland. Dark blue represents endocrine cells and light blue represents support cells. Size of the circle represents the number of DEGs in each cluster for all three tissues. (G) DEGs across tissues (intertissue analysis). Sixteen DEGs in common (PVN, pituitary, and adrenal), 3 DEGs (PVN and pituitary), 6 DEGs (PVN and adrenal), and 97 DEGs (pituitary and adrenal). Fourteen DEGs exclusively in the PVN (purple), 162 DEGs exclusively in the pituitary (green), and 343 DEGs exclusively in the adrenal gland (blue). Size of the circle represents the total number of DEGs in each cluster for all three tissues. (H) Expression patterns of dysregulation across DEGs per tissue. Heatmaps represent the percentage of up- and down-regulated DEGs per cluster within the PVN (purple), pituitary (green), and the adrenal (blue). Heatmap scale, 0% (gray); 50% (white); 100% (dark purple/green/blue).

Subsequently, we performed differential expression analyses to evaluate cell typespecific molecular signatures of chronic stress. We compared differentially expressed genes (DEGs) within tissues (intratissue analysis) and found that no single gene was differentially expressed (DE) across all cell types for any of the three tissues (tables S4 to S9), suggesting that cell typespecific effects of chronic stress could be masked or diluted in alternative studies using bulk RNA-seq. In contrast, when gene expression was analyzed within cell types, interesting effects emerged. In the PVN, we identified a total of 66 DEGs in 10 of the 18 cell types (Fig. 3D). In the pituitary, our analysis revealed a total of 692 DEGs in 17 of the 22 pituitary clusters (Fig. 3E). Consistent with cell distribution changes by condition, in the adrenal gland, we also observed the largest number of DEGs. Specifically, we identified 922 DEGs in 10 of the 16 adrenal clusters, ranging from 21 to 171 DE transcripts per cell type (Fig. 3F). A full list of DEGs per cell type across all three tissues can be found in tables S4 to S6.

We further compared DEGs across tissues (intertissue analysis). First, we collapsed all DEGs per tissue and identified 39 unique DEGs in the PVN, 278 in the pituitary, and 462 in the adrenal. We then looked for common genes and found 16 DEGs across all tissues (Fig. 3G). There were also 6 DEGs in common between the PVN and the adrenal, 3 DEGs between the PVN and the pituitary, and 97 DEGs between the pituitary and the adrenal glands. In addition, there were 14 genes exclusively DE in the PVN, 162 in the pituitary, and 343 in the adrenal gland (Fig. 3G and table S10). Among the genes dysregulated across the three tissues, we found several genes coding for protein members of the GC receptor (GR) chaperone complex known to play key roles in the stress response (21). Among these were HSP90 (Hsp90aa1 and Hsp90ab1), which is responsible for the direct binding of GR to the chaperone complex; HSP70 (Hspa1a and Hspa8), which encodes the first protein that recognizes and binds newly synthesized GR; and HSP40 (Dnaja1 and Dnajb1), which mediates the interaction between GR and its chaperones (22). We also observed consistent differences between the PVN and the adrenal gland for the transcription factor Nfkbia (NFB), known to interact with GCs due to their strong anti-inflammatory properties (22, 23), and Fkbp4 (encoding for the FKBP52 protein), a major regulator of GR activity (table S10) (23, 24).

Moreover, we found that most cell populations in the PVN and the pituitary showed an up-regulation of DEGs after exposure to chronic stress, except for microglial cells (PVN), macrophages, and vascular cells (pituitary), where DEGs were down-regulated (Fig. 3H). In the adrenal gland, we noticed a different pattern of regulation with several cell types, including macrophages and adrenocortical cells, showing a down-regulation of DEGs after exposure to chronic stress (Fig. 3H), suggesting a larger range of transcriptional plasticity after chronic stress at the adrenal level. Overall, these results suggest that the most profound differences due to chronic stress in the HPA axis occur in the adrenal gland, where our intra- and intertissue analyses identified the largest number of DEGs and the most significant changes at the cell population level.

The adrenal gland is a highly dynamic organ, which can quickly adapt and regenerate in response to different types of stimuli (25). For example, the adrenal significantly increases its weight in response to chronic stress, a phenomenon that has been documented in rodents, as well as human psychiatric patients (2628). In our study, we confirmed a significant increase of the adrenal weight of mice exposed to chronic social stress (Fig. 1, D and E), and single-cell transcriptomic analyses of the adrenal gland revealed a specific population of overrepresented zona fasciculata cells within the stressed group (zFasc1) (Fig. 3C). In an attempt to further investigate zFasc1 cells and identify what makes them unique, we compared their molecular profiles against all other cells in the adrenal. Because this population was so strongly driven by stress, we reasoned that the genes defining this cell type are also important responders to chronic stress. We found that the top three genes that defined the zFasc1 population were the adenosine 5-triphosphate (ATP)binding cassette subfamily B member 1B (Abcb1b) [qval: 3.27 10146; fold change (FC): 7.4], Suprabasin (Sbsn) (qval: 4.08 1084; FC: 6.6), and the 5-reductase (Srd5a2) (qval: 7.78 1082; FC: 5.8) (Fig. 4A, table S3, and fig. S5D). These genes have been previously associated with GC transport (29, 30), cell proliferation (31), and glucose metabolism (32). To validate our findings and to rule out any potential bias introduced by single-cell dissociation methods that can affect the proportions of cells in the original intact tissue, we performed mRNA in situ hybridization of Abcb1b, Sbsn, and Srd5a2 using RNAscope in adrenal glands obtained from nave or chronically stressed mice. Consistent with our single-cell results, the expression of these genes was restricted to adrenocortical cells from the zona fasciculata (Fig. 4B). Moreover, we observed a significant increase of Abcb1b and Sbsn, but not Srd5a2, mRNA expression in the zona fasciculata of stressed mice as compared to controls (Fig. 4, C to E).

(A) UMAP plot showing the expression of the top three genes that differentiate zFasc1 from other zFasc clusters: Abcb1b, Sbsn, and Srd5a2. Cyp11b1 is expressed in all zona fasciculata cells (zFasc1 to zFasc5). (B) Expression of Abcb1b, Sbsn, and Srd5a2 is restricted to adrenocortical cells from zona fasciculata. Representative adrenal glands from control and stressed mice, showing mRNA expression (brown) of Abcb1b, Sbsn, and Srd5a2 by RNAscope. Nuclei were stained with vector hematoxylin QS (purple). Scale bars, 500 m. (C to E) Chronic stress leads to a significant increase of Abcb1b (P < 0.0002) and Sbsn (P < 0.0005), but not Srd5a2 (P = 0.9715), mRNA expression in the zona fasciculata of stressed (n = 14) as compared to control (n = 14) mice. Representative images show the percentage of mRNA expression (brown) and nuclei (purple). Scale bars, 50 m. (F) CSDS leads to cellular hypertrophy in the adrenal cortex of chronically stressed mice (P < 0.0001). Bar graphs represent the average number of nuclei from the zona fasciculata. Average cell area was calculated by dividing the number of nuclei by the total area. Values are multiplied by 1000 for graphical representation. (G and H) Hypertrophy in the adrenal cortex is associated with higher levels of Abcb1b mRNA expression. Bar graphs represent the average number of nuclei present in areas of high Abcb1b (P < 0.0001) and Sbsn (P = 0.9628) mRNA expression as compared to low expressing regions in zona fasciculata of stressed mice (n = 11). All unpaired t tests, two-tailed. ***P < 0.001, ****P < 0.0001.

Subsequently, we tested whether the increase in adrenal weight after chronic stress exposure was due to an increase in the number of cells (hyperplasia) or an increase in the size of cells (hypertrophy) at the adrenal cortex. Our analysis revealed that, in stressed mice, the number of nuclei present in the zona fasciculata was significantly lower as compared to controls (Fig. 4F), suggesting cellular hypertrophy in the adrenal cortex of chronically stressed mice. Last, we evaluated whether growth characteristics of zona fasciculata cells with high expression levels of Abcb1b or Sbsn were different from low-expressing cells. Unexpectedly, our analysis revealed that the number of nuclei present in areas with high Abcb1b expression was significantly lower than in regions with low Abcb1b expression (Fig. 4G). We did not find any differences in nuclei density between regions of high or low Sbsn expression (Fig. 4H). These results suggest that hypertrophy in the adrenal cortex is associated with higher levels of Abcb1b mRNA expression.

Next, we investigated how the adrenal expression levels of Abcb1b, Sbsn, and Srd5a2 change over time, during 21 days of chronic stress exposure. Therefore, we exposed six groups of mice to a different number of social defeat sessions (0, 3, 5, 10, or 21 days). An additional group of mice received 21 days of social defeat, followed by 48 hours of recovery time to match the end point of our original CSDS paradigm (23 days) (Fig. 5A). We observed a significant and gradual increase in adrenal weight across time points (Fig. 5B). Three days of social defeat were sufficient to stimulate a significant increase in adrenal weight, which continued steadily and plateaued between days 10 and 21. We then quantified bulk mRNA expression levels of Abcb1b, Sbsn, and Srd5a2 in the adrenal cortex from these mice using quantitative real-time polymerase chain reaction (qRT-PCR). We found a significant increase of Abcb1b mRNA levels after 5 days of social defeat, while an increase for Sbsn was present only after 21 days (Fig. 5C). Consistent with our in situ nuclei quantification, we also identified a significant correlation between adrenal weight gain and the expression levels of Abcb1b (r = 0.73; P < 0.0001) and Sbsn (r = 0.51; P = 0.004) (Fig. 5D), suggesting that increases in the expression of these genes were proportional to increases in adrenal weight. In contrast, Srd5a2 did not yield any significant results in these experiments (Fig. 5, C and D). We did not find any significant differences in adrenal weight or mRNA expression levels of these genes between days 21 and 23, suggesting that the long-lasting effects of the CSDS paradigm are still present 48 hours after the last defeat session. Last, our results suggest that chronic stress exposure causes zona fasciculata cells to enlarge and increase their expression of Abcb1b, perhaps as a mechanism to cope with the increased production of GCs in the system.

(A) Experimental timeline. Six different groups of mice (n = 5) were exposed to a different number of social defeat sessions. (i) Controlno defeat, (ii) 3 days, (iii) 5 days, (iv) 10 days, (v) 21 days, and (vi) 21 days, followed by 48 hours of recovery time. (B) Three days of social defeat are sufficient to stimulate a significant increase in adrenal weight, which continued steadily and plateaued between days 10 and 21 (P < 0.0001). (C) Social defeat exposure leads to a significant increase of Abcb1b mRNA levels after 5 days of social defeat (P < 0.0001), an increase of Sbsn after 21 days (P < 0.001), while no significant changes in Srd5a2 expression (P = 0.12). Bar graphs represent mRNA levels of Abcb1b, Sbsn, and Srd5a2 normalized to Hprt. qRT-PCR, quantitative real-time polymerase chain reaction. (D) Social defeat leads to a significant correlation between adrenal weight gain and mRNA levels of Abcb1b (Pearson r = 0.73; P < 0.0001) and Sbsn (r = 0.51; P = 0.004), but no correlation with Srd5a2 expression (r = 0.29; N.S., no significance. P > 0.05). ***P < 0.001, ****P < 0.0001. CTRL, controls; SD, social defeat; D, day.

The Abcb1 gene, also known as multidrug resistance protein 1 (MDR1) or P-glycoprotein 1 (P-gp), is a well-characterized, ATP-dependent efflux pump, whose role is to transport xenobiotics and endogenous cellular metabolites across cellular membranes (33). The protein product of Abcb1 is encoded by two gene variants in mice (Abcb1a and Abcb1b) but only one gene in humans (ABCB1) (34). Moreover, it has been hypothesized that this gene modulates HPA axis activity and mediates antidepressant treatment response by regulating access of GCs and antidepressants into the brain (35). Most of the current literature in biological psychiatry has been primarily focused to understand the activity of Abcb1a in the brain, based on early observations that, in humans, the ABCB1 gene is highly expressed in endothelial cells of the blood-brain barrier (36). However, translational studies in rodents have not been successful in explaining how Abcb1 regulates the response to stress or antidepressant treatment (37). One of the reasons might be that most of these studies were carried out under the assumption that Abcb1a and Abcb1b have similar patterns of expression in the brain. Our single-cell analysis shows a very different picture with limited coexpression among the two variants. Abcb1a is specifically expressed in endothelial cells from the PVN and the pituitary (Fig. 6A), while Abcb1b is expressed in microglia and macrophages of all three tissues, in lactotropes and somatotropes of the pituitary, and in a subsection of zona fasciculata cells (zFasc1), where it shows its highest expression (Fig. 6A). Furthermore, we quantified the expression of Abcb1a and Abcb1b using publicly available bulk RNA-seq data from 35 different mouse tissues (38) and found that their expression also differs considerably in other peripheral organs. Abcb1a is lowly expressed in the periphery, while Abcb1b is the predominant variant showing high expression levels among multiple tissues, particularly in the adrenal gland where the expression of Abcb1b is several magnitudes higher as compared to every other tissue tested (Fig. 6B). These findings suggest that the adrenal gland is an important site for Abcb1 activity.

(A) UMAP plots representing cell typespecific mRNA expression of Abcb1a and Abcb1b in the PVN, pituitary, and adrenal gland of mice. (B) Bulk RNA sequencing data from 35 different mouse tissues showing mRNA expression levels of Abcb1a and Abcb1b. Heatmaps represent expression levels (0 to 12). Red, high expression; white, low expression. Expression values are displayed as Transcripts per Kilobase Million (TPM) and are log2-transformed.

Previous studies in rodents have shown that in vivo inhibition of Abcb1 by intraperitoneal injection of tariquidar, a highly specific and potent Abcb1a/b inhibitor (39), leads to a decrease in CORT levels after acute stress (40). Others have shown that mutant mice lacking both variants (Abcb1a/b) have lower baseline CORT levels as compared to wild-type controls (41). However, these studies could not attribute changes in CORT to a specific Abcb1 variant (Abcb1a or Abcb1b), nor could they conclude that the effects are modulated at the level of the brain or any of the peripheral tissues where Abcb1a and Abcb1b are expressed. To specifically explore the function of Abcb1b in the adrenal gland, we examined whether pharmacological inhibition by tariquidar modulates secretion of CORT in vitro, using an adrenocortical cell line. Mouse Y1 cells were stimulated for 24 hours with 10 nM forskolin alone, or in combination with different concentrations of tariquidar. Forskolin induces secretion of CORT by stimulation of adenylate cyclase (42). While 24 hours of forskolin treatment significantly increased CORT levels as compared to controls, we found a dose-dependent decrease of CORT with increasing concentrations of tariquidar (Fig. 7A), suggesting that GC secretion from adrenocortical cells might be dependent on Abcb1b function. In an attempt to translate our findings to humans, we assessed the modulatory role of ABCB1 on GCs, using human NCI-H295R adrenocortical cells, a validated in vitro model for steroid profiling based on their ability to produce and secrete the major steroidogenic enzymes of the adrenal cortex (43). In line with our previous results, treatment of NCI-H295R cells with 10 nM forskolin led to a significant increase of medium cortisol levels, as compared to vehicle-treated controls (Fig. 7B). Treatment with increasing concentrations of tariquidar led to a significant decrease of media cortisol levels (Fig. 7B). Together, our results show that in vitro pharmacological manipulation of Abcb1 in adrenocortical cell lines leads to a decrease in GC secretion, suggesting that modulation of Abcb1b in adrenocortical cells affects GC secretion in both mice and humans.

(A) Pharmacological inhibition of Abcb1 by tariquidar in mouse Y1 adrenocortical cells. CORT levels (ng/ml) after 24-hour treatment with forskolin (0 and 10 nM) or tariquidar (0, 10, 50, 125, 250, 500, and 1000 nM). (B) Pharmacological inhibition of ABCB1 by tariquidar in human NCI-H295R adrenocortical cells. Cortisol levels (ng/ml) after 24-hour treatment with forskolin (0 and 10 nM) or tariquidar (0, 10, 50, 125, 250, 500, and 1000 nM). One-way ANOVA, ****P < 0.0001.

In humans, chronic endogenous oversecretion of ACTH due to a pituitary or ectopic tumor results in excessive GC secretion and enlargement of the adrenal glands (Fig. 8A) (44). Thereby, this disease stage overlaps with the chronic activation of the HPA axis and hypersecretion of GCs in stress-related disorders. In cases of unsuccessful pituitary surgery or in those patients in whom the ectopic source of ACTH remains obscure, bilateral adrenalectomy is required to treat steroid excess. This opened the possibility to study adrenal glands that had been chronically stimulated and to compare those with controls in the absence of ACTH oversecretion. We quantified mRNA expression of ABCB1 and SBSN, using RNAscope in cases (n = 8) and controls (n = 6). SBSN mRNA was not detectable in human adrenocortical samples (Fig. 8B). Following this approach, we identified a significant up-regulation of ABCB1 mRNA in Cushings adrenocortical samples, as compared to controls (Fig. 8C). These results are consistent with our initial findings in chronically stressed mice and reinforce our evidence for a role of ABCB1 as a modulator of GC activity in the adrenal gland. In addition, our results highlight ABCB1 as a potential regulator of the detrimental effects of impaired glucose metabolism associated with patients with Cushings syndrome.

(A) Graphical representation of the effects of Cushings disease on the adrenal gland. (B and C) Expression of ABCB1 (P = 0.0056) and SBSN (P = 1.0) mRNA, using RNAscope in adrenal glands from patients with ACTH-dependent Cushings syndrome (n = 8) and controls (n = 6). Representative images show the percentage of mRNA expression (brown) and nuclei (purple) normalized by total area. Scale bars, 50 m, **P < 0.01.

Individuals who lack ABCB1, as it occurs in some breeds of dogs with the ABCB1-1 mutation (45), have severe adverse reactions to common medications that act as substrates of this transporter, such as immunosuppressants and steroid hormones (46). Previous studies have shown that dogs and rodents lacking a functional Abcb1/ABCB1 gene have a blunted HPA axis response compared to wild-type animals (41, 47). In humans, multiple single-nucleotide polymorphisms (SNPs) map to the ABCB1 gene locus, and some of these variants have been associated with reduced protein function and activity (48). One of the most studied ABCB1 polymorphisms is the rs2032582 (G2677T), which is a nonsynonymous variant on exon 21 that has been linked to major depressive disorder and treatment response (49). To investigate the relevance of our findings in depressed human patients, we examined whether the ABCB1 polymorphism rs2032582 is associated with an altered HPA axis response, using peripheral plasma samples from 154 treatment-nave, depressed patients. We measured plasma ACTH and cortisol concentrations in depressed patients at baseline, following CRF stimulation, and 15-min intervals for the following hour (Fig. 9A). The genotype and allele distributions of rs2032582 in patients are shown in Fig. 9A. At baseline, patients with the minor allele (TT) showed a decrease in cortisol levels as compared to the major (GG) and heterozygote (TG) alleles; however, this effect did not reach statistical significance after Bonferroni correction (Fig. 9B). After CRF stimulation, we found a significant genotype-by-time interaction in patients cortisol levels (qval: 0.033). More specifically, patients with the minor (TT) allele showed a dampened cortisol response after CRF stimulation (Fig. 9C). We did not find any statistical differences in ACTH levels after CRF stimulation (Fig. 9D), suggesting that the effects of rs2032582 on the ABCB1 gene might be taking place at the level of the adrenal gland. These results are consistent with our mouse and cell culture findings and support the idea that Abcb1/ABCB1 function may regulate HPA axis response.

(A) Experimental design. Predictors of remission in depression to individual and combined treatments (PReDICT) cohort (N = 154) to investigate effects of the ABCB1 variant rs2032582 on HPA axis function. CRF stim, CRF stimulation test; SNP, single-nucleotide polymorphism; HWE-P, Hardy-Weinberg equilibrium P value. (B) Baseline cortisol levels (g/ml) for treatment-nave, depressed patients carrying the rs2032582 SNP genotype. (C and D) Cortisol (g/ml) and ACTH (pg/ml) levels after CRF stimulation (log-transformed). Only completers were included in the analysis. There are no dropouts in sample sizes over time for cortisol or ACTH. GG = major homozygotes (n = 56), GT = heterozygotes (n = 74), TT = minor homozygotes (n = 24). Mixed effects models, Bonferroni-corrected *P < 0.05.

Despite decades of research, the molecular and cellular identity of the HPA axis components, their inter-relationships, and their function after chronic stress exposure are still only partially understood. Here, using scRNA-seq, we describe cell typespecific molecular signatures of chronic stress in all three components of the HPA axis, providing a level of resolution never before reached.

The PVN integrates and coordinates the neuroendocrine HPA axis response to stressful stimuli. However, aside from containing the neuroendocrine neurons that control the synthesis and release of CRF, the PVN also exhibits a significant degree of cellular and molecular complexity, with multiple types of neuronal and nonneuronal subtypes. In this study, we characterized and described the cellular heterogeneity and identity of all cell types in the PVN. We identified many DEGs that are involved in the intracellular trafficking of the GC and mineralocorticoid receptors and play key roles in the response to chronic stress, such as heat shock proteins and Fkbp4 across multiple cell types (24). We also found groups of genes that were DE in unique cell types, such as the cysteine-rich angiogenic inducer 61 gene (Cyr61), which was only found DE in ependymal cells. Cyr61 is a target gene of the hippo signaling pathway, which regulates tissue homeostasis, regeneration, proliferation, and growth and has recently been linked to the pathophysiology of stress-related psychiatric disorders (50). In the neuropeptide cluster, we found a down-regulation of corticotropin-releasing factor (Crf) and vasopressin (Avp), as well as an up-regulation of oxytocin (Oxt) and somatostatin (Sst); however, these changes did not survive correction for multiple testing. We did not find any significant dysregulation of GR (Nr3c1) mRNA in any of the clusters of the PVN. Nevertheless, we did find a significant difference in the total number of Nr3c1+ cells in some of the cell clusters of the PVN (fig. S2D), suggesting that the GR mRNA differences reported in the literature (4, 6, 9) could be due to a decrease in the total number of Nr3c1+ cells after chronic stress, rather than lower expression levels of the existing cells. A decrease in the total number of Nr3c1+ cells is not found across all cell types of the PVN but is rather limited to specific cell populations. These populations could represent the cell types where stress exerts its main effects in the PVN via GR. However, these findings would need to be further validated and replicated in other studies. Last, we found that most cell populations across the PVN showed an up-regulation of DEGs after exposure to chronic stress, except for microglial cells where most DEGs were down-regulated. These changes in microglial cells in combination with gene expression differences (across multiple cell types) of several genes involved in the intracellular trafficking of GCs are possibly the result of overexposure to GCs during a prolonged (chronic) stress paradigm. GCs are released during the stress response and are well known for their immunosuppressive and anti-inflammatory properties. In addition, growing evidence suggests that changes in neuroendocrine function and metabolism are significant triggers of inflammation, which has been linked to the development of neuropsychiatric disorders. Ultimately, while this is an important issue in the field, it is logistically challenging to address considering that the effects or stress, GCs, and inflammation are closely intertwined, likely powering each other in a bidirectional way.

The second component of the HPA axis, the pituitary gland, is a complex organ and an important regulator of major physiological processes, including the neuroendocrine stress response (51). It is composed of a heterogeneous mix of endocrine, general support, and stem cells (17, 18). Despite a significant body of research characterizing attributes of pituitary activity, the cell typespecific regulation of chronic stress at the pituitary level is still poorly understood. Here, we characterized cell typespecific molecular signatures of chronic stress in the pituitary gland. Among many, our DE analysis revealed several genes that were consistently dysregulated in multiple endocrine cells, such as somatotropes, gonadotropes, lactotropes, and corticotropes. Specifically, we found an up-regulation of Cd63, Hsp90aa1, and Hsp90ab1, as well as a down-regulation of several ribosomal genes in all four cell types, suggesting altered GC and ribosomal activity. Moreover, corticotropes are directly stimulated by CRF and are responsible for the release of ACTH into circulation. In our analysis, we found 32 DEGs in this population. However, we did not find any significant differences in the expression of the corticotropin-releasing hormone receptor 1 (Crhr1) or the GR (Nr3c1) gene. Furthermore, and consistent with our findings in the PVN, we found that most cell types across the pituitary showed an up-regulation of DEGs after exposure to chronic stress, except for macrophages and vascular cells, where most DEGs are down-regulated. In our single-cell data, we found a large number of DEGs across multiple types of endocrine cells, suggesting that the stress response in the pituitary gland is a dynamic and complex process that is not limited to the effect that CRF exerts on corticotropes. In our analyses, somatotropes were the population of pituitary cells that showed the biggest changes after chronic stress, both in terms of number of DEGs and changes in proportions of cells. Somatotropes produce and release growth hormone, and they play an important role in the regulation of GC synthesis and adrenal growth and have been shown to positively affect adrenal cell size and number of adrenocortical cells (52). However, the role that somatotropes play in chronic stress and the development of stress-related psychiatric disorders are still poorly understood. Our high-throughput, cell typespecific findings of the effects of chronic stress on the pituitary and somatotropes are both novel and a significant advancement to our understanding of the mechanisms of stress adaptation in the pituitary gland.

Last, the adrenal gland is a major effector of the HPA axis, where interplay between several types of specialized cells takes place to coordinate a complex endocrine, immune, and metabolic response to stress. It is composed of the adrenal medulla and the adrenal cortex, two embryonically different endocrine tissues (25). The adrenal cortex is further divided into three major zones: zona glomerulosa (zG), zona fasciculata (zF), and zona reticularis (zR), each responsible for the synthesis and release of mineralocorticoids, GCs, and androgens, respectively (27). Zona reticularis has been shown to be absent in mice (25). Until now, our understanding of the mechanisms responsible for chronic stress adaptation in the adrenal has been limited. Our study is the first to provide a cell typespecific, unbiased, molecular characterization of the adult adrenal gland (under baseline or chronic stress conditions). Across several cell types, we found a significant dysregulation of genes coding for steroidogenic enzymes responsible for the biosynthesis of corticosteroids, such as GCs and mineralocorticoids. More specifically, we found a dysregulation of Star, Fdx1, Cyp11b1, Cyp21a1, Cyp11a1, Hsd3b1, Nr4a1, and Agtr1a after exposure to chronic stress. In contrast to what we found in the PVN and pituitary, cell types in the adrenal showed both up-regulation and down-regulation of DEGs after exposure to chronic stress, suggesting a larger range of transcriptional plasticity after chronic stress at the adrenal level. Although the changes in the expression of genes coding for steroidogenic enzymes are consistent with the current literature (27), our results offer a new level of resolution by describing the specific cell types where these changes take place in the adrenal. Furthermore, our results highlight that changes after chronic stress in the adrenal are not limited to the endocrine cells of the adrenal cortex or adrenal medulla. In our data, we also find significant changes in the number of macrophages, as well as the number of DEGs in this cluster, after chronic stress. Macrophages are modulated by GCs to secrete cytokines and regulate inflammation and the immune system (53). To the best of our knowledge, this study is the first to show a significant effect of chronic stress in macrophages of the adrenal gland. In addition, our results show a global dysregulation of transcriptional activity in macrophages across all three components of the HPA axis (PVN, pituitary gland, and adrenal gland), suggesting that this cell population is part of a common, multilevel and multitissue signaling network that regulates adaptation to chronic stress.

One of the main findings from our study is the identification of a novel population of overrepresented Abcb1b+ cells within the zona fasciculata of the stressed group. The identification of this novel and specialized cell type in the adrenal gland could not have been possible using standard bulk RNA-seq methods. All previous transcriptomic studies examining the effects of chronic stress in the adrenal gland have been limited to adrenocortical, adreno-medullar, or whole tissue homogenates that average out the signature of thousands of cells, which can mask, dilute, or even distort signals of interest coming from specialized cell populations. Hence, one can expect that any cell typespecific signature of chronic stress (as is the case for zFasc1 cells) has been diluted or even lost in these studies. Here, through a series of complementary experiments, we validated this novel subpopulation of Abcb1b+ cells in the adrenal cortex, which play an important role in stress adaptation. Our experiments showed that increased mRNA expression of Abcb1b+ cells in the adrenal gland is associated with increased adrenal weight and cellular hypertrophy in the adrenal cortex of stressed mice, suggesting that chronic stress exposure causes zona fasciculata cells to enlarge and increase their expression of Abcb1b, perhaps as a mechanism to cope with the increased and sustained production of GCs in the system. The Abcb1 gene is a well-characterized efflux pump whose role is to transport substances, deemed as harmful, across membranes. However, most of the work to study this gene in psychiatry has been primarily focused on understanding the activity of the variant Abcb1a in the brain. Our single-cell analysis in combination with bulk RNA-seq data from 35 different mouse tissues showed that Abcb1b is the predominant variant in the periphery showing high expression levels among multiple tissues, particularly in the adrenal gland, suggesting that the adrenal is an important site for Abcb1 activity. Furthermore, to disentangle the effects of Abcb1a versus Abcb1b in the response to stress, we performed a series of in vitro experiments in mouse and human adrenocortical cells. Our results showed that pharmacological inhibition of Abcb1b in adrenocortical cell lines leads to a decrease in GC secretion, suggesting that modulation of Abcb1b in adrenocortical cells affects GC activity in both mice and humans. Moreover, in an attempt to translate our findings to humans, we investigated the expression of ABCB1 in adrenal cortical tissues from patients diagnosed with ACTH-dependent Cushings syndrome. These patients suffer from excessive GC secretion and adrenal hypertrophy due to a pituitary or ectopic tumor. Thus, this disease stage overlaps with the chronic activation of the HPA axis and hypersecretion of GCs in stress-related disorders. We found a significant up-regulation of ABCB1 in cases, as compared to controls. In addition to being consistent with our findings in chronically stressed mice, these results highlight the role of ABCB1 as a modulator of GC activity in the adrenal gland and postulate ABCB1 as a potential regulator of the impaired glucose metabolism associated with Cushings syndrome. Last, we investigated the relevance of our findings in depressed human patients by examining whether the ABCB1 polymorphism rs2032582 (G2677T) is associated with an altered HPA axis response in peripheral plasma samples from treatment-nave, depressed patients. In humans, the rs2032582 polymorphism has been associated with reduced protein function and activity and has been linked to major depressive disorder, suicidal ideation, and treatment response (49). Consistent with our findings in mice, adrenocortical cell lines, and adrenocortical samples from human Cushings patients, we found that, after CRF stimulation, patients with the minor (TT) allele showed a dampened cortisol but normal ACTH response, suggesting that the effects of rs2032582 on the ABCB1 gene might be taking place at the level of the adrenal gland. In addition, our results support the idea that Abcb1/ABCB1 function may regulate HPA axis response.

Together, our data offer new insights into how chronic stress regulates transcriptional activity in a multilevel, cell typespecific fashion. We identified hundreds of novel genes that are dysregulated across all tissues and levels of the HPA axis. On the basis of our intra- and intertissue analyses, we found the most profound differences due to chronic stress in the adrenal gland, which had the highest number of DEGs and the most significant changes at the cell population level. Through a series of complementary behavioral, molecular, cellular, and functional experiments, we identified a novel subpopulation of Abcb1b+ cells in the adrenal cortex, which play an important role in the adaptation process and plasticity associated with chronic stress exposure. The exact mechanism underlying the effect of ABCB1 on GC regulation and secretion in the adrenal cortex still needs to be further explored. However, previous studies have shown that transcriptional regulation of the Abcb1 genes can be mediated through a putative GC response element (GRE) identified in the promoter region of these genes in both rodents and humans (54, 55). At least in mice, this GRE binding site is only found in the promoter region of the Abcb1b variant, suggesting that Abcb1b is directly regulated by GCs in the periphery, predominantly in the adrenal glands. Therefore, we propose that the Abcb1b/ABCB1 gene and protein are involved in mediating chronic stress adaptation through regulation and control of GCs in the adrenal gland. Our findings raise the prospect that modulating ABCB1 function may be important in the treatment of patients suffering from neuropsychiatric and metabolic disorders, such as stress-related disorders and Cushings syndrome. They further suggest that adrenal ABCB1 activity could be used to stratify patients and tailor treatment strategies. Ultimately, our results provide a deeper understanding of the complex mechanisms of HPA axis regulation.

All experiments were performed in accordance with the European Communities Council Directive 2010/63/EU. All protocols were approved by the Ethics Committee for the Care and Use of Laboratory Animals of the government of Upper Bavaria, Germany. Male mice aged between 7 and 10 weeks old were used for all experiments. Mice were bred in the animal facility of the Max Planck Institute of Biochemistry (Martinsried, Germany) and group-housed (four to five mice per cage) until 1 week before the start of the experiments, when mice were single-housed. Mice were kept in individually ventilated cages (IVCs; 30 cm by 16 cm by 16 cm; 501 cm2), serviced by a central airflow system (Tecniplast, IVC Green LineGM500), according to institutional guidelines. IVCs had sufficient bedding and nesting material as well as a wooden tunnel for environmental enrichment. Animals were maintained under pathogen-free, temperature-controlled (23 1C), and constant humidity (55 10%) conditions on a 12-hour light/12-hour dark cycle (lights on at 7:00 a.m.) with food and water provided ad libitum, at the Max Planck Institute of Psychiatry (Munich, Germany).

C57BL/6N males (7 weeks old) were exposed to the CSDS paradigm for 21 consecutive days, as previously described (12). Briefly, experimental mice were introduced daily into the home cage of a dominant CD1 resident mouse, which rapidly recognized and attacked the intruders within 2 min. To avoid serious injuries, the subordinate mouse was separated immediately after being attacked by the CD1 aggressor. After the physical encounter, mice were separated by a perforated metal partition, allowing the mice to keep continuous sensory but not physical contact for the next 24 hours. Every day, for a total of 21 days, mice were defeated by another unfamiliar, CD1 resident mouse, to exclude a repeated encounter throughout the experiment. Defeat encounters were randomized, with variations in starting time (between 9:00 a.m. and 6:00 p.m.) to decrease the predictability to the stressor and minimize habituation effects. Control mice were single-housed, in the same room as the stressed mice, throughout the course of the experiment. All animals were handled daily and weighed every 4 days. Coat state was scored on a scale of 0 to 3 according to the following criteria: (0) No wounds, well-groomed and bright coat, and clean eyes; (1) no wounds, less groomed and shiny coat, OR unclean eyes; (2) small wounds, AND/OR dull and dirty coat, and not clear eyes; (3) extensive wounds, OR broad piloerection, alopecia, or crusted eyes. End point and tissue collection were performed in the morning (9:00 a.m.) and 48 hours after the last social defeat session (day 23). This was done to capture the cumulative effects of chronic stress, rather than the acute effects of the last defeat session. The SAT was conducted during the last week of the CSDS paradigm, and based on their performance, five mice from each group were selected for molecular characterization, thus avoiding potentially stress-resilient animals. For end point, all mice were deeply anesthetized with isoflurane and perfused with cold phosphate-buffered saline (PBS), and target tissues were quickly dissected for molecular experiments. Cardiac blood was collected for the assessment of basal CORT levels. Adrenal glands were dissected from fat and weighed. The brains, pituitary, and adrenal glands from selected mice were immediately processed for RNA single-cell analysis. Tissues from all remaining mice were collected for downstream validation experiments.

Social avoidance behavior was assessed with a novel CD1 mouse in a two-stage social interaction test. In the first 2.5-min test (nontarget), the experimental mouse was allowed to freely explore the open-field arena containing an empty wire mash cage against one wall of the arena (labeled as the interaction zone). In the second 2.5-min test (target), the experimental mouse was returned to the arena with an unfamiliar male CD1 mouse enclosed in the wire mash cage. The ratio between the time in the interaction zone of the nontarget trial and the time in the interaction zone of the target trial was calculated and deemed as the interaction time ratio.

Blood sampling was performed during end point (9:00 a.m.) by collecting blood from the heart of each mouse before perfusion with PBS. All blood samples were kept on ice and centrifuged at 4C, and 10 l of plasma was removed for measurement of CORT. All plasma samples were stored at 20C until CORT measurement. CORT concentrations were quantified by radioimmunoassay (RIA) using a CORT double antibody 125I RIA kit (sensitivity: 25 ng/ml; MP Biomedicals Inc.) following the manufacturers instructions. Radioactivity of the pellet was measured with a gamma counter (Wizard2 2470 Automatic Gamma Counter; Perkin Elmer). All samples were measured in duplicate, and the intra- and interassay coefficients of variation were both below 10%. Final CORT levels were derived from the standard curve.

Six different groups (each N = 5) of C57BL/6N males were exposed to a different number of social defeat sessions to assess the cumulative effects of stress and its correlation with changes in mRNA levels of Abcb1b, Sbsn, and Srd5a2. The groups were defined as follows: (i) controlno defeat, (ii) 3 days of social defeat, (iii) 5 days of social defeat, (iv) 10 days of social defeat, (v) 21 days of social defeat, and (vi) 21 days of social defeat, followed by 48 hours of recovery time. The last group was introduced to match the end point of our original chronic social defeat paradigm cohort (23 days). All mice were 7 weeks old at the beginning of the experiment. Individual social defeat encounters were carried out exactly as previously explained in the CSDS paradigm section of the methods. End point and tissue collection were performed in the morning (9:00 a.m.) of day 23. All mice were deeply anesthetized with isoflurane, and the adrenal glands were quickly dissected for molecular experiments. Adrenal glands were further dissected from fat and weighed. Trunk blood was collected for the assessment of basal CORT levels.

Tissue dissociation. Mice were anesthetized lethally using isoflurane and perfused with cold PBS to get rid of undesired blood cells in target tissues. Brains, pituitaries, and adrenal glands were quickly dissected and immediately transferred to ice-cold oxygenated artificial cerebral spinal fluid (aCSF) (brains), ice-cold Hanks balanced salt solution (HBSS) (pituitaries), or ice-cold PBS (adrenals) and kept in the same solutions during dissection and dissociation. The aCSF was oxygenated throughout the experiment with a mixture of 5% CO2 in O2. Sectioning of the brain was performed using a 0.5-mm stainless steel adult mouse brain matrix (Kent Scientific) and a Personna Double Edge Prep Razor Blade. A slide (approximately 0.58 mm Bregma to 1.22 mm Bregma) was obtained from each brain, and the extended PVN was manually dissected under the microscope. Two cell suspensions were prepared for each of the three tissues with one pool for controls and one pool for stressed mice. The PVN from five different mice was pooled and dissociated for 35 min using the Papain Dissociation System (Worthington) following the manufacturers instructions. Similarly, the pituitaries from five mice were pooled and dissociated for 15 min using papain. Last, the adrenal glands from five mice were pooled and dissociated for 55 min using a 0.2% collagenase II solution. All cell suspensions were incubated at 37C using a shaking water bath. After this, cell suspensions were filtered with 30-m filters (Partec) and kept in cold aCSF, HBSS, or PBS.

Cell capture, library preparation, and high-throughput sequencing. Cell suspensions with approximately 1,000,000 cells/l were used. Each pool was loaded onto individual lanes of a 10X Genomics Chromium chip, as per factory recommendations. For all three tissues, the control and stress cell suspensions were loaded and processed together in the same chip to avoid batch effects by condition. Reverse transcription and library preparation were performed using the 10X Genomics Single-Cell v2.0 kit following the 10X Genomics protocol. Molar concentration and fragment length of libraries were quantified by qPCR using KAPA Library Quant (Kapa Biosystems) and Bioanalyzer (Agilent High Sensitivity DNA kit), respectively. Each library was sequenced on a single lane of an Illumina HiSeq4000 System generating 100base pair paired-end reads at a depth of ~340 million reads per sample.

Preprocessing and quality control. Preprocessing of the data was done using the 10X Genomics Cell Ranger software version 2.1.1 in default mode. The 10X Genomics supplied reference data for the mm10 assembly and corresponding gene annotation was used for alignment and quantification. All further analyses were performed using Scanpy (version 1.4.4.post1) (13), following guidelines from an established best practices workflow (14). For quality control, we looked at the distribution of count depth, number of genes, and mitochondrial read fraction per sample. Because distributions were fairly homogeneous, we chose to pick the same thresholds for all samples (tissues and conditions). Specifically, we filtered out (i) cells with less than 1000 counts, (ii) less than 400 genes detected, and (iii) percentage of mitochondrial gene counts higher than 25%. In addition, genes expressed in less than 20 cells were removed as well. Quality control (QC) plots can be found in fig. S5 (E to G). This resulted in a dataset of 21,723 single cells, of which 6966 cells and 16,168 genes were from the PVN, 9879 cells and 15,437 genes were from the pituitary, and 4878 cells and 13,997 genes were from the adrenal gland. The size factors used for normalization were obtained using Scran (version 1.14.5) (56), and the data were log1 Ptransformed. Each dataset was batch-corrected using Combat (57), available in Scanpy. For each tissue, we selected the top 4000 highly variable genes using the highly_variable_genes function. Dimensionality reduction was performed using principal components analysis computed on highly variable genes and taking the first 50 PCs. Last, we computed a k-nearest neighbor graph (KNN)graph (k = 15) on the low-dimensional embedding, necessary for UMAP visualization.

Clustering, marker gene identification, and cluster annotation. Data were clustered using the Louvain (version 0.6.1) algorithm implemented in Scanpy (13). This is a graph-based clustering method that relies on the KNN-graph discussed above. We clustered at two different resolution levels (r = 0.5 and r = 1). After inspection of the cell clusters, we observed that those obtained using a finer resolution (r = 1) aligned better with our annotations and therefore used them for visualization and downstream analyses. Marker genes for each cluster were detected using a Welchs t test between cells in the cluster and all cells outside of it as reference. This was done using the rank_genes_groups function implemented in Scanpy and computed on log-normalized nonbatch-corrected data. Cell types were determined using a combination of marker genes identified from the literature and Gene Ontology for cell types using the web-based tool: mousebrain.org (58).

Differential expression analysis. Differential expression analyses were performed using MAST (59) implemented in R, which models scRNA-seq data using a generalized linear model (GLM). The computation was performed on log-normalized nonbatch-corrected data, and, for each cell cluster, we fit the following model: [Y ~ 1 + condition + n_genes], where Y is the log-normalized nonbatch-corrected data, 1 is the intercept term, condition is the covariate that accounts whether the mouse was stressed or not, and n_genes is used as a technical covariate as a proxy for technical and biological factor that might influence gene expression. The test produced a P value for each gene in each cell cluster and a q value, which is the P value after adjustment for multiple testing, using false discovery rate (FDR) correction. Furthermore, the mean expression of each gene for the two different conditions was computed.

Ambient RNA assessment. After QC analyses, we noticed the presence of highly expressed genes across all cells, despite being known marker genes of specific cell types. We noticed that most of these genes coded for neuropeptides or hormones and decided to assess whether we could explain this as ambient RNA contamination. To investigate which genes were expressed as ambient RNA, we analyzed the unfiltered datasets for the three tissues. We once again looked at the count depth distribution for what we conclude are empty droplets and selected cells with counts between 100 and 300 for the PVN, between 300 and 600 for the pituitary, and between 50 and 200 for the adrenal gland. We also removed genes that are expressed in less than 20 cells. After these steps, we obtained a dataset of 120,320 droplets and 14,129 genes for the PVN, 113,043 droplets and 13,777 genes for the pituitary, and 107,698 droplets and 11,684 genes for the adrenal gland. Because these are empty droplets and we do not expect any meaningful clustering of the data, we used a less sophisticated normalization technique, normalizing each cell by total counts over all genes, thus obtaining the same total count per cell after this step. The number of counts per cell to obtain was selected automatically as the median count per cell before normalization. For all tissues, we computed the mean expression of each gene across all droplets by condition (stress versus control). Furthermore, to exclude from our list of significantly DEGs those that are detected owing to differential ambient RNA expression, we performed differential expression testing using MAST across all droplets using the same GLM formulation defined above (note that, in our previous analysis, we tested within each cluster).

For paraffin embedding, adrenal glands were dissected and the surrounding fat was removed and fixed in 10% neutral buffered formalin (Sigma-Aldrich, HT501128) overnight at room temperature. Tissue was embedded manually over 3 days. All washes were carried out for 1 hour at room temperature unless indicated. Day 1: three times PBS, 25% EtOH, 50% EtOH, 70% EtOH, and 70% EtOH overnight at 4C. Day 2: 80% EtOH, 90% EtOH, 95% EtOH, and 100% EtOH overnight at 4C. Day 3: 100% EtOH, Neoclear (Sigma-Aldrich, 109843) I for 10 min at room temperature; Neoclear II for 10 min at 60C; Neoclear: paraffin 1:1 for 15 min at 60C, paraffin I for 1 hour at 60C, paraffin II for 1 hour at 60C, and paraffin III for 1 hour at 60C. Samples were sectioned at 5 m. RNAscope was carried out on paraffin-embedded sections with the RNAscope 2.5 HD Kit-BROWN (ACD bio 322300) assay following the manufacturers protocols, with Standard timings for retrieval and protease treatment. The following probes were used (all ACD bio): Mm-Abcb1b (422191), Mm-Sbsn (564441), Mm-Srd5a2 (431361), Hs-ABCB1 (401191), and Hs-SBSN (447411). Positive control Mm-Ppib (313911), Hs-UBC (310041), and negative control dapB (310043) were also used. Nuclei were stained with Vector Hematoxylin QS (Vector Laboratories, H-3404), and slides were mounted in VectaMount Permanent Mounting Medium (Vector Laboratories, H-5000).

Paraffin sections were deparaffinized and rehydrated as per immunohistochemistry. Slides were incubated for 30 s with Hematoxylin QS, washed with running water, incubated for 30 s with eosin, washed with running water, and mounted in VectaMount Permanent Mounting Medium.

Hematoxylin and eosin and RNAscope slides were scanned with a NanoZoomer-XR digital slide scanner (Hamamatsu). Images were processed with NanoZoomer digital pathology view (Hamamatsu), and quantification was done with Fiji.

Four areas of the same dimensions (252 252 pixels) were selected from the zona fasciculata of the cortex. Nuclei were counted, and the average cell area was calculated by dividing the number of nuclei by the total area. Values were multiplied by 1000 for graphical representation.

Quantification of messenger RNA levels of Abcb1b, Sbsn, and Srd5a2 in the adrenal glands was carried out using qRT-PCR. Total RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). RT-PCR reactions were run in triplicate using the ABI QuantStudio 6 Flex RT-PCR System and data were collected using the QuantStudio RT-PCR software (Applied Biosystems). Expression levels were calculated using the standard curve, absolute quantification method. The endogenous expressed gene Hprt was used to normalize the data. The following Taqman probes were used: Abcb1b: Mm00440736_m1, Sbsn: Mm00552057_m1, Srd5a2: Mm00446421_m1, and Hprt: Mm00446968_m1.

Mouse Y1 cells and human NCI H295R adrenocortical cells were seeded into 12-well plates and incubated overnight using Dulbeccos modified Eagles medium high glucose (4.5 g/liter) (Gibco) with 7.5% horse serum (Gibco), 2.5% fetal bovine serum (FBS) (Gibco), and 1% penicillin-streptomycin (Gibco) and RPMI 16/40 + GlutaMax (Gibco) with 10% FBS (Gibco), 1% Insulin-Transferrin-Selenium-Ethanolamine (ITS) (Thermo Fisher Scientific), and 1% penicillin-streptomycin (Gibco), respectively. In this experiment, 100,000 Y1 and NCI H295R adrenocortical cells per well were used. Cells were then stimulated for 24 hours with 10 nM forskolin and subsequently treated with different concentrations of tariquidar (0, 10, 50, 125, 250, 500, and 1000 nM) and incubated for 24 hours. Last, supernatants and cell pellets were collected and harvested for further analyses and measurement of CORT (ng/ml) and cortisol (g/liter) levels. Media CORT levels in Y1 cells were quantified by RIA using a CORT double antibody 125I RIA kit, as previously described in the animal experiments. Media cortisol levels in NCI H295R adrenocortical cells were determined using an enzyme-linked immunosorbent assay (ELISA) kit (RE52061, TECAN, IBL Hamburg, Germany). The standard range was 20 to 800 ng/ml.

The study was approved by the Ethics Committee of the University of Wuerzburg (Germany) (#88/11), and written informed consent was obtained from all subjects. Eight patients with biochemically confirmed persistent ACTH-dependent Cushings syndrome were studied. Cushings syndrome was established according to current guidelines (60). Half of the patients (n = 4) had pituitary-dependent Cushings syndrome, while, in the other patients (n = 4), ectopic Cushings syndrome had been diagnosed. The patients underwent bilateral adrenalectomy as ultima ratio to control life-threatening hypercortisolism after other therapies had failed. Formalin-fixed paraffin-embedded sections were stained as described above. The normal adrenal tissue was derived from adrenal glands removed as part of tumor nephrectomy (n = 6). They were histologically proven normal adrenal glands without neoplastic tissue.

Data of the Emory Predictors of Remission in Depression to Individual and Combined Treatments (PReDICT) (61, 62) study was used to investigate effects of the ABCB1 variant rs2032582 on HPA axis function in 154 unmedicated patients with a current Diagnostic and Statistical Manual of Mental Disorders (DSM)IV diagnosis of major depressive disorder. The PReDICT study was designed and conducted in accord with the latest version of the Declaration of Helsinki. The Emory Institutional Review Board (IRB) and the Grady Hospital Research Oversight Committee gave ethical approval for the study design, procedures, and recruitment strategies (Emory IRB numbers 00024975 and 00004719). The PReDICT study is registered at ClinicalTrials.gov Identifier: NCT03226912 and NCT00360399. DNA was extracted from whole blood, and genome-wide SNP genotyping was performed using HumanOmniExpress BeadChips. Quality control was performed in PLINK. Samples with low genotyping rate (<98%) were removed. SNPs with a high rate of missing data (>2%), significant deviation from the Hardy-Weinberg equilibrium (HWE, P < 105), or a low minor allele frequency (<5%) were excluded from further analyses. SNP genotypes were coded as 0 for major homozygotes (GG, n = 56), 1 for heterozygotes (TG, n = 74), and 2 for minor homozygotes (TT, n = 24) and did not deviate from HWE (2 = 0.27, P = 0.60). HPA axis function was assessed using the dexamethasone/corticotropin-releasing hormone (Dex/CRF) test, consisting of an oral administration of 1.5 mg of Dex at 11:00 p.m. and an infusion of ovine CRF (1 g/kg) at 3:00 p.m. on the next day. Cortisol and ACTH levels were measured from plasma samples taken immediately before CRF administration (pre-CRF) (i.e., at 3:00 p.m.) and again at 3:30 p.m. (30 min), 3:45 p.m. (45 min), 4:00 p.m. (60 min), and 4:15 p.m. (75 min). Baseline cortisol levels were available for all 154 patients with genotype data for the SNP rs2032582. Only completers were included in the analysis, so there are no dropouts in sample sizes over time. Linear regression models were used to test for effects of the SNP genotype on baseline cortisol levels using R version 3.6.2. To assess differences in cortisol and ACTH levels after the Dex/CRF test over time, linear mixed effects models with a random intercept for each patient were applied. All models included gender, age, and baseline depression severity sum scores on the 17-item Hamilton Depression Rating Scale (63) and the first five genetic ancestry (multidimensional scaling) components as covariates.

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Single-cell molecular profiling of all three components of the HPA axis reveals adrenal ABCB1 as a regulator of stress adaptation - Science Advances

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