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3 Effective Fasting Methods for Weight Loss, Science Says Eat This Not That – Eat This, Not That

Posted: January 20, 2022 at 2:22 am

Since it's been a trendy eating habit for a while now, you've likely already heard of intermittent fasting, or IF for short. If you're trying to lower the number on the scale, variations of this diet offer minimalist approaches to losing weight.

"Any time we have a calorie deficit, there will be weight loss," Jenny Fontana, NT, NCRC, NCFAC, who instructs certified nutrition coaches, tells Eat This, Not That!.

There are several different forms of intermittent fasting, some of which may be more practicalweight loss interventionsthan others. A studyrecently published in JAMA Network highlighted three methods found to be particularly effective.

Related: Intermittent Fasting Can Lead to "Significant" Weight Loss, New Research Says

"This study is essentially a review of review articles. It demonstrates that the different forms of intermittent fasting (i.e., alternate day fasting, the 5:2 diet, and time-restricted feeding) are all effective weight loss interventions for people with obesity," Krista Varady, PhD, co-author of the study and a nutrition professor at the University of Illinois, Chicago, told Medical News Today.

Beyond weight loss, IF also offers a wealth of science-backed health benefitshere are seven to be aware of. But first, here's a breakdown of the three forms of intermittent fasting name-checked by Varady.

Followers of alternate day fasting adhere to a schedule that switches back and forth between eating and fasting. Those who abide by this diet alternate between one feast day and one fast day, which typically includes a 500-calorie meal.

"The trick is to make sure you are still eating healthy on days when you are not fasting," Heather Hanks, MS CAM, nutritionist and medical advisor at Medical Solutions BCN, tells Eat This, Not That!. "If you overindulge in calories from refined sugars and carbs, you'll throw your insulin and hunger hormone levels off and wind up storing more glycogen than you're burning."

Healthline notes that "the most popular version of this diet is called 'The Every Other Day Diet' by [Varady], who has conducted most of the studies on ADF."

Followers of the 5:2 diet eat an unrestricted diet for five days of the week and fast for the remaining two.

When it comes to long term compliance with the first two diets on this list, Shadi Vahdat, MD, the medical director at LiveWell Integrative Medicine, offers a word of caution.

"While the alternate day fasting, 5:2 diet, and time-restricted feeding can all be effective in weight loss, reduction of body fat and the associated metabolic improvements that come with them, long term compliance with the alternate-day fasting or 5:2 diet can become very challenging," Vahdat tells Eat This, Not That!.

There aren't calorie restrictions in time-restricted feeding, but followers of this method only eat during a daily window of four to 10 hours. Fasting occurs outside of that designated eating window.

"The most practical and feasible option for a lot of people turns out to be time-restricted feeding," Vahdat tells Eat This, Not That!. "If done correctly with daily overnight fasting of anywhere between 12-14 hours, then the benefits can be obtained long-term with none to minimal side effects."

To learn more about fasting for weight loss, check out Doing This Before Fasting Can Speed Up Your Fat Burn, New Study Says. Then, don't forget to sign up for our newsletter for more of the latest health and food news!

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8 Ways to Live a Longer Life Eat This Not That – Eat This, Not That

Posted: January 20, 2022 at 2:22 am

A premature death is considered to be a loss of life before the age of 75 according to America's Health Rankings. While early deaths happen for various reasons, many can be prevented, the Centers for Disease Control and Prevention stated. "Each year, nearly 900,000 Americans die prematurely from the five leading causes of death yet 20 percent to 40 percent of the deaths from each cause could be prevented." The CDC said, "The five leading causes of death in the United States are heart disease, cancer, chronic lower respiratory diseases, stroke, and unintentional injuries." With that said: Life doesn't need to be cut short. Living a healthy lifestyle, getting annual check ups and keeping a positive outlook all help prolong our time here. In addition, here's a few ways to help avoid a premature death according to experts Eat This, Not That! Health spoke with. Read onand to ensure your health and the health of others, don't miss these Sure Signs You've Already Had COVID.

Dr. S. Adam Ramin, MD, urologic surgeon and medical director of Urology Cancer Specialists in Los Angeles says, "The immune system is our first line of defense against cancerous cells. Long before a particular type of cancer has grown and multiplied enough times to become detectable by blood tests, imaging, or screening tools, the cancer had initially started with a tiny microscopically sized colony of individual cells. This small population of cancer cells are not detectable by even modern methods of cancer screening. However the amazing human body's immune system is capable of identifying and mounting a strike force against these unwanted mutant cells. By maintaining a healthy vibrant immune system, we give our own bodies a fighting chance at irradiating the cancer cells at their infancy. How do we promote a healthy immune system?

Dr. Ramin explains, "Foods that are treated with preservatives and are found in a can, a box, or prepared to last a long time may cause cancer. The preservative products and treatment of these foods with heat/radiation may alter their natural chemical makeup. This in turn may contribute to formation of products in our digestive system that cause DNA mutations and eventually cancer."

RELATED: Signs You Must Lose Your Abdominal Now

Dr. Carmen Echols, MD certified Family Medicine Physician says, "Although 1 in 8 women are diagnosed with invasive breast cancer in the United States, early detection plays a key role in prognosis and survival. The earlier the detection, the better the outcome, because the cancer is detected at an earlier stage. Additionally, Black women are more likely to be diagnosed with more aggressive breast cancer and more advanced staged breast cancer than their White and Hispanic counterparts; they are also diagnosed at earlier ages as well. Therefore, it is absolutely important to get a mammogram as soon as you are able to based on your age and family history of breast cancer."

RELATED: I'm an ER Doctor and Wish Everyone Knew This One Thing

"The recommended age for colon cancer screening is 45 years old," Dr. Echols states. "Some people avoid the colonoscopy because they dread the required bowel prep process before the procedure. Still, other people avoid getting a colonoscopy because they have regular bowel movements or do not see blood in their stool, and feel it is unnecessary. However, someone can still have precancerous cells or blood in their stool that they are not able to see with the naked eye. A colonoscopy is a procedure that combines diagnosis, prevention and treatment. If there are any polyps found during a colonoscopy, they are removed and sent for biopsy to determine if they are cancerous or not. If precancerous cells are found, then you may need to get a colonoscopy more often than if non cancerous cells were found. Like with breast cancer, when you start colon cancer screening can also be impacted by your age and family history."

RELATED: Omicron Symptoms Patients Mention the Most

Dr. Steve Vasilev MD, quadruple board certified integrative gynecologic oncologist and medical director of Integrative Gynecologic Oncology at Providence Saint John's Health Center and Professor at Saint John's Cancer Institute in Santa Monica, CA recommends, "Strongly consider getting genetic counseling if you have cancer, have a family history of cancer or are at high risk based on ancestry, such as the case in Ashkenazi Jewish women. Based on this counseling a determination is made regarding whether or not genetic testing is required and what to do depending upon the results. The most widely known test is for mutations of the BRCA gene and its relationship with ovarian, breast, uterine and prostate cancer. These are not the only mutations that are available for testing and the list is growing. In some situations, like that of Angelina Jolie, testing results may even lead to considering prophylactic removal of breast tissue, Fallopian tubes and ovaries. This is highly individualized and is usually not recommended until childbearing is complete at approximately age 40 and depends upon your personal history of cancer, family history and the exact type of gene mutation.

It's important to avoid genetic testing that may be commercially available prematurely (e.g. self-testing sent in assays) but not well worked out yet in terms of meaning. This can lead to unnecessary worry about test results that are questionable or tests that may increase risk of cancer but for which there are no good screening or prevention strategies."

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Dr. Mark Dylewski, Chief of Thoracic Surgery with Miami Cancer Institute, part of Baptist Health South Florida states, "One of the biggest issues is that many people believe that lung cancer occurs as a result of bad habits, such as smoking. So many people believe that it is an acquired illness and that if people do not smoke, lung cancer will not happen. Unfortunately, about 17% of lung cancer that occurs in the US occurs in non-smokers. So lung cancer is not always a smoker's disease. I believe tobacco is probably one of the worst things someone can put into their body. It contributes to so many other cancers and other illnesses we see everyday such as hypertension, stroke, coronary artery disease, etc. Lung cancers that occur in nonsmokers usually tend to occur in females between the age of 50 70 years old. We see lung cancer, unrelated to tobacco use, increasing year after year in non-smokers. There may be environmental triggers or secondary exposure leading to this such as chemicals or environmental toxins. If you have a family history of lung cancer, particularly if the family member was a non- smoker, that is important. This would imply that related family members may be at risk for lung cancer, and this should prompt them to talk to their primary care physician about lung cancer screening. My recommendation would be for patients to ask their doctor if they are at risk for developing lung cancer and if screening should be part of their routine. Most patients are familiar with breast cancer and prostate cancer screenings, but not so much with lung cancer. If someone has ever smoked, you should ask your primary doctor or contact a screening program. They can best provide insight into which patients are best candidates for lung cancer screening."

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According to Dr. Naiara Braghiroli, Chief of Skin Cancer and Pigmented Lesions Clinic at Miami Cancer Institute, part of Baptist Health South Florida, "Approximately 75% of skin cancers diagnosed in people of color are in areas that are not exposed to the sun, such as the palms of the hands, nail beds, soles of the feet, inside the mouth and/or the genitalia area. Due to the locations of these skin cancers, there is a higher mortality rate for people of color as diagnosis is often delayed. As such, self-exams are extremely important. It's important to conduct a self-exam at least once a month, using a mirror and, if possible, have a partner help you, paying close attention to areas not exposed to the sun, looking for new black/brown areas, asymmetrical moles, open wounds that don't heal and old scars that develop open wounds. In addition to self-examinations, be sure to visit your dermatologist annually to potentially catch any areas you might have missed. Early detection is key in curing melanoma, so if you find an unusual spot, mole or skin area, it's critical to see your dermatologist right away.

It's also important to know your family history when it comes to skin cancer. Each individual with a first-degree relative diagnosed with melanoma has a 50% greater chance of developing melanoma in the future than those without a family history of the disease. Additional risk factors to be mindful of are having a lot of moles, scars from a previous trauma and chronic/open wounds. Those who have HPV, an autoimmune disease or who are immunosuppressed are also at greater risk."

Follow the public health fundamentals and help end this pandemic, no matter where you liveget vaccinated or boosted ASAP; if you live in an area with low vaccination rates, wear an N95 face mask, don't travel, social distance, avoid large crowds, don't go indoors with people you're not sheltering with (especially in bars), practice good hand hygiene, and to protect your life and the lives of others, don't visit any of these 35 Places You're Most Likely to Catch COVID.

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COVID Symptoms to Watch For This Month Eat This Not That – Eat This, Not That

Posted: January 20, 2022 at 2:22 am

The latest COVID surge is fueled by the spread of Omicron and has caused the U.S. to reach bleak milestones of record-high cases. The most COVID cases were reported in a week since the beginning of the pandemic according to World Health Organization Director-General Tedros Adhanom Ghebreyesus."Last week, more than 15 million new cases of Covid-19 were reported to WHO from around the world by far the most cases reported in a single week and we know this is an underestimate," Tedros said during a news briefing in Geneva. "This huge spike in infections is being driven by the Omicron variant, which is rapidly replacing Delta in almost all countries." Taking precautions and staying healthy is vital during this time and Eat This, Not That! Health talked with Robert G. Lahita MD, Ph.D. ("Dr. Bob"), Director of the Institute for Autoimmune and Rheumatic Disease at Saint Joseph Health and author of Immunity Strong who explained what symptoms to watch out for and why the surge is happening. Read onand to ensure your health and the health of others, don't miss these Sure Signs You've Already Had COVID.

Dr. Bob explains, "A COVID surge is when we see a large spike in new cases. We've seen this happen after big traveling and gathering periods such as holidays. Winter is particularly bad because it's also flu season, so we have people getting sick with the common cold, the flu, and COVID at the same time. There are a lot of employee shortages with so many out sick at the same time with different infections. Additionally, the holidays can often stress people out, and stress can dampen your immune system's ability to fight viruses."

According to Dr. Bob, "People should watch for fever, shortness of breath, and loss of taste and smell as these are all possible Covid symptoms."Dr. Teresa Bartlett, senior medical officer at Sedgwick says, "The majority of people are exhibiting a severe sore throat and describing it as swallowing razor blades, stuffy nose, fever, body aches and a cough. Often the virus starts with a headache and many think they have a sinus infection. Be on the lookout for these symptoms. I have spoken to so many patients who think if they did not lose taste or smell they can't possibly have COVID but that simply is not true."

Dr. Shadi Vahdat, an assistant clinical professor at UCLA and medical director at LiveWell Integrative Medicine adds, "For a lot of people infected with Omicron the symptoms will be much like the common cold. In one study from Norway where most people infected with the Omicron variant were vaccinated they reported the most common symptoms as cough (83%), runny nose/stuffy nose (78%), fatigue/lethargy (74%), sore throat (72%), headache (68%) and fever (54%), reduced taste (23%), reduced smell (12%). 42% reported mild to moderate symptoms and none required hospitalization. For many front line workers who are taking care of COVID patients in the emergency rooms and hospitals it seems evident that most of the severely impacted and sick who require hospitalization and ICU admissions the vast majority continue to be those who are unvaccinated."

RELATED: Dr. Fauci Says if You Have COVID, Do This

According to Dr. Bob, "People are still going to the hospital due to COVID, but a smaller proportion of those patients are in ICU or on ventilators. Some data show roughly 50 to 65 percent of admissions in some New York hospitals come in for other issues and then test positive for the virus. The thing with Omicron is that it is EXTREMELY transmissible. If you're in a room with someone who has it, you will probably get it. It's spreading like wildfire, which means a lot of people are going to get it at once, and that means hospitals who are already facing staff shortages and burnt out workers are going to be filling up."

RELATED: The Best Things to Take If You Get COVID

While nobody can know for sure, experts are estimating it could happen later this month, but the next couple of weeks are critical. Dr. Ashish Jha, dean of the Brown University School of Public Health, told ABC's This Week."We're seeing two sets of things happening: A lot of vaccinated people getting infected. We're doing fine. Largely avoiding getting particularly sick, avoiding the hospital; a lot of unvaccinated people and high-risk people who have not gotten boosted and they're really filling up the hospitals, and so our hospital systems are under a lot of stress." He continued, "Then we have to start thinking about a long-term strategy for how do we manage this virus and not go from surge to surge feeling like we don't really have a longer-termed approach."

Jha added, "I expect this surge to peak in the next couple of weeks. It'll peak in different places of America at different times, but once we get into February, I really do expect much, much lower case numbers."

"It's simple: They can get vaccinated and boosted. This is the best way to keep yourself out of the hospital and to stay alive," Dr. Bob states. The Centers for Disease Control and Prevention lists the following ways to help stay healthy" "Wear a mask; Stay 6 Feet From Others; Inside your home, avoid close contact with people who are sick; Outside of your home, remember that some people without symptoms may be able to spread virus." So follow the public health fundamentals and help end this pandemic, no matter where you liveget vaccinated or boosted ASAP; if you live in an area with low vaccination rates, wear an N95 face mask, don't travel, social distance, avoid large crowds, don't go indoors with people you're not sheltering with (especially in bars), practice good hand hygiene, and to protect your life and the lives of others, don't visit any of these 35 Places You're Most Likely to Catch COVID.

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How a wellness nonprofit for Houston’s BIPOC community is making moves this week – Houston Chronicle

Posted: January 20, 2022 at 2:22 am

Davina Davidson is making moves this week.

Her nonprofit, Melanin Moves Project, which seeks to educate members of the BIPOC Black, Indigenous, people of color community about the benefits of overall wellness, is hosting a one-day wellness event, complete with workshops and seminars about movement, meditation and healthy living.

The Wellness Expo is scheduled from 9:30 a.m. to 1:30 p.m. Jan. 22 at Yoga Tres, 5427 Bellaire. The cost to attend is on a sliding scale, ranging from $30 to $100.

During the event, guests can catch a stretch therapy demo with Davidson, founder and president of Melanin Moves Project. Attendees will also learn about restorative yoga with instructor Shawn Moore and try a sweat date with high-intensity interval training coach Bertha Rials.

MORE FROM LINDSAY PEYTON: 'He came in like Superman': A Clear Lake hospital chaplain's COVID mission

Chiropractor Kiva Davis will provide a playful rocket yoga class, healer Raquel Nweze will lead a session on essential oils and chakras, and instructor Maria Powell will offer a joint efficiency workshop.

To register for the Wellness Expo, visit melaninmovesproject.org.

Other presenters include Zevi Ramos, James Elvis Lynn III, Jordan Lynn, Denetrya Brookins and Acufunkture Integrative Medicine.

The expo is twofold, Davidson said. One, it is a celebration of melanated teachers and educators with a wealth of knowledge, and two, it is so fun and exciting to meet new people along your movement journey.

Her sister Amaris Davidson, community outreach program chair for Melanin Moves Project, said previous iterations of the expo were held online due to the pandemic. This year, shes looking forward to the event being held in person again.

Theres something so powerful about being around other people on their wellness journey, she said. Theyre interested in the same tools and resources. To be able to provide that is awesome.

The Wellness Expo is just one of the ways Melanin Moves Project seeks to build awareness of health.

We educate from childhood all the way to adulthood, said Davina, whose main goal is to encourage movement. Theres not one right way but you have to move.

The organization offers workshops year-round on various topics, as well as teacher training and mentorship for yoga instructors. Currently, courses are offered virtually. Before the pandemic, spaces were rented for in-person events.

Last fall, Melanin Moves Project began a collaboration with chef Denetrya Brookins to add a greater focus on nutrition. This month, the nonprofit is launching a mens program.

We strive to create a space where men can fully embrace themselves, Davina said.

The program will center on mental, physical and spiritual well-being, incorporating activism and service, while also providing BIPOC men with the tools and resources needed to reach those goals.

Melanin Moves Project also works with youth, joining with leadership development organization Train Up A Champion to offer a curriculum.

Finding ways to connect and collaborate to reach greater heights is a priority for the program. Melanin Moves Project works with a number of BIPOC-owned businesses that are also interested in wellness. Partners range from the art haven Project Row Houses to Shape Community Center, which offers programs and activities to all individuals of African descent.

The organization began in 2017 as the Melanin Yoga Project.

Davina had gone from working as a school teacher to a yoga instructor. The realization that yoga classes were often filled with affluent, white students made her want to change the equation: According to a 2002 survey, 85 percent of yoga practitioners were white.

Creating Melanin Yoga Project was Davinas response and the nonprofit made yoga available to BIPOC communities, at school and in community centers.

She also started training instructors and offering courses to studios.

Amaris recalled taking Davina to her first yoga class, then watching as her sister became a teacher and started Melanin Yoga Project. Amaris, who was living in LA at the time, would sign up for Davinas classes whenever she visited Houston. She also took the Melanin Yoga Projects online teacher certification, with a final in-person session at the conclusion.

Davina started online well before the pandemic, Amaris said with a smile.

When Davina reached out in February 2020 to offer her sister a role in the growth of the organization, Amaris was unsure what she could contribute. But Davina convinced her that the seven years she spent on her churchs connection team, building community relations, provided just the right experience.

Its the same thing, Davina reminded her.

Amaris jumped on board. Ive been here ever since, she said.

Soon after, Davina broadened the focus of Melanin Yoga Project to include other types of movement and wellness in general.

Its important for us to create a space for everyone whether youre a yoga person or youve never even practiced yoga, she said. We can do all the things. We dont have to do just one.

In June 2021, the nonprofits name changed to Melanin Moves Project with a more expanded mission.

The expansion came from many realizations, Davina said. Yoga is not the only path to healing or liberation for melanated folks. We were missing important pillars, like social justice initiatives, nutrition and mental health awareness.

Her board approved the new vision.

Yoga is a niche market, Amaris said. Now were adding mental health, social justice and nutrition. You can see so many doors opening.

The nonprofit has been moving full steam ahead since its summer pivot.

Each week, Amaris said, theres a Monday brainstorming meeting. Then there are quarterly meetups with community partners.

She and Davina are always talking strategy.

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She likes to have a long vision, Amaris said. She wants to have a community center one day, where everyone can come from everywhere. And Im down for the journey.

Davina envisions a brick-and-mortar center in the future. And not just one, we could have pop-ups all around the U.S., she said. I know that I have to dream big.

In the meantime, she is launching Melanin Moves TV, which can be accessed from anywhere.

In 2018, she started the platform as Yoga with Davina. After the pandemic, and the expansion, she changed the channels name and focus. There are a number of live courses, as well as a video library.

We are back recording, Davina said. There will be cooking classes, talks and programs for kids as well.

The nonprofit is run completely by volunteers and needs more to move forward. Currently, Davina is focused on sharing the Melanin Movement Project with the community.

I want people to know about our offerings, she said. This exists in your city. And were here to stay. We have a desire to impact as many people as we can.

Amaris agreed. Were making wellness accessible and you dont have to do it alone, she said. Were creating a community. And this can go as far as we want it to.

We are so ready to just go, Davina added. Were making an imprint. It might be small, but its just the beginning.

Lindsay Peyton is a Houston-based freelance writer.

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Adapt the Frequency of COVID-19 Testing Depending on Transmission Rate and Community Immunity, Study Finds – UT News – UT News | The University of…

Posted: January 20, 2022 at 2:22 am

AUSTIN, Texas Expanding rapid testing stands out as an affordable way to help mitigate risks associated with COVID-19 and emerging variants. Infectious disease researchers at The University of Texas at Austin have developed a new model that tailors testing recommendations to new variants and likely immunity levels in a community, offering a new strategy as public health leaders seek a way out of a pandemic that has so far thwarted the best efforts to end its spread. It is the first study to identify optimal levels of testing in a partially immunized population.

Analysis from the UT Covid-19 Modeling Consortium, published in The Lancet Regional Health Americas, describes cost-effective testing for people without symptoms and recommends isolation strategies to help policymakers safeguard against COVID-19 resurgences linked to new variants. A prior study from the team published in The Lancet Public Health provided optimal testing strategies for a fully unvaccinated population.

As COVID-19 continues to evolve and cause waves of infections worldwide, rapid testing is an economic strategy for slowing spread and saving lives. Our study helps decision makers determine whether and how often to test, said Lauren Ancel Meyers, director of the consortium and a professor of integrative biology and statistics and data sciences at UT Austin. Frequent testing is recommended when the virus is spreading rapidly in a population with low levels of immunity.

The consortium developed a multiscale model that uses how much the virus is circulating in a local population, how much of the population is immunized against COVID-19, and other factors to determine how often people without symptoms should be tested in order to help reduce the spread of the virus.

The study recommends a staged strategy that tracks the changing risks as new variants emerge and subside. If a rapidly spreading variant emerges in a partially immunized population, the researchers recommend testing everyone at least once per week combined with a 10-day isolation of people who test positive and their households. As the level of immunity increases in a population, testing can be rolled back to once per month and eventually suspended. For example, for a variant as infectious and immune-evasive as omicron, daily testing is advised until 70% of the population is immunized against the variant, followed by monthly testing until 80% are immunized.

The U.S. may face future waves of transmission caused by vaccine-evasive variants. The study suggests that proactive testing will remain a cost-effective strategy for reducing risks and avoiding burdensome restrictions as new threats arise. The recommended testing strategies balance the costs associated with administering tests and missing school or work during isolation with the benefits of preventing COVID-19 hospitalizations and deaths.

As COVID-19 continues to evolve, so does our arsenal of effective countermeasures. Our research shows that mass use of rapid tests coupled with voluntary isolation and household quarantine can be both life saving and cost saving, if tailored to local risks, Meyers said. Now is the time to prepare for yet unknown COVID-19 variants and future pandemics. Proactive testing and isolation can be key to keeping schools and businesses open while preventing overwhelming surges in our hospitals.

Co-corresponding authors are Zhanwei Du, previously of Meyers lab, Yan Bai of The University of Hong Kong and Lin Wang of the University of Cambridge. Other authors are Xutong Wang of The University of Texas at Austin; Abhishek Pandey, Meagan Fitzpatrick and Alison P. Galvani of Yale School of Public Health; Matteo Chinazzi, Ana Pastore y Piontti and Alessandro Vespignani of Northeastern University; Nathaniel Hupert of Weill Cornell Medicine and Cornell Institute for Disease and Disaster Preparedness; Michael Lachmann of Santa Fe Institute; and Benjamin J. Cowling of Hong Kong University. Meyers is the Cooley Centennial Professor of Integrative Biology and Statistics & Data Sciences at The University of Texas at Austin.

The research was supported by the National Institutes of Health, Centers for Disease Control and Prevention, HK Innovation and Technology Commission, China National Natural Science Foundation, European Research Council and EPSRC Impact Acceleration Grant.

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Adapt the Frequency of COVID-19 Testing Depending on Transmission Rate and Community Immunity, Study Finds - UT News - UT News | The University of...

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Strange COVID Symptoms No One Talks About Eat This Not That – Eat This, Not That

Posted: January 20, 2022 at 2:22 am

By now we should all know the common signs of COVIDcough, fever, fatigue, body aches, muscle aches and shortness of breath, among other symptoms. But in addition to the typical woes, some people experience unusual effects of the virus that aren't widely known about. Researchers are still learning about why strange symptoms happen to some people and not others. Eat This, Not That! Health talked to doctors who revealed odd signs of COVID they've seen and explained why symptoms vary so much. Read onand to ensure your health and the health of others, don't miss these Sure Signs You've Already Had COVID.

Dr. Ali Jamehdor, DO Medical Director, Emergency Department at Dignity Health St. Mary's Medical Center, says, "Most of us know about the 'usual' sign and symptoms that come with COVID infections. Runny nose, cough, sore throat, headache, body ache and fever. But we have seen some symptoms that are associated with COVID-19that aren't usually associated with an upper respiratory viral infection. Remember in the beginning when we started to hear about people losing their sense of smell or taste? This seems to be mainly associated with the original COVID-19 virusless so with the Delta variantand as the Omicron variant has become the superior variantwe're not seeing it at all these days. While it's not that unusual to lose some sense of smell or taste with a regular cold because of congestion and other inflammatory processes that occur on the oral nasopharyngeal cavity, this loss of smell and or taste was occurring in patients with absolutely no other complaints. Some people would lose these senses for hours, some days and there are still cases that have not regained these important senses for over a year. There has not been any true medical explanation for this, there does not seem to be any remedies at this time but luckily this symptom seems to be losing its potency and we are seeing fewer and fewer of this complaint."

Dr. Michael Hirt, a Board Certified Nutrition from Harvard University and Board Certified in Internal Medicine and is with The Center for Integrative Medicine in Tarzana California says, "Without the other classic COVID symptoms occurring simultaneously, most people who get COVID diarrhea think they have a mild case of food poisoning or ate something that just didn't 'agree' with them. Viral shedding in the stool is so common with this virus that 'COVID hunting' public health officials often sample community wastewater to track the levels of the virus in cities, neighborhoods, and even college dorms."

RELATED: "Most People are Going to Get COVID," but You Can Cheat It. Here's How.

Dr. Teresa Bartlett, senior medical officer at Sedgwick explains, "The skin rashes are really an odd symptom we are seeing in some cases. Rashes on the body, head and even COVID toe that appears like bruising on the toes. Many people with the Omnicron variant are getting a really bad sore throat that was not the case with the prior variants. It appears people either have a minor stuffy nose and scratchy throat or they are really sick with headache, dizziness, cough, fever and severe fatigue."

RELATED: Dr. Fauci Identifies "Possible Cause" of Long COVID

Dr. Hadassah Kupfer, Audiologist and Hearing Aid Specialist says, "Tinnitus, or ringing in the ear, has been reported on occasion, either following Covid-19 infection or even the Covid-19 vaccination and boosters. We have seen multiple cases in our NYC audiology practice, where there has been high community spread of COVID infection, and COVID vaccination is highly prevalent and/or mandated across most work sectors. Tinnitus is usually a sign of damage to the inner ear- which ultimately builds up into a noticeable hearing loss. Although the direct link between COVID and tinnitus is still unknown, one possibility is attributed to the cytokine storm that occurs in COVID patients, which may attack the very delicate cochlea (inner ear). If this is correct, then patients with underlying risk factors for developing hearing loss (diabetes, heart conditions, prior noise exposure, prior diagnosis of hearing loss) will be more vulnerable to auditory symptoms from COVID, as this may speed up the pathological process. The delicate cochlea would also be more vulnerable in those with autoimmune conditions, since their body is more likely to attack itself."

RELATED: Signs You've Developed Diabetes Without Knowing It

According to Maryland based dentist Dr. Mansi Oza, "COVID virus binds to the cells inside the mouth. By entering through the blood and swallowing from the mouth, it spreads to the lower lobes of the lungs, gut and other parts of the body." As a result, Dr. Oza explains that the following symptoms can appear in the mouth.

RELATED: The #1 Cause of Heart Attack, Experts Find

"The symptoms people exhibit largely depend on their immune status and how their body responds to the virus," Dr. Bartlett states. "It also could depend on the variant you are exposed to and whether or not you have any immunity either through vaccines or prior infections to COVID 19. Your immune status directly correlates with how well you take care of yourself by eating a well-balanced diet, maintaining a regular exercise program and getting enough sleep."

RELATED: Supplements That Really Do Boost Immunity

Dr. Bartlett says, "The symptoms vary as does the actual contraction of the virus. I have seen cases of 2 people driving in a car for hours together without masks, when the next day one of them becomes sick with the virus and the other person does not get it. Keep in mind there are asymptomatic carriers out there who unknowingly could spread the virus. It is speculated that all this variation is dependent on age, underlying health conditions, blood type, race, social determinants of health, prior vaccinations you may have had in your lifetime, prior diseases you had and how much of the virus you were exposed to."

"Not really. While scientists continue to study all the various symptoms and implications, unless there is something that continues to bother you after you have "recovered" there is no need to be concerned. As always, talk to your doctor if you are worried about something," Dr. Bartlett states.

Follow the public health fundamentals and help end this pandemic, no matter where you liveget vaccinated or boosted ASAP; if you live in an area with low vaccination rates, wear an N95 face mask, don't travel, social distance, avoid large crowds, don't go indoors with people you're not sheltering with (especially in bars), practice good hand hygiene, and to protect your life and the lives of others, don't visit any of these 35 Places You're Most Likely to Catch COVID.

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Strange COVID Symptoms No One Talks About Eat This Not That - Eat This, Not That

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What Is Integrative Medicine for ADHD? A Holistic Health & Wellness Guide – ADDitude

Posted: January 20, 2022 at 2:22 am

ADHD doesnt only affect attention. Better considered an executive function and self-regulation deficit, ADHD affects the whole person the mental, emotional, physical, spiritual, and social self. It increases daily stress and chips away at a positive sense of self. It interferes with self-care and makes it hard to keep healthy habits.

This helps to explain why ADHD is linked to chronic stress, burnout, anxiety, mood disorder, sleep problems, substance use, and other conditions and issues. The reverse is also true: chronic stress and anxiety can worsen ADHD symptoms.

ADHD impacts the whole self, so is treatments must likewise target more than inattention and impulsivity. Integrative medicine is growing in popularity because its a treatment approach that addresses symptoms and promotes general health and wellness.

Integrative medicine considers the whole person and leverages all options holistic thinking, complementary therapies, and conventional treatments in devising a patients care plan.

Studies exploring the effectiveness of integrative approaches for ADHD specifically are limited. Moreover, the most common treatments for ADHD are the conventional medication and psychotherapy. The American Academy of Pediatrics (AAP) recommends treating ADHD in children and adolescents aged 6 to 18 with FDA-approved medications, plus parent training in behavior modification and behavioral classroom interventions. Research studies have found that stimulant medications are most effective, and combined medication and psychosocial treatment is the most beneficial treatment option for most adult patients with ADHD.

Still, just as ADHD affects many aspects of wellbeing, a variety of treatments and approaches can do the same.

[Get This Free Download: Natural ADHD Treatment Options]

As an integrative practitioner, my approach for treating patients with ADHD is this: If the ADHD symptoms are significantly impairing, I start with medication, and then phase in other strategies, often outside of conventional care. If the ADHD symptoms are mild to moderate, the non-medication and lifestyle approaches can be tried first.

Over time, as the other skills and strategies are employed, the need for medication can be re-evaluated and the dose reduced.

An example of an integrative medicine plan for ADHD may combine psychotherapy (a conventional strategy), stress-management skills (holistic thinking), and omega-3 fatty acids (a complementary supplement). All ADHD treatment decisions should be made in consultation and coordination with a licensed medical provider.

[Read: How Nutrition, Exercise & Sleep Curb ADHD]

Most of the following approaches address ADHDs secondary symptoms namely stress, anxiety, mood, low self-esteem, and emotional dysregulation. Treating these factors can help decrease the severity and impairment of ADHDs core symptoms.

Cognitive behavioral therapy (CBT) helps patients develop a greater understanding of their ADHD symptoms and teaches skills that help with executive dysfunction.

CBT aims to improve patients problem-solving and stress-management skills by setting realistic goals and teaching organizational and time-management skills to achieve them. This type of psychotherapy can also improve balanced thinking and communication skills by focusing on ones unique challenges (e.g., history of trauma or other comorbid mental health conditions).

Like CBT, coaching helps individuals meet their goals and develop skills to address ADHD-related barriers along the way.

Mindfulness a practice that includes meditation as well as awareness shifts in daily activities has been shown to improve both inattentive and hyperactive/impulsive symptoms, as well as selected measures of attention, emotion regulation, and executive functions1.

By analysis of automatic habits, the practice allows you to change them in the moment. For example, mindful awareness may help you realize that you are procrastinating, and help you tune in to the emotions that are driving the procrastination.

A facet of mindfulness, practicing self-compassion is particularly important for mental health. Offering yourself some validation and kindness This is hard. Im stressed. Im struggling will make a difference in how stress is experienced.

As you observe your reaction and create inner pause, you can ask: What can I do to help this situation? and find possibilities to do so. The answer may be I need to take a few deep breaths or I need to prioritize my tasks.

Sometimes one can reframe the situation or focus on the positive (e.g., gratitude) to see what is working versus what is not. By making such shifts in awareness and response, you can begin to self-regulate and enhance your resilience.

Seeing ADHD symptoms as neurobiologically driven ways of responding versus the idea that you are defective in some way fosters self-acceptance. The important thing is to see ADHD as a biological difference and condition that needs extra support or accommodation.

Regular sleep, adequate hydration, prioritized self-care, and avoidance of excessive alcohol and other substances can help manage ADHD symptoms. At the same time, the ability to keep up with these practices is often compromised by ADHD itself. It is best for patients and clinicians to identify and target the most problematic areas first.

Exercise has wide-ranging health benefits (physical, cognitive, and emotional) both acutely and when done regularly over time. In particular, aerobic exercise has been shown to improve executive functions, attention, and behavioral symptoms in ADHD2. Other types of mind-body movement, such as yoga or tai chi, can also be helpful for ADHD symptoms.

Stress and anxiety typically make breathing faster and shallower (i.e., chest breathing). Slower and deeper breathing (i.e., belly breathing) is the ideal. Breathing regulates the sympathetic-parasympathetic nervous system balance, so breathwork can counteract stress and change your body state.

Examples of breathing exercises:

Acupuncture, derived from Chinese medicine, aims to treat a variety of conditions by stimulating diverse points on the body (acupoints). This approach focuses on regulating the body organ system to lower inattention and hyperactivity. Some research supporting the use of acupuncture for ADHD is available from Asian countries3. But this approach to ADHD has not been studied widely in Western cultures.For general wellness, acupuncture is often used to treat pain and stress-related conditions. There is also some evidence supporting it as an adjunctive treatment for anxiety. I have found it helpful for those who struggle with chronic stress and pain.

Poor nutrition and lifestyle habits can increase the level of impairment from ADHD. While specific nutritional approaches for ADHD symptoms dont have strong research evidence, we do know that processed foods, refined grains, excessive sugar, and high fat worsen mental health.

Eat the foods that support health and mood. Foods like whole grains, fruits, vegetables, lean proteins, fish, and nuts has been shown to improve depressive symptoms4. Colorful fruit and vegetables (high in flavonoids and antioxidants) appear to protect against cognitive decline5, and may support modulation of neurotransmitters, such as dopamine6. Eating protein at each meal and low-glycemic foods (which dont spike blood sugar quickly) enhance steady blood glucose and cognitive function.

The gut-brain axis refers to the two-way link between these parts of the body (i.e. the emotional and cognitive centers of the brain with intestinal functions). Research tells us that the foods we eat affect the microbiome in the gut, which influences this connection7. Healthy gut flora, for example, can reduce anxiety and serum cortisol levels8. Prebiotic and probiotic foods, like kimchi and sauerkraut, can support gut health.

A variety of supplements and herbs have been studied for their use in treating ADHD. In using supplements, two paths can be taken:

The thinking in using a combination of supplements for ADHD is that multiple nutrients will be involved in the important processes in the brain, such as modulation of key neurotransmitters.Since ADHD symptoms exist on a spectrum from mild to severe, supplementation can be individualized and used with or without medications. When using supplements, practical considerations, like cost or the number of pills needed per day, should be considered.

There are many kinds of integrative providers, with different training backgrounds and attitudes about treating ADHD. A good integrative provider will understand conventional mental health and wont sell only one approach. They should be willing to work collaboratively with you and your other clinicians.

I recommend beginning your search for integrative providers with these national organizations.

Talk with the provider before making an appointment to understand their approach and to see if they are a good fit for your needs. Many of the providers found here have knowledge of integrative approaches, and are willing to collaborate with other integrative clinicians on a holistic treatment plan.

Keep in mind that medication is a very helpful tool to support the brain processing differences due to ADHD. It is important to collaborate with your doctor to find the most effective medication and dosage for you. There may be times when more medication is needed, and times when it can be decreased or eliminated. We know that the level of impairment that comes with ADHD can fluctuate over a lifespan. The change can happen because ones environment (school or job tasks) changes, if lifestyle habits are optimized, or if treatment tools are used successfully.

The whole-person approach looks at how ADHD affects all of ones health and lifestyle, and vice versa. It is important to start treatment gradually and to have support family, ADHD community, nutritionist, coach, or clinician along the way to better wellbeing.

SUPPORT ADDITUDEThank you for reading ADDitude. To support our mission of providing ADHD education and support, please consider subscribing. Your readership and support help make our content and outreach possible. Thank you.

1 Zylowska, L., Ackerman, D. L., Yang, M. H., Futrell, J. L., Horton, N. L., Hale, T. S., Pataki, C., & Smalley, S. L. (2008). Mindfulness meditation training in adults and adolescents with ADHD: a feasibility study. Journal of attention disorders, 11(6), 737746. https://doi.org/10.1177/1087054707308502

2 Mehren, A., zyurt, J., Lam, A. P., Brandes, M., Mller, H., Thiel, C. M., & Philipsen, A. (2019). Acute Effects of Aerobic Exercise on Executive Function and Attention in Adult Patients With ADHD. Frontiers in psychiatry, 10, 132. https://doi.org/10.3389/fpsyt.2019.00132

3 Hong, S. S., & Cho, S. H. (2011). Acupuncture for attention deficit hyperactivity disorder (ADHD): study protocol for a randomised controlled trial. Trials, 12, 173. https://doi.org/10.1186/1745-6215-12-173

4 Ventriglio, A., Sancassiani, F., Contu, M. P., Latorre, M., Di Slavatore, M., Fornaro, M., & Bhugra, D. (2020). Mediterranean Diet and its Benefits on Health and Mental Health: A Literature Review. Clinical practice and epidemiology in mental health : CP & EMH, 16(Suppl-1), 156164. https://doi.org/10.2174/1745017902016010156

5 Yeh, TS, Yuan, C., et. al. (July 28, 2021). Long-term dietary flavonoid intake and subjective cognitive decline in US Men and Women. Neurology, 97(10). https://doi.org/10.1212/WNL.0000000000012454

6 Meireles, M., Moura, E., Vieira-Coelho, M. A., Santos-Buelga, C., Gonzalez-Manzano, S., Dueas, M., Mateus, N., Faria, A., & Calhau, C. (2016). Flavonoids as dopaminergic neuromodulators. Molecular nutrition & food research, 60(3), 495501. https://doi.org/10.1002/mnfr.201500557

7 Carabotti, M., Scirocco, A., Maselli, M. A., & Severi, C. (2015). The gut-brain axis: interactions between enteric microbiota, central and enteric nervous systems. Annals of gastroenterology, 28(2), 203209.

8 Foster, J. A., Rinaman, L., & Cryan, J. F. (2017). Stress & the gut-brain axis: Regulation by the microbiome. Neurobiology of stress, 7, 124136. https://doi.org/10.1016/j.ynstr.2017.03.001

9 Darling, K. A., Eggleston, M., Retallick-Brown, H., & Rucklidge, J. J. (2019). Mineral-Vitamin Treatment Associated with Remission in Attention-Deficit/Hyperactivity Disorder Symptoms and Related Problems: 1-Year Naturalistic Outcomes of a 10-Week Randomized Placebo-Controlled Trial. Journal of child and adolescent psychopharmacology, 29(9), 688704. https://doi.org/10.1089/cap.2019.0036

10 Johnstone JM, Hatsu I, Tost G, et al. Micronutrients for Attention-Deficit/Hyperactivity Disorder in Youths: A Placebo-Controlled Randomized Clinical Trial. Journal of the American Academy of Child and Adolescent Psychiatry. 2021 Jul. DOI: 10.1016/j.jaac.2021.07.005. PMID: 34303786.

11 Ghoreishy, S.M., Ebrahimi Mousavi, S., Asoudeh, F. et al. Zinc status in attention-deficit/hyperactivity disorder: a systematic review and meta-analysis of observational studies. Sci Rep 11, 14612 (2021).

12 Bener, A., Kamal, M., Bener, H., & Bhugra, D. (2014). Higher prevalence of iron deficiency as strong predictor of attention deficit hyperactivity disorder in children. Annals of medical and health sciences research, 4(Suppl 3), S291S297. https://doi.org/10.4103/2141-9248.141974

13 Effatpanah, M., Rezaei, M., Effatpanah, H., Effatpanah, Z., Varkaneh, H. K., Mousavi, S. M., Fatahi, S., Rinaldi, G., & Hashemi, R. (2019). Magnesium status and attention deficit hyperactivity disorder (ADHD): A meta-analysis. Psychiatry research, 274, 228234. https://doi.org/10.1016/j.psychres.2019.02.043

14 Derbyshire E. (2017). Do Omega-3/6 Fatty Acids Have a Therapeutic Role in Children and Young People with ADHD?. Journal of lipids, 2017, 6285218. https://doi.org/10.1155/2017/6285218

15 Dimpfel, W., Schombert, L., Keplinger-Dimpfel, I. K., & Panossian, A. (2020). Effects of an Adaptogenic Extract on Electrical Activity of the Brain in Elderly Subjects with Mild Cognitive Impairment: A Randomized, Double-Blind, Placebo-Controlled, Two-Armed Cross-Over Study. Pharmaceuticals (Basel, Switzerland), 13(3), 45. https://doi.org/10.3390/ph13030045

16 Akhondzadeh, S. et.al. (2005). Passiflora incarnata in the treatment of attention-deficit hyperactivity disorder in children and adolescents. Therapy 2(4);609-614. 10.1586/14750708.2.4.609

17 Lee, S. H., Park, W. S., & Lim, M. H. (2011). Clinical effects of korean red ginseng on attention deficit hyperactivity disorder in children: an observational study. Journal of ginseng research, 35(2), 226234. https://doi.org/10.5142/jgr.2011.35.2.226

18 Shakibaei, Fereshteh, et al. Ginkgo Biloba in the Treatment of Attention-Deficit/Hyperactivity Disorder in Children and Adolescents. A Randomized, Placebo-Controlled, Trial. Complementary Therapies in Clinical Practice, vol. 21, no. 2, Apr. 2015, pp. 6167., doi:10.1016/j.ctcp.2015.04.001.

19 Trebatick, J., Kopasov, S., Hradecn, Z., Cinovsk, K., Skodcek, I., Suba, J., Muchov, J., Zitnanov, I., Waczulkov, I., Rohdewald, P., & Durackov, Z. (2006). Treatment of ADHD with French maritime pine bark extract, Pycnogenol. European child & adolescent psychiatry, 15(6), 329335. https://doi.org/10.1007/s00787-006-0538-3

20 Dave, U. P., Dingankar, S. R., Saxena, V. S., Joseph, J. A., Bethapudi, B., Agarwal, A., & Kudiganti, V. (2014). An open-label study to elucidate the effects of standardized Bacopa monnieri extract in the management of symptoms of attention-deficit hyperactivity disorder in children. Advances in mind-body medicine, 28(2), 1015.

21 Katz, M., Levine, A. A., Kol-Degani, H., & Kav-Venaki, L. (2010). A compound herbal preparation (CHP) in the treatment of children with ADHD: a randomized controlled trial. Journal of attention disorders, 14(3), 281291. https://doi.org/10.1177/1087054709356388

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When Is the Best Time to Exercise? – The New York Times

Posted: January 20, 2022 at 2:22 am

Morning exercise has very different effects on metabolism than the same workout later in the day, according to an ambitious new animal study of exercise timing. The study, which involved healthy lab mice jogging on tiny treadmills, mapped hundreds of disparities in the numbers and activities of molecules and genes throughout the rodents bodies, depending on whether they ran first thing in the morning or deeper in the evening.

Many of these changes related to fat burning and other aspects of the animals metabolisms. Over time, such changes could substantially influence their disease risks and well-being. And though the study featured rodents, its findings likely have relevance for any of us who wonder if it is better to work out before work, or if we might get as much or more health benefit from after-hours exercise.

As anyone with a body knows, our internal operations and those of almost all living creatures follow a well-orchestrated and pervasive 24-hour circadian rhythm. Recent studies in animals and people show that almost every cell in our bodies contains a version of a molecular clock that coordinates with a broader, full-body timing system to direct most biological operations. Thanks to these internal clocks, our body temperature, blood sugar, blood pressure, hunger, heart rate, hormone levels, sleepiness, cell division, energy expenditure and many other processes surge and slow in repeated patterns throughout the day.

These internal rhythms, while predictable, are also malleable. Our internal clocks can recalibrate themselves, research shows, based on complex cues from inside and outside of us. In particular, they respond to light and dark but are likewise affected by our sleep habits and when we eat.

Recent research suggests that the time of day that we exercise also tunes our internal clocks. In past studies in mice, running at different hours affected the animals body temperatures, cardiac function and energy expenditure throughout the day and altered the activity of genes related to circadian rhythm and aging.

Results in people have been inconsistent, though. In a small 2019 study of men who joined an exercise program to lose weight, for instance, those who worked out in the morning shed more pounds than those exercising later in the day, even though everyone completed the same exercise routine. But in a 2020 study, men at high risk for Type 2 diabetes who began exercising three times a week developed better insulin sensitivity and blood-sugar control if they worked out in the afternoon than in the morning. Those results echoed similar findings from 2019, in which men with Type 2 diabetes who worked out intensely first thing in the morning showed unexpected and undesirable spikes in their blood-sugar levels following the exercise, while the same workouts in the afternoon improved their blood-sugar control.

Few of these studies ventured deep beneath the surface, though, to look into the molecular changes driving the health and circadian outcomes, which might help to explain some of the discrepancies from one study to the next. Those experiments that did examine exercises effects on a microscopic level, usually in mice, tended to concentrate on a single tissue, such as blood or muscle. But scientists who study physical activity, metabolism and chronobiology suspected the impacts of exercise timing would extend to many other parts of the body and involve intricate interplay between multiple cells and organs.

So, for the new study, published this month as the cover article in Cell Metabolism, an international consortium of researchers decided to try quantifying almost every molecular change related to metabolism that occurs during exercise at different times of day. Using healthy, male mice, they had some jog moderately on wheels for an hour early in the day and others run the same amount in the evening. An additional group of mice sat on locked wheels for an hour during these same times and served as a sedentary control group.

Beginning about an hour after the workouts, researchers took repeated samples from each animals muscle, liver, heart, hypothalamus, white fat, brown fat and blood and used sophisticated machinery to identify and enumerate almost every molecule in those tissues related to energy usage. They also checked markers of activity from genes related to metabolism. Then they tabulated totals between the tissues and between the groups of mice.

Interesting patterns emerged. Since mice are nocturnal, they wake and grow active in the evening and prepare to sleep in the morning, a schedule opposite of ours (unless we are vampires or teenagers). When the mice jogged at the start of their active time equivalent to morning for us the researchers counted hundreds of molecules that increased or dropped in number after the exercise, and that differed from levels seen in mice running closer to their bedtimes or not exercising at all.

Furthermore, some of these changes occurred almost identically in different parts of the body, suggesting to the researchers that various organs and tissues were, in effect, communicating with one another. The rodents muscles and livers, for instance, shared many molecular changes when the animals ran in their morning, but fewer when they jogged soon before bed.

It was quite remarkable to see how extensively exercise timing affected the levels and activities of so many molecules throughout the animals bodies, said Juleen Zierath, a professor of clinical integrative physiology at the Karolinska Institute in Stockholm, Sweden, and executive director of the Novo Nordisk Foundation Center for Basic Metabolic Research at the University of Copenhagen, who oversaw the new study.

Overall, the differences in molecular profiles between morning workouts (in mouse terms) and those later in their days tended to signal greater reliance on fat than blood sugar to fuel the early exercise. The opposite occurred when the mice ran in their evening. If those patterns held true in people, it might suggest morning exercise contributes more to fat loss, whereas late-day workouts might be better for blood-sugar control.

But mice are not people, and we do not know yet if the molecular patterns hold true in us. The studys researchers are working on a comparable experiment involving people, Dr. Zierath said.

This study was also limited in scope, examining a single session of moderate aerobic exercise in male mice. It does not show how other kinds of exercise in the morning or evening affect the inner workings of mice or people. Nor does it tell us if what we eat or the time of day we eat, and whether chronotypes whether we tend to be morning or evening people play into these effects, or if being female matters.

But even with its limitations, this is a very important study, said Dr. Lisa Chow, a professor of medicine and endocrinology at the University of Minnesota, who was not involved in this research. It underscores the potency of exercise at any time of day.

It also suggests that, as additional studies build on this ones results, we may become better able to time our workouts to achieve specific health goals. Follow-up studies likely will tell us, for instance, if an evening bike ride or run might stave off diabetes more effectively than a morning brisk walk or swim.

But for now, Dr. Chow said, the best time for people to exercise would be whenever they can get a chance to exercise.

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Injectable amnion hydrogel-mediated delivery of adipose-derived stem cells for osteoarthritis treatment – pnas.org

Posted: January 20, 2022 at 2:20 am

Significance

Osteoarthritis (OA) is a chronic disease affecting millions of people worldwide with no curative solution. In the present study, we developed a minimally invasive injectable system using amnion membrane (AM) from the human placenta as a carrier for fat tissue-derived stem cells (adipose-derived stem cells [ADSCs]) to treat OA. Both AM and ADSCs are rich sources of bioactive molecules that can target the sites of inflammation and reduce the inflammation-driven articular cartilage damage. Our study demonstrated the disease-modifying and regenerative potential of AM hydrogel, a comparable regenerative and disease-modifying effect of AM hydrogel and ADSCs, and the synergistic effect of AM with ADSCs in regenerating cartilage and attenuating OA.

Current treatment strategies for osteoarthritis (OA) predominantly address symptoms with limited disease-modifying potential. There is a growing interest in the use of adipose-derived stem cells (ADSCs) for OA treatment and developing biomimetic injectable hydrogels as cell delivery systems. Biomimetic injectable hydrogels can simulate the native tissue microenvironment by providing appropriate biological and chemical cues for tissue regeneration. A biomimetic injectable hydrogel using amnion membrane (AM) was developed which can self-assemble in situ and retain the stem cells at the target site. In the present study, we evaluated the efficacy of intraarticular injections of AM hydrogels with and without ADSCs in reducing inflammation and cartilage degeneration in a collagenase-induced OA rat model. A week after the induction of OA, rats were treated with control (phosphate-buffered saline), ADSCs, AM gel, and AM-ADSCs. Inflammation and cartilage regeneration was evaluated by joint swelling, analysis of serum by cytokine profiling and Raman spectroscopy, gross appearance, and histology. Both AM and ADSC possess antiinflammatory and chondroprotective properties to target the sites of inflammation in an osteoarthritic joint, thereby reducing the inflammation-mediated damage to the articular cartilage. The present study demonstrated the potential of AM hydrogel to foster cartilage tissue regeneration, a comparable regenerative effect of AM hydrogel and ADSCs, and the synergistic antiinflammatory and chondroprotective effects of AM and ADSC to regenerate cartilage tissue in a rat OA model.

Osteoarthritis (OA) affects the entire synovial joint, including the articular cartilage, synovium, and subchondral bone (1). In the United States alone, 27 million people are affected by this disease, and the associated healthcare cost has been estimated at more than $185 billion annually (2). Recent studies have shown that inflammation and its induced catabolism play an important role in promoting OA symptoms and accelerating the disease (3). The best-known and critical inflammatory mediators are tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1) expressed during the early stages of OA (4, 5). The catabolic effects of proinflammatory cytokines lead to reduced cartilage cellularity, changes in chondrocyte functions, and further breakdown of cartilage extracellular matrix (ECM) (6, 7). Both nonsurgical and surgical therapies are currently being used in OA treatment which provide temporary relief but have failed to treat OA pathogenesis.

Since inflammation is the key factor in OA progression, developing novel therapies that can suppress inflammation and promote regenerative pathways may prevent/delay OA progression and thus hold promise for OA treatment. Along this line, a variety of studies investigated the potential role of adipose-derived stem cells (ADSCs) in treating OA (3). The therapeutic properties of ADSCs are multifaceted (7, 8) as they contain several antiinflammatory and chondroprotective agents which inhibit inflammation, suppress immune recognition, and reduce apoptosis and dedifferentiation of chondrocytes (911). Recently, the beneficial effects of ADSCs on OA treatment via intraarticular injections have been studied in different animal models (1214) as well as clinical trials (15, 16). Although stem cell therapy has achieved promising results in OA treatment, the long-term therapeutic application of stem cells remains limited. Previous studies have shown that stem cells provide a temporary/early-stage effect rather than prolonged effectiveness in treating OA, indicating the need for higher cell numbers and multiple cell injections. The short-term effects of stem cells are mainly due to limited long-term cell survival/retention, extensive cell death and poor cellular function, and inadequate cellular distribution following injection in the target site (17). To overcome these challenges, large doses of cells and multiple injections have been tried; however, these approaches are not economically viable and are associated with the risk of cell overexpansion (17).

To overcome these limitations, delivery systems capable of sustaining the survival and maintaining functions of implanted cells are needed to stimulate endogenous regeneration through interactions of transplanted cells and the host tissue. In our previous study (18), we developed an injectable amnion membrane (AM)based hydrogel as a stem cell delivery system. These AM hydrogels supported cellular functionalities such as cell viability, proliferation, and stemness. Also, our studies showed the ability of both AM hydrogels and AM hydrogelADSC combinations to provide an immunomodulatory and chondroprotective environment in an invitro osteoarthritic model (18). AM is the innermost layer of placental tissue which is easily accessible and includes collagens (types I, III, IV, V, and VI), fibronectin, laminin, proteoglycans, and hyaluronan. AM has been shown to suppress the expression of potent proinflammatory cytokines, such as IL-1 and IL-1 and decrease matrix metalloproteinase (MMP) levels through the expression of natural MMP inhibitors present in the membrane. AM also contains IL-1Ra, a receptor antagonist for IL-1, a proinflammatory cytokine that has been shown to up-regulate in OA (19). Placenta also plays an important role in reducing host immune response in case of allogeneic transplantation as it possesses the unique function of preventing the fetal allograft from being rejected (19). The feasibility of using AM as a carrier system for chondrocytes which promoted cell proliferation, cellular phenotype invitro, and cartilage regeneration invivo has been demonstrated in various studies (20, 21). However, the major limitation of these studies is the use of AM in the form of sheets, which would require invasive surgical procedures. To overcome the surgery-associated complications, minimally invasive therapies using micronized AM in saline were developed which also have demonstrated efficacy in attenuating OA invivo (22, 23). However, intraarticular injections experience rapid clearance of therapeutics, which may limit efficacy and produce nonsignificant effects (24). Thus, localization and prolonged retention are critical for a sustained release of therapeutics to act efficiently with minimal injections.

The overall goal of this study was to improve the efficacy of stem cell therapy to treat OA using our previously designed cell-protective and cell-supporting injectable AM hydrogel as a stem cell delivery system. We investigated the potential of AM hydrogel as a delivery system for ADSCs and evaluated the effect of AM hydrogels with and without ADSCs to prevent inflammation and cartilage degeneration and promote cartilage tissue regeneration in a collagenase-induced knee OA rat model.

In total, 376 proteins were identified in the AM gel by liquid chromatographymass spectrometry (LC-MS) (Dataset S1). The important proteins which might impart the beneficial effects of amnion for tissue regeneration include collagen, laminin, fibronectin, small leucine-rich proteoglycans (SLRPs), proteoglycans, and tissue inhibitors of metalloproteinases (TIMPs) (19). The proteins identified in AM gel were categorized according to their predominant location and functions using the Human Proteome Reference Database and UniProt database (Fig. 1). The identified proteins were found to be largely located in the extracellular environment of AM tissue (Fig. 1A). The predominant functions of these proteins were to facilitate ECM and cytoskeleton organization (EC + SC + SM: >85%; Fig. 1B). To further identify the biological implications of these proteins observed in AM gel, the proteins were associated with their Gene Ontology (GO) terms. (Dataset S2). The top five GO terms for the biological process of protein observed in AM gel were extracellular matrix organization (GO:0030198), cell adhesion (GO:0007155), collagen fibril organization (GO:0030199), neutrophil degranulation (GO:0043312), and cornification (GO:0070268)/keratinization (GO:0031424) (Fig. 1C). Table 1 summarizes the list of proteins identified in AM gel related to cartilage tissue regeneration.

Relative abundance of proteins identified in the AM. Pie chart representing the distribution of all identified proteins in the AM according to their subcellular location (A) and function (B). Assignments were made according to their primary location and function as reported in the Human Protein Reference (http://www.hprd.org/) and UniProt (https://www.uniprot.org/) databases. Primary subcellular location: CP, cytoplasm and cytosol; CS, cytoskeleton; EC, extracellular environment; ER, endoplasmic reticulum; G, Golgi apparatus; M, mitochondrion; MB, integral to membrane and plasma membrane; N, nucleus or nucleolus; R, ribosome; V, vesicles including cytoplasmic vesicle, endosome, and lysosome. Function: EC, ECM structural constituent; I, immune response; M, metabolism and energy pathways; RN, regulation of nucleotide; SC, structural constituent of cytoskeleton; SM, structural molecule activity; ST, signal transduction; T, transporter activity; O, other functions including apoptosis, cell cycle, cell growth, motor activity, organization, extracellular ligands, and unknown. The value was rounded off to one decimal place. (C) Top five GO annotations for biological processes of the detected proteins in AM gel.

The identification of proteins related to cartilage tissue regeneration in AM

Pathogenesis of OA in the rat knee joints occurred after 1 wk of collagenase II injection. The hematoxylin and eosin (H&E) images indicated that the sham knee did not show signs of synovial inflammation (Fig. 2A). The collagenase-injected group showed a high degree of synovial inflammation (Fig. 2B, black arrows) with an increase in the number of synovial lining cell layers and infiltration of inflammatory cells. The Safranin O image reflected smooth joint surfaces of the sham (Fig. 2C), while the collagenase-injected group showed erosion of the articular cartilage showing signs of OA development (Fig. 2D, black arrow). The knee diameter data showed that the OA group had significantly higher joint swelling on day 7 compared to the sham group (Fig. 2E).

H&E staining images of rat knee joint: (A) Sham group with saline injection, (B) OA group treated with collagenase enzyme. Safranin-O staining images of rat knee joint: (C) Sham group with saline injection, (D) OA group treated with collagenase enzyme. (E) Joint swelling evaluation of sham and OA groups. n = 6 showing mean and SD (****P<0.0001). (Scale bars: A and B, 200 m; C and D, 2 mm.)

After 1 wk of collagenase injection, the rats were divided into four groups: control (phosphate-buffered saline [PBS]), ADSC, AM gel, and AM-ADSC.

Joint swelling analysis at day 7 after collagenase injection showed a significant increase in joint diameter compared to day-0 groups (before collagenase injection), indicating synovitis of the knee joint. No significant difference was observed in the groups at days 3 and 7 posttreatment. At days 14 and 21 posttreatments, knee swelling of the AM-ADSC group was lower compared to control, AM, and ADSC groups. At 28 d posttreatment, ADSC (0.8 0.3, P<0.05), AM gel (0.5 0.1, P <0.0001), and AM-ADSC (0.4 0.1, P <0.0001) treatment groups showed a significant decrease in joint diameter compared to the control group (1.4 0.8). Importantly, the knee diameter in the AM-ADSC group was found to be significantly lower than ADSC (P <0.001) and AM groups (P <0.05), indicating decreased synovial inflammation in the combination group AM-ADSC (Fig. 3).

Joint inflammation for different treatment groups (n = 6) showing mean and SD (****P<0.0001, ***P<0.001, **P<0.01, *P < 0.05). All treatments groups showed significant decrease in joint swelling 28 d posttreatment compared to control group. The combination group (AM-ADSC) showed significant decrease in joint swelling compared to ADSCs alone and AM gel alone.

The cytokine profiling analysis of serum showed an increase in intercellular adhesion molecule 1 (ICAM-1), leptin, selectin, and monocyte chemoattractant protein-1 (MCP-1) and a decrease in TIMP-1 7 d after collagenase treatment (Fig. 4 AE). The level of TIMP-1 was found to significantly increase on day 28 in AM-ADSC (39,536 pg/mL 2,277) compared to control (28,485 pg/mL 4,087, P <0.0001) and ADSC (26,520 pg/mL 3,416, P <0.0001) groups (Fig. 4A). No significant difference was found in ICAM-1 and MCP-1 levels from day 3 to 14 posttreatment. On day 21 the AM-ADSC group (5,778 pg/mL 549) showed a significant decrease (P < 0.05) in ICAM-1 compared to the control group (8,641 pg/mL 1,439). A significant decrease in MCP-1 levels was also noted in AM-ADSC group (5,089 pg/mL 810) compared to control groups (7,748 pg/mL 304, P < 0.0001) after day 21. Both AM and ADSC groups showed a comparable decrease in the levels of MCP-1, ICAM-1, leptin, and selectin and an increase in TIMP-1, though not significant compared to control on day 28. The AM-ADSC group further showed a significant reduction of ICAM- 1, leptin, and MCP-1 levels compared to all other groups on day 28 (Fig. 4 B, C, and E), indicating a synergistic antiinflammatory effect (SI Appendix, Table S1). Selectin level was found to decrease for all groups compared to the control group from day 21; however, the effect was not significant (Fig. 4D). An increase in up-regulation of other proinflammatory cytokines was not detected as they were below the detection limit by the enzyme-linked immunosorbent assay (ELISA) multiplex array.

Cytokine profiling of serum isolated from whole blood from different groups: (A) TIMP-1, (B) ICAM-1, (C) leptin, (D) selectin, and (E) MCP-1; n = 6 showing mean and SD (****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

Raman spectroscopic changes of serum were measured by quantifying the integrated peak area in the spectral region 1 (SR1, 1,372 cm1 to 1,599 cm1) and region 2 (SR2, 1,601 cm1 to 1,776 cm1). Upon normalization of the area of these two spectral regions SR1 and SR2 with the integrated peak area of phenylalanine band at 1,004 cm1, it was observed that the normalized integrated areas for both the regions increased upon collagenase treatment (day 7 after collagenase injection) compared to day-0 serum samples. As presented in Fig. 5, the normalized integrated area in SR1 decreased in all the treatment groups with a significant decrease in the AM-ADSC group (2.4 0.6) compared to the control group (7.5 0.5, P < 0.0001) and AM gel group (5.5 1.8, P < 0.05). The normalized integrated area in SR2 also showed a similar trend with a significant reduction in all treatment groups compared to the control group. It is also important to note that the AM-ADSC group for 28-d treatment showed a significant decrease in the peak area (4.4 0.7) compared to both the ADSC group (9.9 1.4, P < 0.05) and AM gel group (10.3 1.7, P < 0.05), indicating a synergistic antiinflammatory effect.

Normalized peak area of (A) region 1 (B) region 2; mean and SD with n = 6 (****P<0.0001, ***P<0.001, *P<0.05).

Fig. 6 shows the gross appearance of the cartilage plateau in all groups. The sham group (Fig. 6A) showed a smooth, glossy cartilage surface. The control group (Fig. 6B) showed cartilage lesions, erosion, and fissures, indicating severe damage. ADSC groups (Fig. 6C) showed improvement compared to the control group; however, the lesions were still prominent. The AM group (Fig. 6D) also showed signs of erosion which was less prominent compared to the ADSC group and control group. The AM-ADSC (Fig. 6E) groups showed slightly damaged cartilage surface which was closer to the sham group, indicating a synergistic chondroprotective effect.

Gross appearance of cartilage surface after 4 wk posttreatment showing smooth surface in (A) sham group, erosion in (B) control group and (C) ADSC group, less erosion in (D) AM group, and fewer signs of lesion in (E) AM-ADSC group with closer appearance to sham group. Yellow arrows indicate the cartilage damage.

Control animals at 4 wk showed pronounced synovial inflammation with an increase in the number of synovial lining cell layers (Fig. 7A, black arrows), and lesions and areas of erosion were prominent along with diminished Safranin O staining for both femoral and tibial surface, suggesting the loss of proteoglycan content (Fig. 7E). ADSC treatment reduced the synovial inflammation (Fig. 7B) and preserved the loss of proteoglycan content of cartilage ECM, but the synovial inflammation, lesion, and areas of erosion were still evident (Fig. 7 B and F). In contrast, histological analysis of AM gel (Fig. 7 C and G) and AM-ADSC (Fig. 7 D and H) treated joints showed a significant reduction in synovial inflammation. Both AM gel and AM-ADSC groups showed smooth cartilage surfaces with no lesions and strong Safranin O staining. However, the AM-ADSC group showed more prominent Safranin O staining with a consistently more uniform cartilage tissue compared to the AM gel group. Safranin O staining of sagittal sections was used to assess the degenerated cartilage matrix area caused by the OA phenotype (Fig. 7I). The calculated total degeneration area was found to be 35.1 13.3% in the control group. AM gel groups showed a significant decrease in the total degenerated area (24.4 7.6%) compared to the control group (P < 0.05), indicating the chondroprotective potential of AM gel. ADSC-injected groups showed a significant decrease in total degeneration area which was found to be 21 6.6% compared to the control group (P < 0.05), indicating a comparable chondroprotective effect. Furthermore, treatment with the AM-ADSC (6.9 3.6%) observed a significant reduction in cartilage degeneration compared to control (P < 0.0001), ADSC group (P < 0.05), and AM group (P < 0.05) and the degenerated area was comparable to the sham (6.6 2.2%), demonstrating the synergistic chondroprotective effect.

H&E staining images of rat knee joints treated with (A) control, (B) ADSC, (C) AM hydrogel, and (D) AM-ADSC after 4 wk. Safranin O staining of rat knee joint treated with (E) control, (F) ADSC, (G) AM gel, and (H) AM-ADSC. (I) Percentage joint degenerated area calculation. n = 6 showing mean and SD (***P<0.001, **P<0.01, *P<0.05). (Scale bars: AD, 200 m; EH, 2 mm.)

Stem cell therapy has emerged as a potential therapy to provide a more reliable and curative solution to treat OA. However, the effectiveness of stem cell therapy is limited by difficulties in achieving the right therapeutic doses within the target site. In view of this, we developed an injectable AM hydrogel as a stem cell delivery system with an aim to localize stem cells at the target site, maintain cellular functionalities and synergistically reduce inflammation, and activate regenerative pathways, thereby attenuating OA progression. The AM hydrogel has been characterized in our previous studies to understand the swelling, degradation, and rheological behavior. Our previous study showed that the stiffness of AM hydrogels ranged between 120 and 1,600 Pa, indicating that the matrix stiffness can be tuned by varying the protein concentration (18). The physical properties of hydrogels play an important role in regulating stem cell fate (25). The AM hydrogels were shown to support ADSC functionalities as softer hydrogels with lower matrix stiffness <1 kPa are known to maintain stem cell viability, proliferation, and stemness (26). Softer hydrogels have also been shown to prevent transplanted cell death after cell delivery, improving the therapeutic efficacy of stem cell delivery at the target site after injection (27). Also, the AM hydrogels exhibited a shear-thinning property which is an important criterion for translating an injectable hydrogel as highly viscous or shear thickening material that may block the syringe while injecting (18). Also, the potential of AM with or without stem cells to present antiinflammatory and chondroprotective effects was demonstrated in an invitro OA model (18). In the present study, a comprehensive proteomic analysis of AM gel was done to understand its composition which may regulate tissue regeneration. LC-MS characterization of AM revealed the presence of proteins such as collagen, laminin, fibronectin, SLRPs, and proteoglycans. Collagen is the most abundant ECM family in the articular cartilage, including mainly collagen II along with IX, X, XI, VI, XII, and XIV collagen (28), which regulates the structurefunction relationship of the cartilage tissue. The presence of collagen VI may play an important role in promoting chondrocyte proliferation (29). Other proteins found in AM such as collagen XII are known to interact with collagen VI, resulting in up-regulation of tissue regeneration (30). The presence of keratin could also be beneficial as studies have shown its role in increasing cellular adhesion and inducing polarization of inflammatory M1 macrophages to antiinflammatory M2 phenotype (31). PLEC is a large cytoskeletal protein that regulates signaling from the extracellular environment to the cell nucleus (32). In cartilage, the OA-associated single-nucleotide polymorphism correlates with differential expression of PLEC and with differential methylation of PLEC CpG dinucleotides (33). Intact vimentin intermediate filament network contributes to the maintenance of the chondrocyte phenotype (34). Heparan sulfate proteoglycans bind to many proteins that regulate cartilage homeostasis. Agrin expression is decreased in OA, and exogenous agrin enhanced cartilage differentiation (35). SLRPs have important effects on cell behavior by interacting with collagens to modulate fibril formation and binding various cell-surface receptors and growth factors. Alterations in the distribution and production of SLRPs could lead to the development of OA (36). TIMPs are the primary endogenous inhibitors of MMPs. TIMP-3 has the broadest inhibition spectrum as it inhibits several members of a disintegrin and metalloprotease (ADAM) and ADAM with thrombospondin motifs (ADAMTS) (37). The proteomic profiling indicates that the AM gel used in our study still retains a rich source of important proteins, which makes it a highly effective biomaterial for OA treatment and cell delivery applications.

A collagenase-induced OA rat model was used to evaluate the effectiveness of AM hydrogel with or without ADSCs to attenuate OA. This is an established model and has been predominantly used to investigate the mechanisms underlying joint damage (38). Collagenase treatment directly digests the collagen from cartilage ECM, resulting in pain, changes in the synovial membrane and subchondral bone, and degeneration of articular cartilage (39). Similar features were observed in our study, which showed inflammation in the synovial membrane and degeneration of cartilage in a collagenase-injected group, thereby reproducing some of the main features associated with onset and development of OA in humans (40). The dosage of collagenase (500 U) was chosen based on a previous study that compared two different dosages (250 U and 500 U) of collagenase to induce OA in the rat model and found 500 U was more effective in inducing inflammation and cartilage degeneration (38).

An early time point of week 1 was chosen to study the effect of the treatments in inhibiting inflammation and cartilage degeneration (38). Intraarticular injection of amnion suspension in a monosodium iodoacetate (MIA) OA model has been previously shown to reduce joint swelling on day 14 (0.7 mm). However, an increase in the joint swelling was noticed on day 21 (1.1 mm) (41). In the present study, all three treatment groups, ADSC (0.8 0.3, P<0.001), AM gel (0.5 0.1, P <0.0001), and AM-ADSC (0.4 0.1, P <0.0001), significantly reduced joint swelling compared to the control group (1.4 0.8) at day 28. The joint swelling was found to comparable in the AM gel group and ADSC group. Moreover, the AM-ADSC group significantly reduced joint swelling compared to other groups. This demonstrated the antiinflammatory properties of AM gel alone and synergistic advantages of combining AM with ADSC in inhibiting inflammation, an early indication of OA onset and progression. This was corroborated by the cytokine profiling wherein AM hydrogel and ADSC showed a comparable decrease in inflammation. AM-ADSC significantly reduced inflammation markers such as MCP-1, leptin, and ICAM-1 and increased TIMP-1 compared to the control group and other treatment groups. MCP-1 is a chemokine produced by synovial cells which attract monocytes to facilitate the OA immune response, leading to clinical symptoms such as redness, swelling, and pain (42). ICAM-1 is a critical mediator of inflammation that mediates activation, migration of leukocytes, and adhesion of antigen-presenting cells to T lymphocytes and has been found to be higher in OA patients (43). Willett etal. (23) previously showed that intraarticular injection of micronized AM reduced MCP-1 levels (132 113 pg/mL) after 21 d in a medial meniscal transection (MMT)induced OA model in rats. However, another study using amnion suspension for OA treatment in an MIA model did not find any significant reduction in MCP-1 or increase in TIMP-1 levels compared to the saline group (38). The present study showed a decrease in the inflammatory markers in AM gel group in a collagenase-induced OA model.

The biomolecular changes in the serological composition were then evaluated by Raman spectroscopy (RS). RS can be used to follow arthritic changes in serum and synovial fluids by determining the changes in the protein secondary structure (44, 45). Studies have also shown the effectiveness of RS in detecting protein changes in inflammatory conditions (45). The spectra regions 1 and 2 used in the present study to detect the changes in serological composition have been previously used to study serum samples from arthritic patients (45). The study showed an increase in the peak area of regions 1 (70 a.u., arbitary unit P < 0.05) and 2 (55 a.u., P < 0.05) in serum samples from arthritis patients compared to the healthy individuals (region 1, 65 a.u.; region 2, 50 a.u.). SR1 reflects signatures from the amide II band and SR2 reflects the changes in the amide I peaks. An increase in SR1 and SR2 peak area indicates more disordered protein secondary structure with altered electrostatic interactions as evident from RS analysis of serum and synovial fluid samples from arthritic patients (44, 45). In the present study, the RS spectra profile showed a significant decrease in the peak area of SR2 in all the three treatment groups compared to control at day 28 posttreatment, which can be attributed to less-disordered protein secondary structure. Overall, the RS observation corroborates the cytokine profile data indicating a comparable effect of AM hydrogel and ADSCs in reducing inflammation and a synergistic antiinflammatory property of AM-ADSC and the unique advantages of combining AM with ADSC to reduce inflammation in an OA environment.

Macroscopic and microscopic evaluations of the treatment groups showed degenerated cartilage tissue in the control group. Compared to the control group, AM hydrogel showed a disease-modifying and regenerative effect by significantly decreasing cartilage degeneration. The disease-modifying and regenerative effect was also found to be comparable in AM and ADSC groups. The disease-modifying and regenerative capability of AM hydrogel was further enhanced upon the addition of ADSCs, indicating a synergistic effect of AM and ADSC. Previous studies have shown that injection of MSC suspension fails to engraft with the cartilage tissue, indicating a short-lived effect of stem cells (46, 47). Sato etal. reported that only a small number of stem cells was detected within the OA cartilage 1 wk posttreatment, which were found to disappear after 5 wk (48). Studies have shown that 90% of cells usually die postinjection at the target site due to physical stress, hypoxia, and inflammation (49). This shows that a single injection of stem cells may not be enough to improve the OA condition, indicating the need for periodic injections (50). While stem cells injected as a suspension do not engraft into the cartilage, MSCs encapsulated in a matrix such as hyaluronic acid (HA) appear to engraft and contribute significantly to cartilage repair. Studies have shown that combining HA and stem cells improves the quality of cartilage compared to cells and HA alone (48, 51, 52). However, recent studies have demonstrated a modest benefit of HA in OA treatment (53). Thus, to develop an alternative solution, AM has been used in the present study as a cell delivery system for OA treatment. It is evident from the present study that ADSCs showed some positive response in reducing inflammation and cartilage degeneration using the collagenase-induced OA model. However, the effect is not as significant as in the combination group, indicating an advantage of combing AM with ADSCs. The use of AM in attenuating OA has also been shown in different studies. The efficacy of intraarticular injection of micronized AM suspension was investigated in a rat MMT model 24 h after MMT surgery and found smaller lesions and fewer defect volume compared to a saline-treated group 21 d postinjection (23). Saline-treated joints showed an average incidence of 2.8 0.2 erosion, 2.4 0.4 lesion, and an average lesion volume of 0.00725 0.005 mm3, whereas the AM suspension showed significant reduction in erosion sites (1.2 0.374) and no lesions (23). Another study using amnion suspension (total joint score, 13.7 0.2) 7 d after OA induction showed no significant improvement in the joint scores compared to the control group (total joint score, 13.5 0.4) (41) in an MIA-induced OA model. Another study demonstrated a dose-dependent benefit of particulate AM along with umbilical cord tissue in attenuating OA. It was noticed that at 4 wk postinjection 100 g/L of particulate AM/UC (umbilical cord) significantly reduced both lesion area and percent lesion area compared to control and 50 g/L of AM/UC group (54). In the present study AM gel (24.4 7.6%) even at a lower concentration of 6 g/L significantly reduce cartilage degeneration compared to control groups (35.1 13.3%) in a collagenase-OA model. The addition of ADSCs further improved the potential of AM gel (6.9 3.6%) to attenuate OA progression. This indicated the advantages of using an AM gel at a lower concentration over particulate AM at a higher concentration in suspension.

The findings of the present study thus indicate that the AM hydrogel can foster cartilage tissue regeneration. The study also demonstrated a comparable effect of AM hydrogel and ADSCs in regenerating the cartilage tissue and synergistic antiinflammatory and chondroprotective properties of AM-ADSC. This indicates the unique advantages of combining AM with ADSC to reduce inflammation and slow down cartilage degeneration and regenerate cartilage tissue in an OA environment. The invivo study also validated our previous study which demonstrated a synergistic antiinflammatory and chondroprotective effect of AM-ADSC in an invitro OA model (18). In addition, since inflammation is a key regulator of OA progression, and as of now there is no solution to modulate the inflammatory processes and prevent OA, the use of AM gel alone or in with ADSC may provide disease-modifying effects to control the disease (3). However, further studies will be needed to evaluate the degradation of AM hydrogel invivo, release kinetics and retention profile of stem cells, and the paracrine effect of cells within the target site.

Cell therapy is widely used to address the current unmet needs of complex degenerative diseases such as OA. However, the lack of an ideal delivery system resulted in inconsistent outcomes, indicating the need for a more reliable strategy. This study demonstrated the feasibility of using a biomimetic injectable hydrogel using AM as a delivery system for ADSCs to attenuate OA and regenerate cartilage tissue in a rat OA model. Our study showed the potential of AM hydrogels for disease modification and regenerating the cartilage tissue. Both AM and ADSC groups showed comparable disease-modifying and cartilage tissue regeneration effects. In addition, the study also confirmed the synergistic effect of the combination group (AM-ADSC) for disease modification and cartilage tissue regeneration. Future studies need to investigate the mechanism of the synergistic effect of AM and ADSCs and the translation potential of AM hydrogels with and without ADSCs using larger animal models.

Discarded, deidentified placental tissues were obtained after getting approval from the institutional ethical committee (University of Connecticut Health). The isolation methods were performed in accordance with the experimental guidelines and regulations approved by the Institutional Review Board, University of Connecticut Health (study number IE-08-310-1). The amnion hydrogel was developed according to a previously published protocol (18). Briefly, AM was decellularized, solubilized in a pepsin solution, and neutralized to form a hydrogel. The neutralized AM was diluted to the desired final AM concentration (6 mg/mL) with PBS on ice. AM was then characterized by LC-MS to evaluate the complex protein composition of AM.

AM was suspended in 5% sodium dodecyl sulfate in 0.1 M TrisHCl (pH 8.5), subjected to sonication, and prepared for downstream proteomics analysis using the S-trap midi column technology (Protifi, LLC). Proteins were subjected to Cys reduction, alkylation using iodoacetamide, and trypsin digestion using Protifis instructions. Eluted tryptic peptides were desalted using Pierce C18 peptide desalting spin columns (P/N 89851) using the manufacturers instructions, dried to completion using a Labconco speedvac concentrator, and resuspended in 0.1% formic acid in water prior to mass spectrometry analysis.

The peptides generated from AM were independently analyzed using ultrahigh-performance LC coupled to tandem MS (UPLC-MS/MS) on a Dionex Ultimate 3000 RSLCnano UPLC system coupled to a Q Exactive HF mass spectrometer (Thermo Scientific). About 1.25 g of each desalted peptide were directly loaded onto a 75-m 25-cm nanoEase m/z Peptide BEH C18 analytical column (Waters Corporation) and separated using a 3-h reversed-phase UPLC gradient at a flow rate of 300 nL/min. Eluted peptides were directly ionized into the Q Exactive HF using positive polarity electrospray ionization. MS/MS data were acquired using a data-dependent Top15 acquisition method. All raw data were searched against the full UniProt Homo sapiens reference proteome (UP000005640, last updated 29 June 2020) using the Andromeda search engine embedded in the MaxQuant software platform (v1.6.43.10) (55, 56). The following modifications were used: fixed carbamidomethyl Cys and variable oxidation of Met, acetylation of protein N termini, deamidation of Asn and Gln, and peptide N-terminal Gln-to-pyro-Glu conversion. Enzyme specificity was set to trypsin, minimum peptide length was set to 5, and all peptide- and protein-level identifications were filtered to a 1% false discovery rate following a target-decoy database search. Label-free quantitation was achieved using the MaxLFQ feature in MaxQuant. All other parameters were kept at default settings. Search results were uploaded into Scaffold v5 (Proteome Software) for visualization and further analysis. All detected proteins were searched and categorized according to their primary location and function using the Human Protein Reference (http://www.hprd.org/) and UniProt (https://www.uniprot.org/) databases. Pie charts were created based on the quantified amounts of detected proteins. GO terms for the biological processes were searched by UniProt database.

ADSCs were isolated from 6- to 8-wk-old Sprague-Dawley rats in accordance with the experimental guidelines and regulations approved by the University of Connecticut Health Center Institutional Animal Care and Use Committee (IACUC)approved protocol. The isolation and characterization by flow cytometry were done according to our previously optimized protocol (18).

Animal experiments were approved by the IACUC at the University of Connecticut Health. Male Sprague-Dawley rats (8 wk old) were used for the study and divided into two groups (sham and collagenase-injected group). Briefly, rats were anesthetized by isoflurane (4% isoflurane for anesthesia induction, 2% for maintenance) and an intraarticular injection was performed with the use a 29-gauge needle inserted through the patella ligament into the joint space of the right knee. They received two injections (day 0 and 3) according to the group. The collagenase-injected group (n = 6) received about 500 U of collagenase type II (Sigma-Aldrich) in 100 L of normal saline after filtering through a 0.22-m membrane (38). The sham group (n = 6) received 100 L of normal saline.

A week after the first collagenase injection, the OA-induced rats were divided into four groups according to the treatments they would receive: control (PBS), ADSCs, AM gel (6mg/mL), and AM-ADSCs combination (n = 6 each group). Using a 29-gauge needle inserted through the patella ligament into the joint space of the right knee, all the OA knees received 100-L injections according to the specific treatment of the group. About 1 106 ADSCs were reconstituted in PBS and AM gel for the ADSC and AM-ADSC groups, respectively.

The knee diameters were measured to determine the extent of joint swelling with a manual caliper. Results were presented as the difference in knee diameter (ipsilateralcontralateral) (35).

Whole blood was collected from the saphenous vein at regular time points. Blood was then allowed to clot for 30 min and the serum was separated by centrifugation at 1,500 g for 10 min. The levels of cytokines in the serum were measured using the Quantibody Rat Cytokine Array 2 multiplex ELISA kit that quantitatively measured 10 rat inflammatory factors: ICAM-1, interferon , IL-1, IL-6, IL-10, leptin, L-selectin, MCP-1, TIMP-1, and TNF-alpha (RayBiotech). All ELISA procedures were performed according to the manufacturers protocols.

Raman measurements were carried out from the serum samples at an excitation wavelength of 785 nm using a free-space custom-built inverted Raman microspectroscopy system as described previously (57). Briefly, the Raman spectrometer consisted of a 193-mm focal length spectrograph (Shamrock 193i; Andor) equipped with a thermoelectric cooled charge-coupled device camera (iDus DU420A-BEX2-DD; Andor). Both excitation and collection were performed using the same 60 objective with the numerical aperture of 1.1 (LUMFLN60XW; Olympus). Serum was isolated and placed onto a quartz coverslip (Ted Pella, Inc.). The average power at the sample was held constant at 25 mW; the integration time for a single Raman measurement was 30 s, and two accumulations were averaged. The raw Raman spectra were preprocessed by removing cosmic rays, subtracting Raman signals from quartz coverslip and smoothing. The area of peaks in region 1 (1,372 cm1 to 1,599 cm1), region 2 (1,601 cm1 to 1,776 cm1) were calculated and normalized with respect to the area of phenylalanine peak at 1,004 cm1 (48) using Origin Pro software.

Animals were killed 4 wk after treatment. The cartilage surface was exposed by carefully removing the surrounding soft tissue including the joint capsule and meniscus. The effect of different treatment groups on osteoarthritic joints was examined macroscopically and photographed using a digital camera.

The dissected knee joints were fixed with 10% neutral-buffered formalin and subsequently decalcified, embedded in paraffin, and cut into 5-m sections. Specimen slides were then deparaffinized and hydrated by soaking them sequentially for the time indicated in xylene, ethanol, and deionized water. For H&E, sections were stained with hematoxylin Harris (Sigma-Aldrich) and counterstained with eosin (Sigma-Aldrich). For Safranin O, sections were stained with Weigerts iron hematoxylin (Sigma-Aldrich) working solution and fast green solution (Sigma-Aldrich) then counterstained with Safranin O solution (Sigma-Aldrich). Slides were viewed with the aid of the light microscope after being cleared with alcohol and xylene (Sigma-Aldrich). Articular surface areas of sagittal joint sections (areas stained red with Safranin O on the articular surface of the tibia and the femur) were quantified using ImageJ image analysis software. Areas of degeneration where there was no red staining by Safranin O were measured using ImageJ and the percent degeneration at the joint was measured using the following formula:%Degeneration=DegeneratedAreasTheoreticalHealthyArticularSurfaceArea*100.

All statistical analysis was done using GraphPad Prism 6. A two-sided ANOVA with 95% confidence interval with Tukeys means comparison was run in GraphPad Prism 6 to evaluate intergroup differences of percent total degenerated areas. For joint swelling, cytokine analysis, and Raman spectroscopic analysis a oneway ANOVA was run with a Tukeys post hoc test to assess statistical significance between groups.

All study data are included in the article and/or supporting information.

We gratefully acknowledge the quantitative proteomics analysis conducted by Dr. Jeremy L. Balsbaugh and Dr. Jennifer C. Liddle of the UConn Proteomics & Metabolomics Facility, a component of the Center for Open Research Resources and Equipment at the University of Connecticut. We are also thankful to Dr. Zhifang Hao, Research Histology core, UConn Health for helping out with the histology studies. We also gratefully acknowledge funding from NIH DP1AR068147 and NIH T32 AR079114.

Author contributions: M.B., L.S.N., and C.T.L. designed research; M.B., J.L.E.I., H.-M.K., R.B., M.B., N.N., R.P., L.S.N., and C.T.L. performed research; M.B., S.S., T.O., R.P., L.S.N., and C.T.L. analyzed data; and M.B., S.S., R.P., L.S.N., and C.T.L. wrote the paper.

Competing interest statement: A patent titled Injectable Amnion Hydrogel as a Cell Delivery System has been filed and published on behalf of the inventors, C.T.L., L.S.N., and M.B. L.S.N. has competing financial interest with Soft Tissue Regeneration/Biorez. C.T.L. has the following competing financial interests: Mimedx (a company that makes amnion-based biologics), Alkermes Company, Biobind, Soft Tissue Regeneration/Biorez, and Healing Orthopaedic Technologies-Bone.

Reviewers: J.G., Johns Hopkins University; and R.L., University of Chicago Division of the Biological Sciences.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2120968119/-/DCSupplemental.

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Mesenchymal Stem CellBased Therapy as a New Approach for the Treatment of Systemic Sclerosis – Newswise

Posted: January 20, 2022 at 2:20 am

Systemic sclerosis (SSc) is an intractable autoimmune disease with unmet medical needs. Conventional immunosuppressive therapies have modest efficacy and obvious side effects. Targeted therapies with small molecules and antibodies remain under investigation in small pilot studies. The major breakthrough was the development of autologous haematopoietic stem cell transplantation (AHSCT) to treat refractory SSc with rapidly progressive internal organ involvement. However, AHSCT is contraindicated in patients with advanced visceral involvement. Mesenchymal stem cells (MSCs) which are characterized by immunosuppressive, antifibrotic and proangiogenic capabilities may be a promising alternative option for the treatment of SSc. Multiple preclinical and clinical studies on the use of MSCs to treat SSc are underway. However, there are several unresolved limitations and safety concerns of MSC transplantation, such as immune rejections and risks of tumour formation, respectively. Since the major therapeutic potential of MSCs has been ascribed to their paracrine signalling, the use of MSC-derived extracellular vesicles (EVs)/secretomes/exosomes as a cell-free therapy might be an alternative option to circumvent the limitations of MSC-based therapies. In the present review, we overview the current knowledge regarding the therapeutic efficacy of MSCs in SSc, focusing on progresses reported in preclinical and clinical studies using MSCs, as well as challenges and future directions of MSC transplantation as a treatment option for patients with SSc.

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Mesenchymal Stem CellBased Therapy as a New Approach for the Treatment of Systemic Sclerosis - Newswise

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