[Los Angeles], [United States], January 2021, The Follicle Stimulating Hormone Test Kit Market research report includes an in-sight study of the key [Title] market prominent players along with the company profiles and planning adopted by them. This helps the buyer of the Follicle Stimulating Hormone Test Kit report to gain a clear view of the competitive landscape and accordingly plan Follicle Stimulating Hormone Test Kit market strategies. An isolated section with top key players is provided in the report, which provides a complete analysis of price, gross, revenue(Mn), Follicle Stimulating Hormone Test Kit specifications, and company profiles. The Follicle Stimulating Hormone Test Kit study is segmented by Module Type, Test Type, And Region.

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In addition, market revenues based on region and country are provided in the Follicle Stimulating Hormone Test Kit report. The authors of the report have also shed light on the common business tactics adopted by players. The leading players of the global Follicle Stimulating Hormone Test Kit market and their complete profiles are included in the report. Besides that, investment opportunities, recommendations, and trends that are trending at present in the global Follicle Stimulating Hormone Test Kit market are mapped by the report. With the help of this report, the key players of the global Follicle Stimulating Hormone Test Kit market will be able to make sound decisions and plan their strategies accordingly to stay ahead of the curve.

Competitive landscape is a critical aspect every key player needs to be familiar with. The report throws light on the competitive scenario of the global Follicle Stimulating Hormone Test Kit market to know the competition at both the domestic and global levels. Market experts have also offered the outline of every leading player of the global Follicle Stimulating Hormone Test Kit market, considering the key aspects such as areas of operation, production, and product portfolio. Additionally, companies in the report are studied based on the key factors such as company size, market share, market growth, revenue, production volume, and profits.

Key Players Mentioned: Easydiagnosis, Wondfo, Daan, Bioscience, BGI, Chivd, AccuBioTech, Ameritek

Market Segmentation by Product: Chemiluminescence ImmunoassayTime-resolved Immunoassay

Market Segmentation by Application: HospitalClinic

The Follicle Stimulating Hormone Test Kit Market report has been segregated based on distinct categories, such as product type, application, end user, and region. Each and every segment is evaluated on the basis of CAGR, share, and growth potential. In the regional analysis, the report highlights the prospective region, which is estimated to generate opportunities in the global Follicle Stimulating Hormone Test Kit market in the forthcoming years. This segmental analysis will surely turn out to be a useful tool for the readers, stakeholders, and market participants to get a complete picture of the global Follicle Stimulating Hormone Test Kit market and its potential to grow in the years to come.

Key questions answered in the report:

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Table of Contents:

1 Follicle Stimulating Hormone Test Kit Market Overview1.1 Product Overview and Scope of Follicle Stimulating Hormone Test Kit1.2 Follicle Stimulating Hormone Test Kit Segment by Type1.2.1 Global Follicle Stimulating Hormone Test Kit Production Growth Rate Comparison by Type 2020 VS 20261.2.2 Chemiluminescence Immunoassay1.2.3 Time-resolved Immunoassay1.3 Follicle Stimulating Hormone Test Kit Segment by Application1.3.1 Follicle Stimulating Hormone Test Kit Consumption Comparison by Application: 2020 VS 20261.3.2 Hospital1.3.3 Clinic1.4 Global Follicle Stimulating Hormone Test Kit Market by Region1.4.1 Global Follicle Stimulating Hormone Test Kit Market Size Estimates and Forecasts by Region: 2020 VS 20261.4.2 North America Estimates and Forecasts (2015-2026)1.4.3 Europe Estimates and Forecasts (2015-2026)1.4.4 China Estimates and Forecasts (2015-2026)1.4.5 Japan Estimates and Forecasts (2015-2026)1.5 Global Follicle Stimulating Hormone Test Kit Growth Prospects1.5.1 Global Follicle Stimulating Hormone Test Kit Revenue Estimates and Forecasts (2015-2026)1.5.2 Global Follicle Stimulating Hormone Test Kit Production Capacity Estimates and Forecasts (2015-2026)1.5.3 Global Follicle Stimulating Hormone Test Kit Production Estimates and Forecasts (2015-2026)1.6 Follicle Stimulating Hormone Test Kit Industry1.7 Follicle Stimulating Hormone Test Kit Market Trends

2 Market Competition by Manufacturers2.1 Global Follicle Stimulating Hormone Test Kit Production Capacity Market Share by Manufacturers (2015-2020)2.2 Global Follicle Stimulating Hormone Test Kit Revenue Share by Manufacturers (2015-2020)2.3 Market Share by Company Type (Tier 1, Tier 2 and Tier 3)2.4 Global Follicle Stimulating Hormone Test Kit Average Price by Manufacturers (2015-2020)2.5 Manufacturers Follicle Stimulating Hormone Test Kit Production Sites, Area Served, Product Types2.6 Follicle Stimulating Hormone Test Kit Market Competitive Situation and Trends2.6.1 Follicle Stimulating Hormone Test Kit Market Concentration Rate2.6.2 Global Top 3 and Top 5 Players Market Share by Revenue2.6.3 Mergers & Acquisitions, Expansion

3 Production and Capacity by Region3.1 Global Production Capacity of Follicle Stimulating Hormone Test Kit Market Share by Regions (2015-2020)3.2 Global Follicle Stimulating Hormone Test Kit Revenue Market Share by Regions (2015-2020)3.3 Global Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)3.4 North America Follicle Stimulating Hormone Test Kit Production3.4.1 North America Follicle Stimulating Hormone Test Kit Production Growth Rate (2015-2020)3.4.2 North America Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)3.5 Europe Follicle Stimulating Hormone Test Kit Production3.5.1 Europe Follicle Stimulating Hormone Test Kit Production Growth Rate (2015-2020)3.5.2 Europe Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)3.6 China Follicle Stimulating Hormone Test Kit Production3.6.1 China Follicle Stimulating Hormone Test Kit Production Growth Rate (2015-2020)3.6.2 China Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)3.7 Japan Follicle Stimulating Hormone Test Kit Production3.7.1 Japan Follicle Stimulating Hormone Test Kit Production Growth Rate (2015-2020)3.7.2 Japan Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)

4 Global Follicle Stimulating Hormone Test Kit Consumption by Regions4.1 Global Follicle Stimulating Hormone Test Kit Consumption by Regions4.1.1 Global Follicle Stimulating Hormone Test Kit Consumption by Region4.1.2 Global Follicle Stimulating Hormone Test Kit Consumption Market Share by Region4.2 North America4.2.1 North America Follicle Stimulating Hormone Test Kit Consumption by Countries4.2.2 U.S.4.2.3 Canada4.3 Europe4.3.1 Europe Follicle Stimulating Hormone Test Kit Consumption by Countries4.3.2 Germany4.3.3 France4.3.4 U.K.4.3.5 Italy4.3.6 Russia4.4 Asia Pacific4.4.1 Asia Pacific Follicle Stimulating Hormone Test Kit Consumption by Region4.4.2 China4.4.3 Japan4.4.4 South Korea4.4.5 Taiwan4.4.6 Southeast Asia4.4.7 India4.4.8 Australia4.5 Latin America4.5.1 Latin America Follicle Stimulating Hormone Test Kit Consumption by Countries4.5.2 Mexico4.5.3 Brazil

5 Follicle Stimulating Hormone Test Kit Production, Revenue, Price Trend by Type5.1 Global Follicle Stimulating Hormone Test Kit Production Market Share by Type (2015-2020)5.2 Global Follicle Stimulating Hormone Test Kit Revenue Market Share by Type (2015-2020)5.3 Global Follicle Stimulating Hormone Test Kit Price by Type (2015-2020)5.4 Global Follicle Stimulating Hormone Test Kit Market Share by Price Tier (2015-2020): Low-End, Mid-Range and High-End

6 Global Follicle Stimulating Hormone Test Kit Market Analysis by Application6.1 Global Follicle Stimulating Hormone Test Kit Consumption Market Share by Application (2015-2020)6.2 Global Follicle Stimulating Hormone Test Kit Consumption Growth Rate by Application (2015-2020)

7 Company Profiles and Key Figures in Follicle Stimulating Hormone Test Kit Business7.1 Easydiagnosis7.1.1 Easydiagnosis Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.1.2 Easydiagnosis Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.1.3 Easydiagnosis Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.1.4 Easydiagnosis Main Business and Markets Served7.2 Wondfo7.2.1 Wondfo Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.2.2 Wondfo Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.2.3 Wondfo Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.2.4 Wondfo Main Business and Markets Served7.3 Daan7.3.1 Daan Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.3.2 Daan Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.3.3 Daan Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.3.4 Daan Main Business and Markets Served7.4 Bioscience7.4.1 Bioscience Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.4.2 Bioscience Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.4.3 Bioscience Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.4.4 Bioscience Main Business and Markets Served7.5 BGI7.5.1 BGI Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.5.2 BGI Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.5.3 BGI Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.5.4 BGI Main Business and Markets Served7.6 Chivd7.6.1 Chivd Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.6.2 Chivd Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.6.3 Chivd Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.6.4 Chivd Main Business and Markets Served7.7 AccuBioTech7.7.1 AccuBioTech Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.7.2 AccuBioTech Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.7.3 AccuBioTech Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.7.4 AccuBioTech Main Business and Markets Served7.8 Ameritek7.8.1 Ameritek Follicle Stimulating Hormone Test Kit Production Sites and Area Served7.8.2 Ameritek Follicle Stimulating Hormone Test Kit Product Introduction, Application and Specification7.8.3 Ameritek Follicle Stimulating Hormone Test Kit Production Capacity, Revenue, Price and Gross Margin (2015-2020)7.8.4 Ameritek Main Business and Markets Served

8 Follicle Stimulating Hormone Test Kit Manufacturing Cost Analysis8.1 Follicle Stimulating Hormone Test Kit Key Raw Materials Analysis8.1.1 Key Raw Materials8.1.2 Key Raw Materials Price Trend8.1.3 Key Suppliers of Raw Materials8.2 Proportion of Manufacturing Cost Structure8.3 Manufacturing Process Analysis of Follicle Stimulating Hormone Test Kit8.4 Follicle Stimulating Hormone Test Kit Industrial Chain Analysis

9 Marketing Channel, Distributors and Customers9.1 Marketing Channel9.2 Follicle Stimulating Hormone Test Kit Distributors List9.3 Follicle Stimulating Hormone Test Kit Customers

10 Market Dynamics10.1 Market Trends10.2 Opportunities and Drivers10.3 Challenges10.4 Porters Five Forces Analysis

11 Production and Supply Forecast11.1 Global Forecasted Production of Follicle Stimulating Hormone Test Kit (2021-2026)11.2 Global Forecasted Revenue of Follicle Stimulating Hormone Test Kit (2021-2026)11.3 Global Forecasted Price of Follicle Stimulating Hormone Test Kit (2021-2026)11.4 Global Follicle Stimulating Hormone Test Kit Production Forecast by Regions (2021-2026)11.4.1 North America Follicle Stimulating Hormone Test Kit Production, Revenue Forecast (2021-2026)11.4.2 Europe Follicle Stimulating Hormone Test Kit Production, Revenue Forecast (2021-2026)11.4.3 China Follicle Stimulating Hormone Test Kit Production, Revenue Forecast (2021-2026)11.4.4 Japan Follicle Stimulating Hormone Test Kit Production, Revenue Forecast (2021-2026)

12 Consumption and Demand Forecast12.1 Global Forecasted and Consumption Demand Analysis of Follicle Stimulating Hormone Test Kit12.2 North America Forecasted Consumption of Follicle Stimulating Hormone Test Kit by Country12.3 Europe Market Forecasted Consumption of Follicle Stimulating Hormone Test Kit by Country12.4 Asia Pacific Market Forecasted Consumption of Follicle Stimulating Hormone Test Kit by Regions12.5 Latin America Forecasted Consumption of Follicle Stimulating Hormone Test Kit13 Forecast by Type and by Application (2021-2026)13.1 Global Production, Revenue and Price Forecast by Type (2021-2026)13.1.1 Global Forecasted Production of Follicle Stimulating Hormone Test Kit by Type (2021-2026)13.1.2 Global Forecasted Revenue of Follicle Stimulating Hormone Test Kit by Type (2021-2026)13.1.3 Global Forecasted Price of Follicle Stimulating Hormone Test Kit by Type (2021-2026)13.2 Global Forecasted Consumption of Follicle Stimulating Hormone Test Kit by Application (2021-2026)14 Research Finding and Conclusion

15 Methodology and Data Source15.1 Methodology/Research Approach15.1.1 Research Programs/Design15.1.2 Market Size Estimation15.1.3 Market Breakdown and Data Triangulation15.2 Data Source15.2.1 Secondary Sources15.2.2 Primary Sources15.3 Author List15.4 Disclaimer

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Follicle Stimulating Hormone Test Kit Market Overview, Trend Analysis, Competitive Landscape, Forecast to 2027 | Easydiagnosis, Wondfo, Daan - Splash...

Recommendation and review posted by Bethany Smith

The Telegraph

Joe Bidens daughter Ashley has said she will not have a job in her father's administration, unlike Ivanka Trump, in her first interview since the election. The only child of President-elect Biden and wife Jill, Ashley, a 39-year-old social worker in Delaware, said she instead wanted to use her new platform to advocate for social justice and mental health. I will not have a job in the administration, she told NBC's Today Show, in what could be seen as a jibe at the current First Daughter, who, along with husband Jared Kushner, had adviser roles in the White House. I do hope to bring awareness and education to some topics, subjects that are, you know, really important. Ms Biden, who is married to plastic surgeon Howard Krein, was active in her father's presidential campaign, speaking at the 2020 Democratic National Convention, and hosting an event for women in Wisconsin.

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Parler reappears on web, still not in app stores - Yahoo News UK

Recommendation and review posted by Bethany Smith

The Telegraph

Joe Bidens daughter Ashley has said she will not have a job in her father's administration, unlike Ivanka Trump, in her first interview since the election. The only child of President-elect Biden and wife Jill, Ashley, a 39-year-old social worker in Delaware, said she instead wanted to use her new platform to advocate for social justice and mental health. I will not have a job in the administration, she told NBC's Today Show, in what could be seen as a jibe at the current First Daughter, who, along with husband Jared Kushner, had adviser roles in the White House. I do hope to bring awareness and education to some topics, subjects that are, you know, really important. Ms Biden, who is married to plastic surgeon Howard Krein, was active in her father's presidential campaign, speaking at the 2020 Democratic National Convention, and hosting an event for women in Wisconsin.

More here:
Trump Administration begins moving out of the White House - Yahoo News UK

Recommendation and review posted by Bethany Smith

When asked to trial the ProLon 5-Day Fasting Mimicking Diet, I was sceptical. As a health and nutrition coach, my advice is based on promoting long-term lifestyle changes no gimmicks, and forget about juice cleanses or diets that cut out whole food groups and instead focusing on making simple, sustainable, and well-balanced food choices for that individual. So I was pleasantly surprised.

ProLon is a patented five-day, plant-based meal plan that provides you with a daily meal kit containing all youll eat for each day. It contains some macro- and micronutrients that are scientifically selected for inclusion, but the low-calorie content and specific nutrient breakdown are designed to mimic fasting, and so provide some of the benefits you can get from fasting.

According to ProLon, its a programme of scientifically designed meals that give you the nourishment you need without activating your bodys food sensing system. Your body thinks that its on a prolonged fast, allowing for fasting gains, but without all the hunger pains.

Each small box, helpfully labelled Day 1 through to Day 5, contains a nut-based energy bar, two soups, a variety of snacks (including kale chips, olives and even the occasional Choco-crisp bar), energy drinks and supplements. You can eat the contents in any order and at any time of the day, but you must stick to the food for that day. Day 1 was slightly higher in calories with approximately 1,200, while Days 2 to 5 were approximately 800 calories. Wearing my nutritionist hat, I quickly realised that the taller you are and the more active you are, the harder this fast would be. For the first time ever, I was grateful for my five-foot frame.

ProLon is a scientific eating plan that directly lowers circulating insulin and as such is an excellent way to lose the most toxic and voluminous fat on the body

ProLon claims its diet can help rejuvenate your body while getting it to eat real food, helps your body reset and rejuvenate, and supports your bodys natural processes of intracellular clean-up and cell renewal. It also helps maintain lean body mass while lowering body fat.

ProLon came on to the market after 20 years and US$36 million in research and development. Initial clinical studies seem promising. Over a three-month period, ProLon was shown over three cycles to help individuals lose an average of 2.6 kg and 4 cm off their waist circumference. In another study on fast-mimicking diets, published in the magazine Science Translational Medicine in 2017, it aided in maintaining healthy systolic blood pressure and helped the body to rejuvenate.

What intrigued me most is that the brain behind the ProLon fast is Dr Valter Longo, director of the Longevity Institute at the University of Southern California, a man well known for research into longevity.

Here in Hong Kong, ProLon is also recommended by Dr Lauren Bramley, MD, LMCHK, MSc Endocrinology, Diabetes and Metabolism at her medical and anti-ageing clinic. Based on her medical expertise, ProLon is a scientific eating plan that directly lowers circulating insulin and as such is an excellent way to lose the most toxic and voluminous fat on the body, which centres around the abdomen, buttocks, chest, and back. As insulin is a hormone that can negatively impact many other hormones, including thyroid, reducing it improves other hormone levels and ratios to be truly anti-ageing. People look and feel much better with lower insulin levels. The sweetener used in ProLon is inulin, a natural substance that actually preserves muscle, burns fat, and improves insulin resistance this cannot be said for any other sweeteners found in weight loss products.

This in addition to the growing body of research showing that fasting can promote cellular regeneration encouraged me to take the first step. And so with an open mind and prompted by the promises of longevity and anti-ageing, I opened my first box.

By Day 4, I felt like I could go on forever; by the start of Day 5, though, I was glad I wasnt

After the elation of committing to a new plan has passed, youll likely feel hungry and (if anything like me) at a bit of a loss with all the extra time on your hands time youd usually spend chatting over a long meal, a glass of wine, grocery shopping or simply making dinner. This hit me around Day 2.

At the halfway point of the plan, I was feeling great. By Day 3, I was clear-headed and more energised, sleeping better and noticing that some of my inflammation and bloating had gone. I was waking up without an alarm and with a spring in my step, feeling that now is a good time to read up on all the benefits of fasting to keep me going! By Day 4, I felt like I could go on forever; by the start of Day 5, though, I was glad I wasnt. This was the hardest day for me, but by the end of the day I felt a real sense of accomplishment, not to mention that I looked and felt great in the days following!

I wont lie, not eating real meals and going low-calorie isnt for the faint-hearted. That said, after the first two days youre on a roll and with the right strategies you can absolutely survive five days and reap all the benefits that come with it.

Choose a week when youre mentally in a good space to undertake the fast no big social events centred around food and wine, and the ability to keep your schedule as flexible as you can.

Start each day with positive affirmations and remind yourself why you chose to do this. Remember that this is entirely your choice, so enjoy the process rather than focus on what you cant have. It will all still be there in five days!

Depending on how you feel, consider delaying your first meal. You dont want to end up at 5pm with no food to look forward to and a long night ahead.

Youre allowed one coffee a day I had more (decaffeinated) and I dont think it was a disadvantage. I also drank tons of herbal teas hot and cold as well as the glycerine drink provided in the meal kit. Its hard to feel hungry when constantly sipping on a flavoured drink.

I also added herbs and salt to spice up the flavour of the soup. Trust me, it really helped with some of the green-vegetable-based soups!

Go easy on exercise and do lots of activities that help switch on your para-sympathetic nervous system, such as yoga, pilates, stretching and gentle walks, as well as anything that helps detoxification, such as hot yin yoga, infra-red saunas and contrast showers.

Use this newly found time to do low-key activities with friends or anything that distracts you from thinking about food! I Marie Kondod my apartment and even managed to sort out my wardrobe, possibly inspired by the knowledge that, post-fast, I might even fit into those jeans Id kept since 2015.

Use the quiet time to catch up on books youve been meaning to read but never got round to.

Go to bed early and focus on a good nights sleep, knowing that sleep is one of the best things you can do to improve health (and is also scientifically proven to aid fat loss).

I also consciously didnt look at any food-related social-media pages or TV programmes why torment yourself unnecessarily?

Transition out of the fast sensibly. On Day 6, light foods, soups and cooked foods are ideal. I tend to be an all-or-nothing person, so the temptation to go out for a slap-up meal and glass of wine was huge. I resisted and was really glad I did!

If youre looking for a quick reboot, a way to recharge yourself or simply to kickstart a new health regime in the New Year, this could be a great option. Its also a good way to shift a few kilograms and mentally get into a good place to start a healthier way of living. If fat loss is a goal, then clinical trials have shown that the three-month protocol can produce some great results. However, its not a long-term solution and Id highly recommend ensuring you have a proper nutrition plan in place for after you finish the fast.

Also, its not for everyone and especially not suitable for those who are pregnant, breast feeding or underweight, or have pre-existing food disorders or any serious medical condition. Its also not a replacement for a proper nutrition plan and should always be done in conjunction with a health-care practitioner.

When I first embarked on the fast, my goal was to see if it lived up to its multiple claims and genuinely to understand how someone would feel during each stage of the five days. I was surprised by how good I felt by the end and can honestly say that I felt rejuvenated and would probably even do it again.

Its simple and effective enough to do whenever you need a reboot, and to create the momentum to springboard yourself into a healthier, happier you. Youll also notice that the results should continue after the fast is completed. With newly sensitised tastebuds, a body thats become used to consuming less food, fewer food cravings and a new appreciation for real food, this usually means that you continue to reap the benefits long after. If anything, Id say the reset benefits far outweigh the five-day fast itself.

Dr Lauren Bramleys clinic is offering a great deal, in which you can book a consultation with a health and nutrition coach, plus either one Prolon Fast or a package of three Prolon Fasts spread over three months. By quoting Prestige, you receive a 15 percent discount off the total package cost.

Beth Wright is a qualified health and nutrition coach focused on creating truly personalised nutrition and lifestyle programmes for her clients. She takes a holistic approach, looking at nutrition, fitness, hormones, supplementation, sleep and stress to help take peoples health, well-being and performance to a new level. Find out more about her programmes at bfit-thewrightway.com.

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The ProLon Fasting Mimicking Diet: Whats the Skinny? - Prestige Online

Recommendation and review posted by Bethany Smith

The University of Glasgow has joined a large-scale European research project investigating the fundamental causes of hypertension, or high blood pressure.

The multidisciplinary research programme termed MINDSHIFT (www.eumindshift.eu) started on January 1 and is made possible by a European grant of over four million euros from the European Unions Horizon 2020 research and innovation programme.

Led by Maastricht UMC+ and Maastricht University and involving the Universitys Institute of Cardiovascular and Medical Sciences, UoG the four-year programme, includes 14 other partners from the United Kingdom, Spain, Italy, France, Austria, Switzerland and Poland.

The collaborative network includes academic institutions, research centres and industry with specific expertise and interest in hypertension that. Over the next few years the network will unravel new mechanisms of hypertension and will advance knowledge in the field.

Hypertension is called the silent killer because there are no symptoms yet it causes almost all cardiovascular diseases and is therefore a main cause of death globally. There is a clearly identifiable cause for high blood pressure in less than 15 per cent of cases, while in the remaining 85 per cent it is unclear. The researchers are keen to understand the underlying mechanisms that cause hypertension.

The University of Glasgow team will be contributing to four projects within the MINDSHIFT programme. Early stage researchers will be supervised by clinical and basic scientists within the College of MVLS and closely collaborate with other consortium partners across Europe.

Prof. Rhian Touyz will study the molecular make-up of small blood vessels and how they contribute to the development of high blood pressure. Prof. Christian Delles will study how hypertension leads to damage of the heart, kidneys and other organs and why some patients are more affected than others.

Prof. Eleanor Davies will dissect the role of aldosterone in the development of hypertension and how this hormone is regulated by small RNA molecules; and Prof. Paul Shiels will put hypertension into the context of ageing, and will develop new treatments to interfere with the ageing process.

Prof. Rhian Touyz, Director of the Institute of Cardiovascular and Medical Sciences, said: Hypertension is the major cause of cardiovascular disease worldwide and despite decades of research, we still do not fully understand how and why it develops, how to prevent it and how to properly treat it.

In the MINDSHIFT Consortium, we aim to address these gaps in knowledge. We will train the next generation of hypertension researchers and provide them with skills in basic and clinical science so that they can translate their research into the clinic and make a real difference to our patients with high blood pressure. The collaborative approach makes this programme particularly attractive and we look forward to working with our colleagues across Europe.

Excerpt from:
UofG PART OF INTERNATIONAL RESEARCH NETWORK STUDYING CAUSES OF HYPERTENSION - India Education Diary

Recommendation and review posted by Bethany Smith

This article by Camila Ferezin was originally published onThe Green Fund, and appears here with permission.

Cannabis sativaL. (hemp, marijuana) produces male and female inflorescences on different plants that produceunisexual flowers.

In commercial production, marijuana plants are all genetically feminized, as female plants accumulate far larger amounts of several cannabinoids in thetrichomes than male plants, which are often removed or killed. Moreover, the presence of male plants among females often leads to the fecundation of female plants; a fecundated female plant will lead to seed formation, reducing flower quality (as they become hermaphrodites) and buds/cannabinoids yield.

Both genetics and environment play a role in the determination of the sex of a cannabis plant. Many growers focus on the growing conditions to ensure that hermaphroditism (when you have both male and female flowers on the same plant) does not take place, but genetics play just asimportant a role.

Scientists have already identified whichgenes are responsible for flowerformation and sex development in other plants in the past.

For instance, loss or gain of function of those genes, due to environmental conditions or genetic manipulation, can have deleterious effects, including elimination (when the plant doesn't develop any flower) or conversion of floral parts (changes from female to male plants, for example).

As previously mentioned, sex determination in the cannabis flower is controlled primarily at the genetic level. Therefore,mappingthe genes responsible for sex differentiation and understanding the molecular basis of male flower development in cannabis is extremely interesting in a marketing perspective.

Male flower poses a problem for farmers growing drug-type female plants for cannabinoids since male flowers have fewer trichomes and lead to limited amounts of cannabinoids.

Nowadays, the rapidly growinghempfarming industry requires sex determination genetics for seed and fibre quality, as even feminized seeds can develop male flowers due to environmental conditions and mutations in the plant's DNA.

In this study, headed by Soheil S. Mahmoud from The University of British Columbia, feminized plants were treated with silver thiosulfate (Ag2S2O3) complex, a chemical compound capable to induce masculinizing effects and development of male flowers in female plants, and employed a comparative RNA-Seq study (a technique that can examine the quantity and sequences ofRNA, and analyzes the gene of the plant) to identify who are the genes involved in sex modification and flower development.

The investigation highlighted a number of genes with potential roles in floral development and sex determination.

Among these are genes involved in flower development, and plant hormone signalling.

The results suggest that stamen (the pollen-producing part of aflower, usually with a slender filament supporting theanther) development in female cannabis plants can be associated with complex networking of diverse genes involved in floral development, phytohormone signalling, sugar/lipid metabolism and other sex-related pathways.

Although the exact roles of these genes must be further investigated in plants, this study could be useful for the understanding of a plant's predisposition to produce opposite sex flowers and help growers to regulate this trait depending on the purpose of the cropping.

Moreover, this could be one step forward into specifically genetic modified cannabis, in order to manipulate certain features of the plant, such as increasing the percentage of female flowers, complete deletion of male flowers and enhanced trichome production per plant.

But the amazing word ofCannabis Genetic Modificationdoes not stop there, and we may face a steep growth on the field, leading to the development of Cannabis plants that arepest resistant, drought resistance, improved yieldsand withselective phenotypes.

Studies such as this not only produce outstanding knowledge about the plant biology but also creates amazing market opportunities for those seeking for specific traits in their cropping production.

Read the original Article onThe Green Fund.

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JEDDAH: Just 30 percent of women worldwide work in science, but Saudis are challenging this long-standing trend.Women represent 58 percent of university students in Saudi Arabia, with many studying in science, technology and engineering and furthering their careers with studies overseas.In a report by the Saudi Education Ministry, women outnumbered men in graduating with a bachelors in biology, information technology, mathematics, statistics, and physics.Universities and research centers have adopted measures to support the inclusion of female scientists.Ambitious, driven and facing challenges along the way to their success, here are the Saudi women scientists who have made a mark in the field for their extraordinary work.Suha KayumResearch engineerWith a career spanning 10 years, Kayum a research engineer with Saudi Aramcos EXPEC Advanced Research Center was tasked with accelerating the evolution of software algorithms to enhance Aramcos reservoir simulator, which helped the company cut costs.Kayum was a developer for the companys in-house basin and seismic simulators. In 2016, she designed and received a patent for an algorithm that enabled the first 1-billion cell basin simulation run.

Dr. Elaf AhmedLab scientistWith a keen research interest in nano-organisms, Ahmeds main focus while conducting postdoctoral work at King Abdullah University for Science and Technology was synthesis of environmental nano materials using electrochemically active biofilms.She later joined the companys Oil and Gas Treatment Division at Aramcos Research and Development Center.Her main focus at the division is to conduct research projects for water treatment technologies and find new ways to treat water found in oil and gas reservoirs.

Dr. Ilham AbuljadayelImmunologistIn what could be one of the most profound achievements by a Saudi scientist, Dr. Ilham discovered the process of retrodifferentiation, a method also known as retrograde differentiation that treats blood diseases.A common process for the maintenance of cell integrity against damaging agents, Dr. Ilham applied her findings in the first preclinical study in 2000 in collaboration with George Washington Medical Center, US, in two animal models of human diseases to study the utility of retrodifferentiated stem cells.Her research has helped treat 390 patients with diseases ranging from sickle cell anaemia, multiple sclerosis, thalassaemia, and hepatitis C among others.Dr. Abeer Al-OlayanPetroleum scientistWith an academic and industrial background in various fields of chemistry spanning over 20 years, Dr. Abeer is a research scientist at Saudi Aramcos EXPEC Advanced Research Center and is responsible for leading its chemicals development initiative.As a fellow at MIT, she submitted a fellowship research abstract that focuses on reducing dependency on food-based chemicals to tackle drilling and subsurface challenges. She has 10 registered patents with the US Patent Office for the development of methods, materials and compositions in drilling and fluid transfer.

Dr. Malak Abed AlthagafiPhysician-scientistDiagnosed with a rare genetic disease at a young age, Althagafi got a first glimpse of what her future could be during her treatment. Her educational path started with the study of genetic diseases in children and led to molecular pathology before she focused on surgical oncology, molecular genetics and neuropathology.Dr. Malak is one of the few American board-certified molecular neuropathologists in the world and has conducted research that focuses on decoding genetic mutations in tumors, specifically brain tumors in children.She became part of the Saudi Human Genome Program in 2014. Her clinical and research interests are mainly in surgical oncology, pathology, molecular genetics pathology and neuropathology, especially its application for treating brain cancers.

Dr. Hind Al-JohaniScientist of physical chemistryHer research interest is in nano-catalysis. In 2017, this Saudi scientist discovered that by using the simple molecule of citrate ions (from citric acid) you could stabilize and control the structure of gold nanoparticles.Using this new discovery, the findings showed that gold can carry drugs through the body without chemical side effects. Attaching antibodies can guide the nanoparticles to specific cells that need treatment. Her findings have had an impact on environmental chemistry where it may also be used for water purification or methods for capturing CO2 emissions.

Dr. Nouf Al-NumairMolecular bioinformatics scientistDubbed the DNA decoder, her research focuses on predicting the early emergence of diseases through genetic mutations.She has achieved this by merging molecular genetics and computer programming to predict the effects of mutations and provide patients with a personalized medical approach to treatment.Using more than seven programming languages to analyze human genes, she has successfully published a number of papers with the findings.Dr. Nouf pursued her career in STEM and is the first Saudi scientist to major in molecular genetics and programming biological information.

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Saudi women making their mark in science - Arab News

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President-elect Joe Biden announced Friday that he has chosen a pioneer in mapping the human genome the so-called book of life to be his chief science adviser and is elevating the top science job to a Cabinet position.

Biden nominated Eric Lander, founding director of the Broad Institute of MIT and Harvard, who was the lead author of the first paper announcing the details of the human genome, as director of Office of Science and Technology Policy and adviser on science. He is the first life scientist to have that job. His predecessor is a meteorologist.

Saying science will always be at the forefront of my administration, Biden said he is boosting the science advisor post to Cabinet level, a first in White House history.

The president-elect also said he is retaining National Institutes of Health Director Dr. Francis Collins, who worked with Lander on the human genome project, and named two prominent female scientists to co-chair the Presidents Council of Advisors on Science and Technology.

Frances Arnold, a California Institute of Technology chemical engineer who won the 2018 Nobel Prize in chemistry, and MIT vice president for research and geophysics professor Maria Zuber will co-chair the outside science advisory council. Lander held that position during Obama administration.

Collins, in an email statement, called Lander brilliant, visionary, exceptionally creative and highly effective in aspiring others.

I predict he will have a profound transformational effect on American science, Collins said.

The job as director of science and technology policy requires Senate confirmation.

Science organizations were also quick to praise Lander and the promotion of the science post.

Elevating (the science adviser) role to member in the Presidents Cabinet clearly signals the administrations intent to involve scientific expertise in every policy discussion, said Sudip Parikh, chief executive officer of the American Association for the Advancement of Science, the worlds largest general scientific society.

Biden chose Princetons Alondra Nelson, a social scientist who studies science, technology and social inequality, as deputy science policy chief.

Lander, also a mathematician, is a professor of biology at both Harvard and MIT and his work has been cited nearly half a million times in scientific literature, one of most among scientists. He has won numerous science prizes, including a MacArthur genius fellowship and a Breakthrough Prize, and is one of Pope Francis scientific advisors.

Lander has said in talks that an opportunity to explain science is his Achilles heel: I love teaching and more than that, I firmly believe that no matter what I do in my own scientific career, the most important impact that I could ever have on the world is going to be through my students.

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Biden picks geneticist as science adviser, puts in Cabinet - The Associated Press

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Just 30 percent of women worldwide work in science, but Saudis are challenging this long-standing trend.

Women represent 58 percent of university students in Saudi Arabia, with many studying in science, technology and engineering and furthering their careers with studies overseas.

In a report by the Saudi Education Ministry, women outnumbered men in graduating with a bachelors in biology, information technology, mathematics, statistics, and physics.

Universities and research centers have adopted measures to support the inclusion of female scientists.

Ambitious, driven and facing challenges along the way to their success, here are the Saudi women scientists who have made a mark in the field for their extraordinary work.

Suha KayumResearch engineer

With a career spanning 10 years, Kayum a research engineer with Saudi Aramcos EXPEC Advanced Research Center was tasked with accelerating the evolution of software algorithms to enhance Aramcos reservoir simulator, which helped the company cut costs.

Kayum was a developer for the companys in-house basin and seismic simulators. In 2016, she designed and received a patent for an algorithm that enabled the first 1-billion cell basin simulation run.

Dr. Elaf AhmedLab scientist

With a keen research interest in nano-organisms, Ahmeds main focus while conducting postdoctoral work at King Abdullah University for Science and Technology was synthesis of environmental nano materials using electrochemically active biofilms.

She later joined the companys Oil and Gas Treatment Division at Aramcos Research and Development Center.

Her main focus at the division is to conduct research projects for water treatment technologies and find new ways to treat water found in oil and gas reservoirs.

Dr. Ilham AbuljadayelImmunologist

In what could be one of the most profound achievements by a Saudi scientist, Dr. Ilham discovered the process of retrodifferentiation, a method also known as retrograde differentiation that treats blood diseases.

A common process for the maintenance of cell integrity against damaging agents, Dr. Ilham applied her findings in the first preclinical study in 2000 in collaboration with George Washington Medical Center, US, in two animal models of human diseases to study the utility of retrodifferentiated stem cells.

Her research has helped treat 390 patients with diseases ranging from sickle cell anaemia, multiple sclerosis, thalassaemia, and hepatitis C among others.

Dr. Abeer Al-OlayanPetroleum scientist

With an academic and industrial background in various fields of chemistry spanning over 20 years, Dr. Abeer is a research scientist at Saudi Aramcos EXPEC Advanced Research Center and is responsible for leading its chemicals development initiative.

As a fellow at MIT, she submitted a fellowship research abstract that focuses on reducing dependency on food-based chemicals to tackle drilling and subsurface challenges. She has 10 registered patents with the US Patent Office for the development of methods, materials and compositions in drilling and fluid transfer.

Dr. Malak Abed AlthagafiPhysician-scientist

Diagnosed with a rare genetic disease at a young age, Althagafi got a first glimpse of what her future could be during her treatment. Her educational path started with the study of genetic diseases in children and led to molecular pathology before she focused on surgical oncology, molecular genetics and neuropathology.

Dr. Malak is one of the few American board-certified molecular neuropathologists in the world and has conducted research that focuses on decoding genetic mutations in tumors, specifically brain tumors in children.

She became part of the Saudi Human Genome Program in 2014. Her clinical and research interests are mainly in surgical oncology, pathology, molecular genetics pathology and neuropathology, especially its application for treating brain cancers.

Dr. Hind Al-JohaniScientist of physical chemistry

Her research interest is in nano-catalysis. In 2017, this Saudi scientist discovered that by using the simple molecule of citrate ions (from citric acid) you could stabilize and control the structure of gold nanoparticles.

Using this new discovery, the findings showed that gold can carry drugs through the body without chemical side effects. Attaching antibodies can guide the nanoparticles to specific cells that need treatment. Her findings have had an impact on environmental chemistry where it may also be used for water purification or methods for capturing CO2 emissions.

Dr. Nouf Al-NumairMolecular bioinformatics scientist

Dubbed the DNA decoder, her research focuses on predicting the early emergence of diseases through genetic mutations.

She has achieved this by merging molecular genetics and computer programming to predict the effects of mutations and provide patients with a personalized medical approach to treatment.

Using more than seven programming languages to analyze human genes, she has successfully published a number of papers with the findings.

Dr. Nouf pursued her career in STEM and is the first Saudi scientist to major in molecular genetics and programming biological information.

This article has been adapted from its original source.

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Challenging The World! Meet The Saudi Women Scientists - Al-Bawaba

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In trying to better understand the relationship between all levels of alcohol intake and the risk of AF, a group of European researchers turned to information contained in the Monica Risk, Genetics, Archiving and Monograph (MONGAM) project database.

This multinational collaborative study was established in the 1990s to explore the relationship among Europeans between the development of cardiovascular disease and classic and genetic risk factors. The dataset contains self-reported information on classic cardiovascular risk factors e.g., BMI, hypertension, smoking status etc, collected from community cohorts across Europe with baseline data available from 1982. For the present study, researchers examined data collected from 1982 to 2010. Individuals self-reported the type of alcohol they drank (e.g., wines, spirits, beer) and drinking patterns. A calculation of the alcohol intake was then undertaken and expressed as grams of alcohol, with one drink approximating to 12g of alcohol. The diagnosis of both AF and heart failure were based on either questionnaire information or from national hospital discharge data. The team also collected data on measurement of the biomarkers N-terminal pro-B-type natriuretic peptide and serum high sensitivity troponin I levels.

FindingsA total of 100,092 individuals without AF at baseline and with a mean age of 47.8 years (51.7% female) were included in the analysis and followed for a median of 13.9 years. Nearly half of all participants (46.3%) reported consuming up to one alcoholic drink per day (12g alcohol). During the follow-up period, there were 5854 incident cases of AF and using regression analysis, the researchers calculated that consumption of one alcoholic drink per day increased the risk of AF by 16% (hazard ratio, HR = 1.16, 95% CI 1.111.22, p < 0.001), irrespective of the type of alcoholic beverage consumed. Furthermore, this association remained (HR = 1.18) after adjustment for cardiovascular risk factors and was similar between the sexes. The association between AF and alcohol intake was also independent of heart failure incidence and remained even after excluding individuals over 80 years of age (who are at a higher risk of AF). While the literature provides some evidence to support a relationship between alcohol consumption and myocardial wall stress, reflected by changes in the two biomarkers measured in the study, the researchers observed only a weak correlation.

They concluded that although it is possible that low intakes of alcohol might offer some degree of protection against cardiovascular disease, their study suggested that this is not the case with AF and that this increased risk occurs independently of classical pathways. They called for a strategy of reducing alcohol intake which might prevent a substantial number of AF cases.

CitationCsengeri Det al. Alcohol consumption, cardiac biomarkers and risk of atrial fibrillation and adverse outcomes. Eur Heart J 2021.

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Low levels of alcohol intake linked to greater risk of atrial fibrillation - Hospital Healthcare Europe

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According to the Global Burden of Disease Study in 2013, migraines were found to be the sixth largest cause of years lost due to disability, worldwide. Further, some sources suggest that migraines constitute the third most common disease in the world with an estimated global prevalence of 14.7% affecting around 1 in 7 people.

However, migraines are more common in women, and according to the NHS, they affect as many as 1 in every 5 women, and only about 1 in every 15 men.

Believed to be a neurological condition, migraines are intense, often pulsing, debilitating headaches that can also be followed by a host of other symptoms.

Most migraine-sufferers experience their first migraine headache between ages of 10 and 40.

Unlike headaches caused by tension, migraines tend to be more severe, and can also be described as a throbbing pain, rather than a consistent, dull one. In addition, migraines are also believed to typically cause headaches affecting only one side the head but the pain may shift from one side to another. In about a third of migraine-sufferers, however, the headaches affect both sides simultaneously.

Also, unlike sinus-related headaches, which are often accompanied by nasal congestion, facial pressure, and fever, migraines have their own set of symptoms.

Related on The Swaddle:

Why Do Some People Get Headaches in the Morning?

The acronym P.O.U.N.D. is considered an easy way to remember the symptoms of migraines. Here, P stands for pulsating pain; O for one-day duration of severe untreated attacks; U for unilateral, i.e., one-sided, pain; N for nausea and vomiting; and D for disabling intensity.

However, migraine symptoms can vary widely among people leading at least half of all migraine-sufferers to think that they have sinus or tension headaches, and not migraines, according to Harvard Health.

In addition to the symptoms contained in P.O.U.N.D., migraines may also be accompanied by:

Moreover, called a migraine hangover, in several cases, tiredness and irritability lasts another two days after the headache subsides. Muscle pain, weakness, and either food cravings or a lack of appetite may also follow the period of intense headaches.

Also, symptoms like constipation, mood swings, food craving, stiffness in the neck, increased thirst and urination, and yawning frequently may also signal the onset of an episode, according to some experts.

Scientists are still trying to understand the underlying cause(s) behind migraines, but neurologists believe that migraines may be caused by changes in the brains nerve cell activities, or blood flow with the latter believed to, at least, worsen migraines, even if it hasnt necessarily caused them. However, what causes these changes is also uncertain, but experts note that they may be associated with a host of environmental and genetic factors.

In fact, 70% of those who experience migraine headaches, have at least one close relative with the problem making a strong case for the influence of genetics on migraines.

Related on The Swaddle:

How the Sharp and Sudden Sensation of Brain Freeze Happens

At the same time, triggers like changing weather, fluctuating sleep patterns, mental or emotional stressors like depression and anxiety, medications or diets, and sensory stressors such as bright lights or strong smells have also been associated with migraines.

Given the higher prevalence of migraine among women, experts are also exploring possible correlations of migraines with sex hormones. About five days prior to the onset of bleeding, thats when the estrogen drops and that drop is related to this triggering of migraine, Jelena Pavlovic, a neurologist at the Montefiore Medical Center in NYC, explained in a Nature report. Its not the absolute levels of hormones, but more the fluctuations in hormones that cause the migraine attacks, Antoinette Maassen van den Brink, a pharmacologist at the Erasmus University Medical Center in Rotterdam, the Netherlands, added. Similarly, a Dutch study from 2004, had also lent credence to the theory had sex hormones may have a role to play in migraines, by finding that 26% of individuals undergoing male-to-female transition had reported experiencing migraines.

Migraines cant be cured yet. But for trigger-induced migraines, the simplest solution is to avoid the trigger. However, since thats not always possible, there are medications to help ease the symptoms of migraines.

But in addition to prescription medications that help manage symptoms better, doctors can also recommend lifestyle changes, or even hormone therapy, to prevent frequent onsets of migraines.

However, experts believe that research on migraines is still lacking, resulting in more than half of those that experience migraines, never diagnosed. According to some migraine-researchers, this is likely because it affects women more, and as we know, womens pain often receives less medical attention than mens. If migraine affected men at the same rate, we would have much better studies A lot of the biases and stigma associated with migraine have to do with it being a disorder of women, Pavlovic remarked.

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Everything You Need To Know About Migraines - The Swaddle

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Anorexia nervosa is a serious mental health condition and a potentially life threatening eating disorder. However, with the right treatment, recovery is possible.

Anorexia nervosa often involves emotional challenges, an unrealistic body image, and an exaggerated fear of gaining weight. However, it can affect people differently.

In some cases, an individual may lose a significant amount of weight and demonstrate the characteristic behaviors of anorexia but not have a very low body weight or body mass index (BMI). Researchers refer to this as atypical anorexia nervosa.

Anorexia nervosa often appears during a persons teenage years or early adulthood, but it can sometimes begin in the preteen years or later in life.

People often think of anorexia nervosa in connection with females, but it can affect people of any sex or gender. Research suggests that the risk of eating disorders may be higher among transgender people than cisgender people.

Statistics show that males represent about 25% of people with anorexia and that the effects are more likely to be life threatening among males than females. The reason for this is that males often receive a later diagnosis due to the mistaken belief that it does not affect them.

Anorexia nervosa is different than anorexia. Anorexia means a loss of appetite or the inability to eat, and it can be a symptom of various diseases.

A person with anorexia nervosa will intentionally restrict their food intake as a way to help them manage emotional challenges. These often involve a fear of gaining weight or a desire to lose weight.

Dietary restrictions can lead to nutritional deficiencies, which can severely affect overall health and result in potentially life threatening complications.

The emotional and psychological challenges of anorexia nervosa can be hard for a person to overcome.

Therapy includes counseling, nutritional advice, and medical care. Some people may need treatment in the hospital.

There are many myths about eating disorders. These can lead to false assumptions and affect a persons chances of seeking and getting help.

Learn more about the myths surrounding eating disorders and the real facts.

Anorexia nervosa is a complex condition. The main sign is significant weight loss or low body weight. In atypical anorexia nervosa, the person may still have a moderate weight despite substantial weight loss.

A lack of nutrients may lead to other physical signs and symptoms, including:

The person may also demonstrate certain behaviors, such as:

The person may associate food and eating with guilt. They may seem unaware that anything is wrong or be unwilling to recognize their issues around eating.

Anorexia nervosa affects people differently. Not everyone with the condition will behave in the same way, and some individuals may experience atypical anorexia nervosa, meaning that they will not have a low body weight.

Concerns about body weight and shape are often features of anorexia nervosa, but they may not be the main cause. Experts do not know exactly why the condition occurs, but genetic, environmental, biological, and other factors may play a role.

Some factors that may increase a persons risk include:

For some people, anorexia nervosa develops as a way of gaining control over an aspect of their life. As the person exerts control over their food intake, this feels like success, and so, the behavior continues.

A person may also have a higher chance of developing an eating disorder if:

In 2015, researchers found that people with anorexia nervosa may have different gut microbial communities than those without the condition. This could contribute to anxiety, depression, and further weight loss.

The COVID-19 pandemic is affecting many people with eating disorders. Learn about some ways to cope.

Early diagnosis and prompt treatment increase the chance of a good outcome.

The doctor may ask the person questions to get an idea of their eating habits, weight, and overall mental and physical health.

They may order tests to rule out other underlying medical conditions with similar signs and symptoms, such as malabsorption, cancer, and hormonal problems.

The National Eating Disorders Association state that the criteria below can help doctors make a diagnosis. However, they note that not everyone with a serious eating disorder will meet all these criteria.

A healthcare professional will make a comprehensive plan to address the individuals specific needs.

It will involve a team of specialists who can help the person overcome the physical, emotional, social, and psychological challenges that they face.

Strategies include:

It can be challenging for a person with anorexia nervosa to engage in treatment. As a result, the persons participation in therapy may fluctuate. Relapses can occur, especially during the first 2 years of treatment.

Family and friends can provide crucial support. If they can understand the condition and identify its signs and symptoms, they can support the individual during recovery and help prevent a relapse.

The person may need to spend time in the hospital if they have:

Treatment will allow for a gradual increase in food intake to restore overall health.

Complications can affect every bodily system, and they can be severe.

They include problems with:

Some of these issues can be life threatening. In addition to the physical effects of poor nutrition, the person may have a high risk of suicide.

A post on the National Institute of Mental Healths website in 2012 described anorexia nervosa as the mental health condition most likely to be fatal.

For this reason, early diagnosis and treatment are essential.

Maria Rago, Ph.D., the president of the National Association of Anorexia Nervosa and Associated Disorders (ANAD), offered Medical News Today the following tips for anyone who thinks that they or a loved one may have anorexia nervosa:

Ms. Rago noted that ANAD have free support groups and mentoring programs for recovery and that they invite people to take advantage of the free services. The right help can change your life, and even save your life, she said.

Anorexia nervosa is an eating disorder and a serious mental health condition. It involves restricting food intake, which can lead to severe nutritional deficiencies.

The effects of anorexia nervosa can be life threatening, but counseling, medication, and treatment for underlying mental health issues can help people with this condition.

If a person has signs of anorexia nervosa, they should seek medical help. Early diagnosis and treatment are more likely to lead to a positive outcome.

Read more:
Anorexia nervosa: Symptoms, causes, and treatment - Medical News Today

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Lynne Malcolm: Hi, Lynne Malcolm with you for All in the Mind. Today, girls and women on the autism spectrum; are they slipping through the net?

Temple Grandin [archival]: My name is Temple Grandin. I'm not like other people. You know, you never get cured of autism, but what you do is you learn more and more things. When I was a little kid I was very autistic, non-verbal, rocking. That's the kind of kid they just put away in institutions. But I had a speech teacher that worked really hard with me, and I can't emphasise enough the importance of young children getting early intervention. You got a two-year-old or three-year-old with no speech, don't wait.

Francesca Happ: One of the best known women with autism is Temple Grandin, and she is an extraordinarily brilliant woman who has I think several degrees and PhDs and designs livestock facilities. And she talks about her ability to visualise the whole of a livestock facility in her head before she even begins to draw the blueprints. And also she talks about her empathy for animals, her ability to see the world as if she was one of the cattle that's going to have to go through this livestock facility and to notice the little shiny things that might scare them or disturb them and so on.

How is she representative of women with autism? Well, she's highly, highly intelligent, she talks about her ability to compensate and to cope, and she also interestingly talks about having made great progress even later in life. So she said at one point that her brain switched on aged 50. So a very important message that it's not just children with autism, it's adults and older adults too with autism.

Lynne Malcolm: That's Francesca Happ, Professor of Cognitive Neuroscience at the Institute of Psychiatry in London, and she's also president of the International Society for Autism Research.

Apart from a few notable exceptions such as the inspiring Temple Grandin, autism or ASD is most often thought of as a male disorder. However, Francesca Happ explains that the statistics tell only part of the story.

Francesca Happ: The general prevalence suggests that about four times as many males as females get an autism diagnosis, and this varies across the spectrum. So the proportions are higher in high functioning or more subtle forms of autism, and more even at the lower end of the spectrum where there is also intellectual impairment.

Lynne Malcolm: So what's the level of understanding of women with ASD generally?

Francesca Happ: I think we know very, very little really about how autism presents in girls and women. There are some studies, but the main problem is that the studies start in a clinic. And so you can see there is a circularity. If we are missing women and girls with autism because we are not good at recognising them, then studying those we do spot isn't going to tell us very much about the ones we miss.

Lynne Malcolm: Dr Janine Manjiviona is a clinical psychologist in Melbourne with 20 years experience of diagnosing girls on the autism spectrum.

Janine Manjiviona: They are being both underdiagnosed I think and misdiagnosed. And when you historically have a look at the whole history of autism, originally in Kanner's descriptions, they were largely focused on males. And Hans Asperger's clinical cases were all male. So across the epidemiological studies throughout the world that provide sex ratios, gender differences are always apparent, and females never outnumber the males, and the reasons for that are not always clear but I think it's plausible that underdiagnosis of females may contribute.

Lynne Malcolm: Janine Manjiviona.

Research is emerging that one of the reasons that ASD in females may be missed by clinicians is that it looks different in girls than it does in boys. Girls may be better at covering up the more obvious characteristics of autism.

Francesca Happ: Some of the differences are that we think the social difficulties in some girls with autism may be less obvious. Some women with autism describe a strategy of copying somebody. They pick somebody in their class or their workplace and they just copy everything about that person, how they dress, how they act, how they talk, and that kind of masking strategy we don't see very much in boys and men with autism.

If you looked at the research on women with autism, most of the work suggests that women with autism may be more impaired and more often have intellectual difficulties and be hit harder by autism, but I suspect that's because the subtler cases are missed by our diagnostic system.

Lynne Malcolm: So why do you think girls often do go undiagnosed?

Francesca Happ: It's interesting question, why we might miss girls with autism, because on the face of it you might think that our expectations of social functioning are even higher for girls than for boys, and so maybe we should be more alert to these difficulties. I think there are a lot of reasons why we might miss autism in women. One is diagnostic overshadowing or diagnostic substitution were, for example, a girl who has eating disorders, when that's picked up as an eating disorder they don't ask any more questions about what else might be different about this young woman. So a girl with autism who has an eating disorder may just be diagnosed with an eating disorder.

I think it's also likely that girls show their autism in a different way, and a way that our diagnostic system isn't ready for. So an example would be that when a clinician is deciding if a child has autism, they will look for rigid and repetitive behaviour, which might include unusual special interests. A boy with autism might have a fascination with electricity pylons and know all the facts about electricity pylons, and the clinician is going to go, aha, that's sounds pretty odd to me. But a girl with autism might be fascinated by a particular pop group, and she learns all the facts about them, and when she says her interest is this pop group, the clinician thinks, well, that's pretty normal. So unless he digs deeper and finds out actually she has no interest in going to hear them perform or even listening to their music, she just collects the facts, then otherwise the clinician is going to be fooled into thinking, okay, this isn't autism.

Lynne Malcolm: Professor Francesca Happ.

Because ASD can be missed in girls, they often don't discover it till later in their lives.

Hannah Belcher had been seeing therapists throughout her adolescence because of her struggles with her psychological health. And a diagnosis of ASD in her early twenties came as a bit of a shock.

Hannah Belcher: I was 23 and I was in art therapy and I'd had a lot of therapy before this that hadn't worked, things like CBT, that get to a point with me where they'd realised that I was really avoidant and quite blocked. She finally said to me, 'I think you've got Asperger's.' I'd never considered it before, it was a real shock. And I went through a whole process of accepting it, denying it, being angry about it, before I got officially diagnosed.

Lynne Malcolm: Why do you think it was unnoticed?

Hannah Belcher: I think the main problem was that my symptoms weren't as obvious as they are in males. So things like eye contact I was perfectly okay with, I actually count out my eye contact, so it was never really a problem. And I think doctors just saw my anxiety and just saw that as the main diagnosis without really considering why is that there. I think they saw that as the challenging behaviour problem and not what was underlying it.

Lynne Malcolm: So, looking back, what were your symptoms?

Hannah Belcher: I had a lot of problems with anxiety, social anxiety in particular, talking to people. So when I started school I found that I just was too anxious to speak so I just couldn't form my words, I couldn't think of what to say to people, and it just became like a really big anxiety problem for me where the more people pressured me, the harder it was to speak. So I dropped out of school when I was 14 because that all became too much for me. But the ones I never really paid attention to, things like sensory problems, I'm quite sensitive to noise and colour and smells and things like that.

Lynne Malcolm: And you were also obsessed with music?

Hannah Belcher: Yes, I still am a little bit, in a kind of typical autistic obsessional away. I just listen to the same song over and over again, and I still do that now, if I get a song I like it's just on repeat constantly until I get sick of it.

Lynne Malcolm: And what about your relationship to food?

Hannah Belcher: I was a very picky eater. I think that was my main problem when I was a child. It had to be in a certain way, it had to be cut in a certain way, put on the plate in a certain way. There was only certain items I would eat and I would just eat them continually until I got sick of them and I'd move onto the next thing. I'm still like that a little bit now but slightly better.

Lynne Malcolm: And what did he think was going on for yourself before you were diagnosed?

Hannah Belcher: I thought it was anxiety problems as well. I never considered autism really. I studied it and I still had never considered that I could have autism because my view of autism was also quite male stereotypical.

Lynne Malcolm: Hannah Belcher thinks that girls and women with autism mask their symptoms because of the social pressure on them as they are growing up. They're taught to be polite, and socially adaptable and they become very good at what she calls social mimicking. She's so passionate about this topic that she's doing a PhD in psychology at Anglia Ruskin University in Cambridge. She's conducting an online survey to improve the diagnosis for females with ASD.

Hannah Belcher: So my research is with Dr Steven Stagg, and we're looking at the number of females that have been undiagnosed with autism possibly, and also looking at if their social mimicking has anything to do with this, whether those females that are better at socially mimicking are being hidden more, and what role that has to play with misdiagnosis as well with other mental health conditions.

Lynne Malcolm: And what are the results that you're seeing?

Hannah Belcher: At the moment it's quite early stages, we haven't really got anything just yet. I've had a lot of females come forward, all saying the same thing, you know, 'I had this problem, I think I'm autistic, I socially mimic really well,' just loads of them seemed to have this same problem.

Lynne Malcolm: So do you think we need different diagnostic measures for testing autism in girls and boys?

Hannah Belcher: I think it's probably important to look at the social adaptions they are making alongside what we already have. So I think something like the social mimicking scale I've come up with, something like that to go alongside the diagnosis, to say, okay, this female is slightly below the threshold for diagnosis but she does score highly on social mimicking, so we have to take that into account, that she could be hiding a lot of her symptoms.

Lynne Malcolm: Hannah Belcher believes that females with ASD present differently because of socialisation.

However, there's growing suspicion that the underlying biology of autism in males and females may also be quite distinct.

Professor Francesca Happ at King's College London studies ASD using brain imaging studies and genetics.

Francesca Happ: I think there are probably both biological differences and social and cultural reasons why we may not be very good at picking up autism in girls. One of the explanations for the unbalanced ratio, why more girls might be affected, is what's called the female protective effect. So we've done some work that suggest that girls who have autism have been hit harder or have a larger genetic dose than boys with autism, and you can tell this because if you look at their brothers and sisters, the brothers and sisters of girls with autism are more likely to have autism or some traits of autism than the brothers and sisters of boys with autism. So that's suggesting there's a bigger genetic hit for those girls with autism than for boys with autism.

Lynne Malcolm: So from a biological perspective, why might females be protected against autism?

Francesca Happ: In general, males are more affected by all neurodevelopmental disorders. So there are more males than females with ADHD, attention deficit hyperactivity disorder, with dyslexia, with dyspraxia or clumsiness. It seems that the male developing foetus takes a longer time in a vulnerable stage and so may be more susceptible to all kinds of things. However, the ratio in autism is even higher than in those other cases. We don't know why exactly that is. Simon Baron-Cohen has a theory that it's about exposure to testosterone in utero, predisposing the male brain to the autistic pattern. That's a very interesting theory. There are other theories as well. The answer is at the moment we don't know.

Lynne Malcolm: So one theory I think you might have been referring to, it is called the extreme male brain theory. Tell me about that theory.

Francesca Happ: So Simon Baron-Cohen has the theory of the extreme male brain, which suggests that all of us have a balance between our ability to understand other people, which he calls empathising, and our ability to understand non-social systems, he calls systemising. And he says in a male brain the systemising is highly developed and the empathising is less good, and that autism can be thought of as a very extreme version of this where social understanding is really very problematic and poor, but understanding of non-social systems can be extremely good.

Lynne Malcolm: So what do you think of this theory?

Francesca Happ: I think it's interesting. I don't fully agree with it. I think that, for example, empathising that Simon Baron-Cohen talks about is actually made up of lots of different abilities, some of which are quite intact in autism, like the ability to empathise with somebody's emotional state or care if somebody is hurt. People with autism have no problem with that. And on the other hand knowing what somebody else is thinking, which is very difficult for people with autism. So I think empathising in Simon's theory really mushes together a whole lot of things that are quite distinct. And I think it's not clear in his theory whether empathising and systemising are in trade-off, so if you are good at one you have to be bad at the other or whether they are really separate dimensions, which some of our work would suggest the non-social assets in autism and the social difficulties in autism are really quite separable and distinct and actually have distinct genetic underpinnings.

Lynne Malcolm: Francesca Happ.

It's All in the Mind with me, Lynne Malcolm, on RN, Radio Australia and online.

We're hearing about the distinct experience of girls and women with autism spectrum disorder. Because they can present differently, they're often not recognised as having ASD, and can be misdiagnosed.

Donna Rigoni has two children, Ayla and Bailey, both with a diagnosis of ASD. Her daughter Ayla is now 5 years old.

Donna Rigoni: When she was about 2 I noticed lots of little things with the way she was playing. She would organise her toys, she wouldn't let anyone touch her toys, it had to be in a certain way. And then with her language she would repeat the same thing over and over. So if we were driving somewhere and she'd see a pattern on the wall she would repeat that every time we drove past that same place. So lots of repetitive information she was giving me.

Lynne Malcolm: So when you are noticing her behaviours, what made you think that they were part of the autism spectrum disorder?

Donna Rigoni: Well, I also have a son who was diagnosed before her, so just following things that he was doing as well, stuff like the sensory issues, walking on her tippy-toes, hands over her ears when there was a loud noise, so even vacuuming was hurtful for her. Things like dressing and washing was all just too traumatic for Ayla, she would adjust scream that I was hurting her every time I'd dry her and wash her. So just putting the play, the language, the sensory problems all together, and it just screamed ASD to me.

Lynne Malcolm: So you have a son with ASD as well?

Donna Rigoni: Yes.

Lynne Malcolm: And what are the differences in the way they behave and the way they present?

Donna Rigoni: It's huge. Ayla's a lot calmer and she talks to me more about her feelings, like she'll say to me, 'Mum, what does that mean? Mum, why is that person looking like that?' Meaning their face expressions. She shuts down when she is overwhelmed, whereas my son will have a meltdown when he is overwhelmed. So he is very loud. He's a bit more angry, sounds a bit more abrupt than her. And even her interests, for my son it's technology, downloads, anything to do with wi-fi and technology interests him, where her interest is animals, horses in particular, dogs, it just seems an interest that more children tend to go to than to what my son's interest was or is. But definitely Bailey was easier to pick.

Lynne Malcolm: Donna Rigoni.

Clinical psychologist Janine Manjiviona diagnosed Donna's daughter Ayla with ASD. She emphasises how important it is to gather comprehensive information from multiple contexts when they're being assessed, to prevent girls from slipping under the radar.

She says that there are a number of other conditions which can overlap with ASD, and girls are most often diagnosed when they hit puberty.

Janine Manjiviona: Well, I think it's at that time when their difficulties become more obvious. Often they are on the periphery of social groups. The girls have often learned their social skills by intellect, not necessarily intuitively or instinctively. Teenage girls are very socially demanding on each other, and the girls with ASD can be marginalised, teased and bullied. And up until then the difficulties may be masked due to a veneer of coping.

Lynne Malcolm: There's discussion in the literature, and you've seen this too in your practice I think, that there is a crossover between the symptoms of ASD and eating disorders such as anorexia nervosa with girls. Can you tell us about that?

Janine Manjiviona: Yes, anorexia is one of the conditions that overlaps with ASD. It's not the only condition and there's lots of comorbidity issues with ASD, but controlling weight can be a way of fitting in, looking right, it can be part of an attempt to gain social acceptance. And I guess another reason might be that when girls hit puberty, many of them have trouble coping with the changes in their body and feelings, menstruation, breast development and so on, and they can develop anorexia as a way of coping. They don't like highlighting their femaleness, and many of them prefer to have a somewhat androgynous look.

They've told me too in my clinical discussions with them that they don't really want to grow up. 'I don't want to grow up,' they say, 'I don't want to have children, I just want to stay home with mum and dad and my brothers and sisters.' So I think it's related in part to that whole issue of change and anxiety.

Lynne Malcolm: Psychologist Janine Manjiviona offers therapy to girls and women with ASD to help them deal with their concept of themselves and the anxiety associated with that.

She also referred Donna Rigoni and her five-year-old daughter Ayla to a therapy program called Social Thinking at Spectrum Services in Melbourne. It's designed to help prevent anxiety developing later.

Donna Rigoni: So she's in a small group of three children which are at the similar level to her on the spectrum. She has been taught things like whole-body listening, so how to listen properly, so learning to stay still, looking at the person who is talking so they know that you are listening. Also we are working on having Ayla's body in the group. She knows that if she is sitting with the group, other people in the group know that she is listening and willing to contribute. If her body is out of the group than the other children in the group will think, oh, Ayla is not really wanting to contribute. So she has used that at school this year. She actually says to her teacher, 'I'm putting my body in the group.' So she is using a lot of the language that she is being taught.

A big one that Ayla's doing at the moment is learning a strategy called the group plan, and what it is is she has to follow what everyone else is doing within the group. So this shows that they are thinking of each other, that Ayla understands that if she is following the group plan, everyone is happy, and they can work together constructively. If she is not in the group plan and wants to do her own thing, well, it might make others unhappy. And this has really helped with things like birthday parties.

We went to a birthday party recently and it was something that Ayla was a little bit stressed about the night before. But the lady in charge of the party explained what they were doing, and I said to Ayla, 'Don't worry Ayla, just follow the group plan.' She said, 'Mum, I know, I'm following the group plan, it's all good.' So she was able to use that in her everyday life. So that has really helped.

She has also learned a phrase called 'words are bumping', and so she knows that if someone is trying to talk on top of her or interrupt, she'll say, 'Hang on, stop, words are bumping, I need to finish what I'm saying.' So this has all been from the speech therapy that she's doing.

We're also doing a lot on thinking thoughts and feeling feelings. So Ayla has been taught that you think with your brain and you feel with your heart. She understands that people have thinking bubbles above their brain. She knows you can't see it but she knows that if they are looking at something, they would probably be thinking about that thing, and that has been really helpful because there are days where Ayla has upset me, and I'll say to her, 'You're hurting my heart, Ayla.' And she'll go, 'Oh, okay.' And so she gets that.

And there'll be times when she'll come home and she'll pick up something and say, 'Mum, what am I thinking about?' And she'll hold something and look at it really intensely, and I'll go, 'You're thinking about having a drink.' And she goes, 'Yeah, that's right!' And then she'll make me look at something and say, 'What are you thinking about?' And we play games like that. So all of that stuff has been an incredible help for Ayla, which is stuff that my son didn't have because he was diagnosed later. So I think the earlier girls are diagnosed, you can give them so much more help. And what I'm hoping is by the time Ayla gets to puberty we will be prepared. So it won't be so stressful, and hopefully she won't be so anxious.

Lynne Malcolm: Donna Rigoni.

Hannah Belcher wasn't diagnosed with ASD until she was 23 years old, and she feels it's really important to get the right diagnosis to prevent inappropriate treatment.

Hannah Belcher: Things like depression, anxiety, OCD and borderline personality disorder are really common misdiagnoses for females with autism, and people don't tend to look at the root of them. So with OCD I say that someone with OCD may have to turn a light switch on and off a certain number of times because they feel like something bad will happen, whereas someone with autism may do that and it's quite a soothing mechanism. And I think with girls in particular this has been misconstrued as a different disorder overall. I think that's the main problem with the misdiagnosis.

Lynne Malcolm: Does that apply to you and behaviours that you display?

Hannah Belcher: I certainly had some OCD traits when I was younger, hand washing in particular, and I do things now that are quite obsessive, movements and things like that, which are more for me soothing than they are obsessive. I don't feel like something bad will happen if I don't clink my fingers a certain number of times, it's a soothing kind of stimming mechanism for me.

Lynne Malcolm: It just makes you feel better?

Hannah Belcher: Yes, which certainly if my psychiatrist saw me doing it he'd think, okay, OCD, but

Lynne Malcolm: Even your psychiatrist doesn't really understand that?

Hannah Belcher: I think it's a general problem with psychiatrists, that they aren't given a thorough training on autism. So the view they have is also quite stereotypically male, so it's still a challenge getting through to the doctors that there is a difference.

Lynne Malcolm: What would you like to see in the future? Where are you going to take your research?

Hannah Belcher: I want to see better diagnosis for females overall, I want to see more females being picked up earlier in their lives when they can get the support at school that they need, and just more awareness about the issue. I had an article out recently and I got accused of sexism and all sorts because the issue is so unknown and so unheard of that it's quite difficult to get the message out there. Certainly I think it's a definite gap in our understanding of autism that needs to be addressed.

Lynne Malcolm: Hannah Belcher.

Francesca Happ: Certainly for the families where there isn't a diagnosis of autism in particular, if your daughter has lifelong social and communication problems, if they don't get on with others in the way that you would expect and they find it hard to know if other people are joking or being sarcastic or telling a lie, if they are socially vulnerable, then you might think about whether this is autism, even though it doesn't fit the stereotype of autism, it doesn't look like Rain Man or one of the portrayals of male autism in the media.

Lynne Malcolm: And Francesca Happ suggests that further study into the gender differences seen in autism may help our overall knowledge of the autism spectrum disorder.

Francesca Happ: I think a lot of researchers believe that if we understood why more males than females are affected with autism we would have a better understanding potentially of either the genetic or environmental influences on autism. So, for example, if Simon Baron-Cohen is right and it's to do with foetal testosterone in utero, then we might have a handle on the mechanisms of brain development that are different in autism. And I suppose the point of all of the genetic work that is going on around the world is to understand the mechanisms in order to improve outcome, to make outcome for people with autism the very best that it can be.

Lynne Malcolm: In the course of her work Francesca Happ has formed friendships with many women with autism and she's struck by the diversity of their experience.

Francesca Happ: Some of my friends with autism will say, 'If I could take a pill tomorrow and wake up without autism I would do it instantly because my autism makes me so frightened of the world and so unable to go out and do the things I want to do that I hate having autism.' And then I have other friends, women with autism, who say, 'Autism is who I am and what I am and I wouldn't change it. It is me.'

Interestingly I have some friends with autism who have transitioned genders from being a woman to being a man and feel much, much more comfortable as a man, which is interesting, and I learn a lot from talking to them. And other women with autism who go and speak about their experiences and are such gifted orators, so funny, so poised on stage, that people really doubt that they even have autism. But then if you could see that same woman trembling at the thought of having to buy a ticket at the train station or having to brush past a dog in the railway carriage to get to her seat, you see this ability of some women with autism to mask their difficulties. But how much it affects their everyday life and how brave they are to struggle to live in our neurotypical world.

Lynne Malcolm: Francesca Happ, Professor of Cognitive Neuroscience at King's College London.

Further information related to today's program can be found on the All in the Mind website, it's easy to navigate your way there from the RN home page.

Thanks to producer Diane Dean and sound engineer Luke Purse. I'm Lynne Malcolm. Great to have your company, see you next week.

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Girls and Autism: One of Lynne Malcolm's favourite programs - ABC News

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This article was originally published here

Stem Cell Res Ther. 2021 Jan 13;12(1):58. doi: 10.1186/s13287-020-02110-x.

ABSTRACT

INTRODUCTION: Critical limb ischemia (CLI) is the most advanced form of peripheral arterial disease (PAD) characterized by ischemic rest pain and non-healing ulcers. Currently, the standard therapy for CLI is the surgical reconstruction and endovascular therapy or limb amputation for patients with no treatment options. Neovasculogenesis induced by mesenchymal stem cells (MSCs) therapy is a promising approach to improve CLI. Owing to their angiogenic and immunomodulatory potential, MSCs are perfect candidates for the treatment of CLI. The purpose of this study was to determine and compare the in vitro and in vivo effects of allogeneic bone marrow mesenchymal stem cells (BM-MSCs) and adipose tissue mesenchymal stem cells (AT-MSCs) on CLI treatment.

METHODS: For the first step, BM-MSCs and AT-MSCs were isolated and characterized for the characteristic MSC phenotypes. Then, femoral artery ligation and total excision of the femoral artery were performed on C57BL/6 mice to create a CLI model. The cells were evaluated for their in vitro and in vivo biological characteristics for CLI cell therapy. In order to determine these characteristics, the following tests were performed: morphology, flow cytometry, differentiation to osteocyte and adipocyte, wound healing assay, and behavioral tests including Tarlov, Ischemia, Modified ischemia, Function and the grade of limb necrosis scores, donor cell survival assay, and histological analysis.

RESULTS: Our cellular and functional tests indicated that during 28 days after cell transplantation, BM-MSCs had a great effect on endothelial cell migration, muscle restructure, functional improvements, and neovascularization in ischemic tissues compared with AT-MSCs and control groups.

CONCLUSIONS: Allogeneic BM-MSC transplantation resulted in a more effective recovery from critical limb ischemia compared to AT-MSCs transplantation. In fact, BM-MSC transplantation could be considered as a promising therapy for diseases with insufficient angiogenesis including hindlimb ischemia.

PMID:33436054 | DOI:10.1186/s13287-020-02110-x

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Comparative analysis of mouse bone marrow and adipose tissue mesenchymal stem cells for critical limb ischemia cell therapy - DocWire News

Recommendation and review posted by Bethany Smith

Brave youngster Evie Hodgson has finally undergone a life-saving bone marrow transplant after one last "twist in the tale" saw the delivery of the stem cells delayed.

Evie, eight, from Sleights near Whitby, began preparations for the transplant earlier this year and has now finally been given the "magic stem cells".

It's not been an easy journey for Evie and her family, who were devastated in August 2020 when a potential donor pulled out at the last minute, and they faced another sudden bump in the road prior to her receiving her first round of treatment today (Friday).

The operation for the eight-year-old was scheduled for 2pm yesterday (Thursday) at The Great Northern Children's Hospital in Newcastle but the cells got stuck in London after coronavirus "caused major issues to the flight schedule".

It meant that Evie and her family had to wait another day for the operation to go ahead, but her mother Tina gave an update today to say that they were "up and running" after what had been an "emotional experience".

She said earlier today: "We have fought so hard to get to this point and Evie is so happy. It really is wonderful.

"Evie's hero donated a phenomenal amount of stem cells so she gets two sittings. The second one will be around dinner time so she gets to do it all again."

The courageous pupil at Fyling Hall School, in Robin Hood's Bay, was diagnosed with aplastic anaemia, also known as bone marrow failure, last year during the start of the covid-19 pandemic.

The youngster captured the attention of the nation when her perfect donor pulled out at the last minute and her transplant hopes were dashed.

But Evie revealed it was " the best Christmas present ever" to find another donor in December.

The family have thanked everybody who has supported them, shared Evie's story and signed up to the stem cell register following their inspirational campaign.

You can join on the DKMS register, here, or through Anthony Nolan register, here.

A dedicated Facebook page has been set up to follow Evie's journey with aplastic anaemia, which you can follow here.

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Brave Evie Hodgson from Sleights finally has bone marrow transplant after one last 'twist in the tale' - Yorkshire Live

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Abstract

Terminally differentiated murine osteocytes and adipocytes can be reprogrammed using platelet-derived growth factorAB and 5-azacytidine into multipotent stem cells with stromal cell characteristics. We have now optimized culture conditions to reprogram human adipocytes into induced multipotent stem (iMS) cells and characterized their molecular and functional properties. Although the basal transcriptomes of adipocyte-derived iMS cells and adipose tissuederived mesenchymal stem cells were similar, there were changes in histone modifications and CpG methylation at cis-regulatory regions consistent with an epigenetic landscape that was primed for tissue development and differentiation. In a non-specific tissue injury xenograft model, iMS cells contributed directly to muscle, bone, cartilage, and blood vessels, with no evidence of teratogenic potential. In a cardiotoxin muscle injury model, iMS cells contributed specifically to satellite cells and myofibers without ectopic tissue formation. Together, human adipocytederived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth.

The goal of regenerative medicine is to restore function by reconstituting dysfunctional tissues. Most tissues have a reservoir of tissue-resident stem cells with restricted cell fates suited to the regeneration of the tissue in which they reside (14). The innate regenerative capacity of a tissue is broadly related to the basal rate of tissue turnover, the health of resident stem cells, and the hostility of the local environment. Bone marrow transplants and tissue grafts are frequently used in clinical practice but for most tissues, harvesting and expanding stem and progenitor cells are currently not a viable option (5, 6). Given these constraints, research efforts have been focused on converting terminally differentiated cells into pluripotent or lineage-restricted stem cells (7, 8). However, tissues are often a complex mix of diverse cell types that are derived from distinct stem cells. Therefore, multipotent stem cells may have advantages over tissue-specific stem cells. To be of use in regenerative medicine, these cells would need to respond appropriately to regional cues and participate in context-dependent tissue regeneration without forming ectopic tissues or teratomas. Mesenchymal stem cells (MSCs) were thought to have some of these characteristics (911), but despite numerous ongoing clinical trials, evidence for their direct contribution to new tissue formation in humans is sparse, either due to the lack of sufficient means to trace cell fate in hosts in vivo or failure of these cells to regenerate tissues (12, 13).

We previously reported a method by which primary terminally differentiated somatic cells could be converted into multipotent stem cells, which we termed as induced multipotent stem (iMS) cells (14). These cells were generated by transiently culturing primary mouse osteocytes in medium supplemented with azacitidine (AZA; 2 days) and platelet-derived growth factorAB (PDGF-AB; 8 days). Although the precise mechanisms by which these agents promoted cell conversion was unclear, the net effect was reduced DNA methylation at the OCT4 promoter and reexpression of pluripotency factors (OCT4, KLF4, SOX2, c-MYC, SSEA-1, and NANOG) in 2 to 4% of treated osteocytes. iMS cells resembled MSCs with comparable morphology, cell surface phenotype, colony-forming unit fibroblast (CFU-F), long-term growth, clonogenicity, and multilineage in vitro differentiation potential. iMS cells also contributed directly to in vivo tissue regeneration and did so in a context-dependent manner without forming teratomas. In proof-of-principle experiments, we also showed that primary mouse and human adipocytes could be converted into long-term repopulating CFU-Fs by this method using a suitably modified protocol (14).

AZA, one of the agents used in this protocol, is a cytidine nucleoside analog and a DNA hypomethylating agent that is routinely used in clinical practice for patients with higher-risk myelodysplastic syndrome (MDS) and for elderly patients with acute myeloid leukemia (AML) who are intolerant to intensive chemotherapy (15, 16). AZA is incorporated primarily into RNA, disrupting transcription and protein synthesis. However, 10 to 35% of drug is incorporated into DNA resulting in the entrapment and depletion of DNA methyltransferases and suppression of DNA methylation (17). Although the relationship between DNA hypomethylation and therapeutic efficacy in MDS/AML is unclear, AZA is known to induce an interferon response and apoptosis in proliferating cells (1820). PDGF-AB, the other critical reprogramming agent, is one of five PDGF isoforms (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD), which bind to one of two PDGF receptors (PDGFR and PDGFR) (21). PDGF isoforms are potent mitogens for mesenchymal cells, and recombinant human (rh)PDGF-BB is used as an osteoinductive agent in the clinic (22). PDGF-AB binds preferentially to PDGFR and induces PDGFR- homodimers or PDGFR- heterodimers. These are activated by autophosphorylation to create docking sites for a variety of downstream signaling molecules (23). Although we have previously demonstrated induction of CFU-Fs from human adipocytes using PDGF-AB/AZA (14), the molecular changes, which underlie conversion, and the multilineage differentiation potential and in vivo regenerative capacity of the converted cells have not been determined.

Here, we report an optimized PDGF-AB/AZA treatment protocol that was used to convert primary human adipocytes, a tissue source that is easily accessible and requires minimal manipulation, from adult donors aged 27 to 66 years into iMS cells with long-term repopulating capacity and multilineage differentiation potential. We also report the molecular landscape of these human iMS cells along with that of MSCs derived from matched adipose tissues and the comparative in vivo regenerative and teratogenic potential of these cells in mouse xenograft models.

Primary mature human adipocytes were harvested from subcutaneous fat (Fig. 1A and table S1) and their purity confirmed by flow cytometry with specific attention to the absence of contaminating adipose-derived MSCs (AdMSCs) (fig. S1, A and B). As previously described (14), plastic adherent adipocytes were cultured in Alpha Minimum Essential Medium (MEM) containing rhPDGF-AB (200 ng/ml) and 20% autologous serum (AS) with and without 10 M AZA for 2 and 23 days, respectively (Fig. 1A). During daily observations, unilocular lipid globules were observed to fragment within adipocytes ~day 10 with progressive extrusion of fat into culture medium, coincident with changes in cell morphology (movie S1). Consistent with these observations, when fixed and stained with Oil Red O, adipocytes that were globular in shape at the start of culture resembled lipid laden stromal cells at day 12 and lipid-free stromal cells at day 25 (Fig. 1B).

(A) Generation and reprogramming of adipocytes. (B) Oil Red Ostained adipocytes (days 0, 12, and 25) during treatment with recombinant human platelet-derived growth factorAB (rhPDGF-AB) and AZA. (C) Flow cytometry plots of LipidTOX and PDGFR in adipocytes cultured as in (A). (D) CFU-F counts from treated and untreated adipocytes during conversion. (E) CFU-F counts from adipocytes treated (Rx) with indicated combinations of rhPDGF-AB, AZA, fetal calf serum (FCS), autologous serum (AS), or serum-free media (SFM). (F) CFU-F counts from adipocytes reprogrammed in the presence of 0, 1, or 10 M PDGFR/ inhibitor AG1296. (G) CFU-F counts per 400 reprogrammed adipocytes from three donor age groups (n = 3 for each) generated using indicated combinations of rhPDGF-AB and AZA. (H) Long-term growth of reprogrammed adipocytes from three donor age groups (n = 3 for each) generated using indicated combinations of rhPDGF-AB and AZA. (I) Long-term growth of iMS cells cultured in SFM or media supplemented with FCS, autologous, or allogeneic serum. Error bars indicate SD, n = 3; *P < 0.05, **P < 0.01, and ***P < 0.0001 calculated using either a Students t test (E and F) or a linear mixed model (H). Photo credit: Avani Yeola, UNSW Sydney.

To evaluate these changes in individual cells, we performed flow cytometry at multiple time points during treatment and probed for adipocyte (LipidTOX) (24) and stromal cell characteristics [PDGFR expression (25); Fig. 1C]. A subpopulation of adipocytes, when cultured in media supplemented with PDGF-AB/AZA and AS (Fig. 1C, top; treated), showed reduced LipidTOX staining intensity at day 10, with progressive reduction and complete absence in all cells by day 19. Adipocytes cultured in the absence of PDGF-AB/AZA retained LipidTOX staining, albeit with reduced intensity (Fig. 1C, bottom; untreated). Adipocytes expressed PDGFR [fig. S1C, (i) and (ii)] but not PDGFR (Fig. 1C) at day 0 but both the frequency and intensity of PDGFR staining increased from day 21. To record these changes in real time, we also continuously live-imaged treated adipocytes from days 15 to 25 and recorded the extrusion of fat globules, change in cell morphology from globular to stromal, and acquisition of cell motility and cell mitosis (movie S1 and fig. S1D). Intracellular fragmentation of fat globules was observed over time in untreated adipocytes (fig. S1E), consistent with variable LipidTOX staining intensity. CFU-F capacity was absent at day 10, present in day 15 cultures, and tripled by day 19 with no substantial increase at days 21, 23, and 25 (Fig. 1D). It is noteworthy that CFU-F potential was acquired before PDGFRA surface expression when adipocytes had started to display stromal cell morphology and had diminished fat content. There was also no CFU-F capacity in adipocytes cultured in MEM with fetal calf serum (FCS) or AS, unless supplemented with both PDGF-AB and AZA. CFU-F capacity was significantly higher with AS than with FCS and absent in serum-free media (SFM) (Fig. 1E and fig. S1F). As previously shown with reprogramming of murine osteocytes, there was dose-dependent inhibition of CFU-F capacity when AG1296, a potent nonselective PDGF receptor tyrosine kinase inhibitor (26), was added to the reprogramming media (Fig. 1F).

To evaluate the impact of patient age and concentrations of PDGF-AB and AZA on the efficiency of human adipocyte conversion, we harvested subcutaneous fat from donors aged 40 (n = 3), 41 to 60 (n = 3), and 61 (n = 3) years and subjected each to three different concentrations of PDGF-AB (100, 200, and 400 ng/ml) and three different concentrations of AZA (5, 10, and 20 M) (Fig. 1G). Although all combinations supported cell conversion in all donors across the three age groups, rhPDGF-AB (400 ng/ml) and 5 M AZA yielded the highest number of CFU-Fs (Fig. 1G). When these cultures were serially passaged in SFM (with no PDGF-AB/AZA supplementation, which was used for cell conversion only), adipocytes converted with reprogramming media containing rhPDGF-AB (400 ng/ml) and 5 M AZA were sustained the longest (Fig. 1H, fig. S2A, and table S2). The growth plateau that was observed even with these cultures [i.e., adipocytes converted with rhPDGF-AB (400 ng/ml) and 5 M AZA when expanded in SFM or FCS] was overcome when cells were expanded in either autologous or allogeneic human serum (Fig. 1I). The genetic stability of human iMS cells (RM0072 and RM0073) was also assessed using single-nucleotide polymorphism arrays and shown to have a normal copy number profile at a resolution of 250 kb (fig. S2B). Together, these data identify an optimized protocol for converting human primary adipocytes from donors across different age groups and show that these can be maintained long term in culture.

Given the stromal characteristics observed in human adipocytes treated with PDGF-AB/AZA (Fig. 1), we performed flow cytometry to evaluate their expression of MSC markers CD73, CD90, CD105, and STRO1 (13) and noted expression levels comparable to AdMSCs extracted from the same subcutaneous fat harvest (Fig. 2A). Primary untreated adipocytes (day 25 in culture) did not express any of these MSC markers (fig. S3A). The global transcriptomes of iMS cells and matched AdMSCs were distinct from untreated control adipocytes but were broadly related to each other [Fig. 2B, (i) and (ii)]. Ingenuity pathway analysis (IPA) using genes that were differentially expressed between AdMSCs versus adipocytes [3307 UP/4351 DOWN in AdMSCs versus adipocytes; false discovery rate (FDR) 0.05] and iMS versus adipocytes (3311 UP/4400 DOWN in iMS versus adipocytes; FDR 0.05) showed changes associated with gene expression, posttranslational modification, and cell survival pathways and organismal survival and systems development [Fig. 2B(iii)]. The number of differentially expressed genes between iMS cells and AdMSCs was limited (2 UP/26 DOWN in iMS versus AdMSCs; FDR 0.05) and too few for confident IPA annotation. All differentially expressed genes and IPA annotations are shown in table S3 (A to E, respectively).

(A) Flow cytometry for stromal markers on AdMSCs (green) and iMS cells (purple) from matched donors. Gray, unstained controls. (B) (i) Principal components analysis (PCA) plot of adipocyte, AdMSC, and iMS transcriptomes. (ii) Hierarchical clustering of differentially expressed genes (DEGs, FDR 0.05). (iii) Ingenuity pathway analysis (IPA) of DEG between AdMSCs/adipocytes (top) or iMS cells/adipocytes (bottom). The most enriched annotated biological functions are shown. (C) (i) Chromatin immunoprecipitation sequencing (ChIP-seq) profiles in AdMSCs and iMS cells from matched donors at a representative locus. Gray bar indicates differential enrichment. (ii) Volcano plots of H3K4me3, H3K27Ac, and H3K27me3 enrichment peaks significantly UP (red) or DOWN (blue) in iMS cells versus AdMSCs. (iii) IPA of corresponding genes. log2FC, log2 fold change. (D) (i) DNA methylation at a representative locus in AdMSCs and iMS cells from matched donors. (ii) Volcano plot of regions with significantly higher (red) or lower (blue) DNA methylation in iMS cells versus AdMSCs. (iii) IPA using genes corresponding to differentially methylated regions (DMRs). (E) OCT4, NANOG, and SOX2 expression in iPS, AdMSCs, and iMS cells. Percentage of cells expressing each protein is indicated. DAPI, 4,6-diamidino-2-phenylindole. (F) AdMSCs and iMS cells differentiated in vitro. Bar graphs quantify staining frequencies, error bars show SD, n = 3. ***P < 0.001 (Students t test). Photo credit: Avani Yeola, UNSW Sydney.

In the absence of significant basal differences in the transcriptomes of AdMSCs and iMS cells, and the use of a hypomethylating agent to induce adipocyte conversion into iMS cells, we examined global enrichment profiles of histone marks associated with transcriptionally active (H3K4me3 and H3K27Ac) and inactive (H3K27me3) chromatin. There were differences in enrichment of specific histone marks in matched AdMSCs versus iMS cells at gene promoters and distal regulatory regions [Fig. 2C(i) and fig. S3, B to D]. H3K4me3, H3K27ac, and H3K27me3 enrichments were significantly higher at 255, 107, and 549 regions and significantly lower at 222, 78, and 98 regions in iMS cells versus AdMSCs [Fig. 2C(ii) and table S4, A to C] and were assigned to 237, 84, and 350 and 191, 58, and 67 genes, respectively. IPA was performed using these gene lists to identify biological functions that may be primed in iMS cells relative to AdMSCs [Fig. 2C(iii) and table S4, D to F]. Among these biological functions, annotations for molecular and cellular function (cellular movement, development, growth, and proliferation) and systems development (general; embryonic and tissue development and specific; cardiovascular, skeletal and muscular, and hematological) featured strongly and overlapped across the different epigenetic marks.

We extended these analyses to also assess global CpG methylation in matched AdMSCs and iMS cells using reduced representation bisulfite sequencing [RRBS; (27)]. Again, there were loci with differentially methylated regions (DMRs) in iMS cells versus AdMSCs [Fig. 2D(i)] with increased methylation at 158 and reduced methylation at 397 regions among all regions assessed [Fig. 2D(ii) and table S4G]. IPA of genes associated with these DMRs showed a notable overlap in annotated biological functions [Fig. 2D(iii) and table S4H] with those associated with differential H3K4me3, H3K27Ac, and H3K27me3 enrichment [Fig. 2C(iii) and table S4, E to G]. Together, these data imply that although basal transcriptomic differences between iMS cells and AdMSCs were limited, there were notable differences in epigenetic profiles at cis-regulatory regions of genes that were associated with cellular growth and systems development.

We next compared iMS cells to adipocytes from which they were derived. Expression of genes associated with adipogenesis was depleted in iMS cells (fig. S4A and table S4I). The promoter regions of these genes in iMS cells had broadly retained an active histone mark (H3K4me3), but, in contrast with adipocytes, many had acquired an inactive mark (H3K27me3) (fig. S4B and table S4J). However, there were examples where iMS cells had lost active histone marks (H3K4me3 and H3K27ac) at gene promoters and potential regulatory regions and gained repressive H3K27me3 [e.g., ADIPOQ; fig. S4C(i)]. In contrast, stromal genes had acquired active histone marks and lost repressive H3K27me3 [e.g. EPH2A; fig. S4C(ii)]. It is noteworthy that promoter regions of genes associated with muscle and pericytes (table S4K) were enriched for active histone marks in iMS cells compared with adipocytes [fig. S4D, (i) and (ii)]. We also compared demethylated CpGs in iMS cells and adipocytes (fig. S4E). There were 7366 sites in 2971 genes that were hypomethylated in iMS cells, of which 236 showed increased expression and were enriched for genes associated with tissue development and cellular growth and proliferation (fig. S4E).

PDGF-AB/AZAtreated murine osteocytes (murine iMS cells), but not bone-derived MSCs, expressed pluripotency associated genes, which were detectable by immunohistochemistry in 1 to 4% of cells (14). To evaluate expression in reprogrammed human cells, PDGF-AB/AZAtreated human adipocytes and matched AdMSCs were stained for OCT4, NANOG, and SOX2 with expression noted in 2, 0.5, and 3.5% of iMS cells respectively, but no expression was detected in AdMSCs (Fig. 2E). In addition to these transcription factors, we also evaluated surface expression of TRA-1-60 and SSEA4. Both proteins were uniformly expressed on iPSCs and absent in AdMSCs [fig. S4F(i)] and adipocytes [fig. S4F(ii)]. Although TRA-1-60 was absent in iMS cells, most (78%) expressed SSEA4 but rarely (<1%) coexpressed OCT4 and NANOG [fig. S4F(i)].

MSCs can be induced to differentiate in vitro into various cell lineages in response to specific cytokines and culture conditions. To evaluate the in vitro plasticity of human iMS cells, we induced their differentiation along with matched AdMSCs and primary adipocytes, into bone, fat, and cartilage, as well as into other mesodermal Matrigel tube-forming assays for endothelial cells (CD31) and pericytes (PDGFR) and muscle (MYH, myosin heavy chain; SMA, smooth muscle actin), endodermal (hepatocyte; HNF4, hepatocyte nuclear factor ), and neuroectodermal (TUJ1; neuron specific class III beta tubulin) lineages (Fig. 2F and fig. S4G). Whereas primary adipocytes remained as such and were resistant to transdifferentiation, iMS cells and AdMSCs showed comparable differentiation potential with the notable exception that only iMS cells generated pericyte-lined endothelial tubes in Matrigel. In keeping with these findings, relative to AdMSCs, iMS cells showed permissive epigenetic marks at pericyte genes [increased H3K4me3 and H3K27Ac; EPHA2 and MCAM; fig. S4H(i); and reduced CpG methylation; NOTCH1, SMAD7, TIMP2, AKT1, and VWF; fig. S4H(ii)]. Together with the notable differences in epigenetic profiles, these functional differences and low-level expression of pluripotency genes in iMS cell subsets suggested that these cells could be more amenable than matched AdMSCs to respond to developmental cues in vivo.

To evaluate spontaneous teratoma formation and in vivo plasticity of iMS cells, we tagged these cells and their matched AdMSCs with a dual lentiviral reporter, LeGO-iG2-Luc2 (28), that expresses both green fluorescent protein (GFP) and luciferase under the control of the cytomegalovirus promoter (Fig. 3A). To test teratoma-initiating capacity, we implanted tagged cells under the right kidney capsules of NOD Scid Gamma (NSG) mice (n = 3 per treatment group) after confirming luciferase/GFP expression in cells in culture (fig. S5, A and B). Weekly bioluminescence imaging (BLI) confirmed retention of cells in situ [Fig. 3B(i)] with progressive reduction in signal over time [Fig. 3B(ii)] and the absence of teratomas in kidneys injected with either AdMSCs or iMS cells [Fig. 3B(iii)]. Injection of equivalent numbers of iPS cells and iPS + iMS cell mixtures (1:49) to approximate iMS fraction expressing pluripotency markers led to spontaneous tumor formation in the same timeframe [Fig. 3B(iii)].

(A) Generation of luciferase/GFP-reporter AdMSCs and iMS cells, and assessment of their in vivo function. (B) Assessment of teratoma initiating capacity; (i) bioluminescence images at 0, 2, 6, and 8 weeks after implantation of 1 106 matched AdMSCs and iMS cells (P2; RM0057; n = 2 per group) under the right kidney capsules. (ii) Quantification of bioluminescence. (iii) Gross kidney morphology 8 weeks following subcapsular implantation of cells (R) or vehicle control (L). (C) Assessment of in vivo plasticity in a posterior-lateral intertransverse lumbar fusion model; (i) bioluminescence images following lumbar implantation of 1 106 matched AdMSCs or iMS cells (P2; RM0038; n = 3 per group) at 1 and 365 days after transplant. (ii) Quantification of bioluminescence. (iii) Tissues (bone, cartilage, muscle, and blood vessels) harvested at 6 months after implantation stained with (left) hematoxylin and eosin or (right) lineage-specific anti-human antibodies circles/arrows indicate regions covering GFP and lineage markerpositive cells. Corresponding graphs show donor cell (GFP+) contributions to bone, cartilage, muscle, and blood vessels as a fraction of total (DAPI+) cells in four to five serial tissue sections. Bars indicate confidence interval, n = 3. Photo Credit: Avani Yeola, UNSW Sydney.

To evaluate whether iMS cells survived and integrated with damaged tissues in vivo, we implanted transduced human iMS cells and matched AdMSCs controls into a posterior-lateral intertransverse lumbar fusion mouse model (Fig. 3A) (29). Cells were loaded into Helistat collagen sponges 24 hours before implantation into the posterior-lateral gutters adjacent to decorticated lumbar vertebrae of NSG mice (n = 9 iMS and n = 9 AdMSC). Cell retention in situ was confirmed by intraperitoneal injection of d-luciferin (150 mg/ml) followed by BLI 24 hours after cell implantation, then weekly for the first 6 weeks and monthly up to 12 months from implantation [Fig. 3C(i)]. The BLI signal gradually decreased with time but persisted at the site of implantation at 12 months, the final assessment time point [Fig. 3C(ii)]. Groups of mice (n = 3 iMS and n = 3 AdMSC) were euthanized at 3, 6, and 12 months and tissues harvested from sites of cell implantation for histology and immunohistochemistry [Fig. 3C(iii)]. Although implanted iMS cells and AdMSCs were present and viable at sites of implantation at 3 months, there was no evidence of lineage-specific gene expression in donor human cells (fig. S5C). By contrast, at 6 months after implantation, GFP+ donor iMS cells and AdMSCs were shown to contribute to new bone (BMP2), cartilage (SOX9), muscle (MYH), and endothelium (CD31) at these sites of tissue injury [Fig. 3C(iii)]. The proportion of donor cells expressing lineage-specific markers in a corresponding tissue section was significantly higher in iMS cells compared with matched AdMSCs at 6 months [Fig. 3C(iii) and table S2] as well as 12 months (fig. S5, E and D, and table S2). There was no evidence of malignant growth in any of the tissue sections or evidence of circulating implanted GFP+ iMS cells or AdMSCs (fig. S5E). Together, these data show that implanted iMS cells were not teratogenic, were retained long term at sites of implantation, and contributed to regenerating tissues in a context-dependent manner with greater efficiency than matched AdMSCs.

Although appropriate to assess in vivo plasticity and teratogenicity of implanted cells, the posterior-lateral intertransverse lumber fusion mouse model is not suited to address the question of tissue-specific differentiation and repair in vivo. To this end, we used a muscle injury model (30) where necrosis was induced by injecting 10 M cardiotoxin (CTX) into the left tibialis anterior (TA) muscle of 3-month-old female severe combined immunodeficient (SCID)/Beige mice. CTX is a myonecrotic agent that spares muscle satellite cells and is amenable to the study of skeletal muscle regeneration. At 24 hours after injury, Matrigel mixed with either 1 106 iMS cells or matched AdMSCs (or no cells as a control) was injected into the damaged TA muscle. The left (injured) and right (uninjured control) TA muscles were harvested at 1, 2, or 4 weeks after injury to assess the ability of donor cells to survive and contribute to muscle regeneration without ectopic tissue formation (Fig. 4A; cohort A). Donor human iMS cells or AdMSCs compete with resident murine muscle satellite cells to regenerate muscle, and their regenerative capacity is expected to be handicapped not only by the species barrier but also by having to undergo muscle satellite cell commitment before productive myogenesis. Recognizing this, a cohort of mice was subject to a second CTX injection, 4 weeks from the first injury/cell implantation followed by TA muscle harvest 4 weeks later (Fig. 4A; cohort B).

(A) Generation of iMS and AdMSCs and their assessment in TA muscle injury model. (B) (i) Confocal images of TA muscle stained for human CD56+ satellite cells (red) and laminin basement membrane protein (green; mouse/human). Graph shows donor hCD56+ satellite cell fraction for each treatment group. (ii) Confocal images of TA muscle harvested at 4 weeks and stained for human spectrin (red) and laminin (green; mouse/human). For each treatment, the left panel shows a tile scan of the TA muscle and the right panel a high magnification confocal image. Graph shows contribution of mouse (M), human (H), or chimeric (C) myofibers in three to five serial TA muscle sections per mouse (n = 3 mice per treatment group). (C) Confocal images of TA muscle 4 weeks following re-injury with CTX, stained for human spectrin (red) and laminin (green; mouse/human). For each treatment, left panel shows a tile scan of the TA muscle, upper right panel a low-magnification image, and lower right panel a high magnification image of the area boxed above. Graph shows contribution of mouse (M), human (H), or chimeric (C) myofibers in three to five serial TA muscle sections per mouse (n = 3 mice per treatment group). Graph bars indicate confidence interval. *P < 0.05, **P < 0.01, and ***P < 0.001 (linear mixed model). Photo credit: Avani Yeola, UNSW Sydney.

In tissue sections harvested from cohort A, donor-derived muscle satellite cells (31) [hCD56 (Thermo Fisher Scientific, MA5-11563)+; red] were evident in muscles implanted with both iMS cells and AdMSCs at each time point but were most numerous at 2 weeks after implantation [Fig. 4B(i) and fig. S6A]. The frequency of hCD56+ cells relative to total satellite cells [sublaminar 4,6-diamidino-2-phenylindolepositive (DAPI+) cells] was quantified in three to five serial sections of TA muscles per mouse in each of three mice per treatment group and was noted to be higher following the implantation of iMS cells compared with AdMSCs at all time points [week 1, 5.6% versus 2.4%; week 2, 43.3% versus 18.2%; and week 4, 30.7% versus 14.6%; Fig. 4B(i), table S2, and fig. S6A]. Donor cell contribution to regenerating muscle fibers was also assessed by measuring human spectrin (32) costaining with mouse/human laminin [(33) at 4 weeks (Fig. 4B(ii)]. At least 1000 myofibers from three to five serial sections of TA muscles for each of three mice in each treatment group were scored for human [H; hSpectrin+ (full circumference); laminin+], murine (M; mouse; hSpectrin; laminin+), or mouse/human chimeric [C; hSpectrin+ (partial circumference); laminin+] myofibers. Although none of the myofibers seen in cross section appeared to be completely human (i.e., donor-derived), both iMS cells and AdMSCs contributed to chimeric myofibers [Fig. 4B(ii)]. iMS cell implants contributed to a substantially higher proportion of chimeric fibers than AdMSC implants (57.7% versus 30.7%; table S2). In cohort B, TA muscles were allowed to regenerate following the initial CTX injection/cell implantation, and re-injured 4 weeks later with a repeat CTX injection. In these mice, although total donor cell contributions to myofibers in TA muscles harvested 4 weeks after re-injury were comparable to that observed in cohort A, there were no myofibers that appeared to be completely human (Fig. 4C). There were substantially more human myofibers following iMS cell implants than with AdMSCs (9.7% versus 5.4%; table S2). There was no evidence of ectopic tissue formation in TA muscles following implantation of either iMS cells or AdMSCs in either cohort.

To assess the physiological properties of muscles regenerated with human myofibers, we performed tetanic force contractions in extensor digitorum longus (EDL) muscles following the schema shown in Fig. 4A. Tetanic forces evoked by electrical pulses of various stimulus frequencies were not significantly different between the experimental cohorts or between the experimental cohorts and control animals [fig. S6B, (i) to (iii)]. However, when challenged with a sustained train of electrical pulses [fig. S6C(i)], the iMS group demonstrated significantly greater absolute [fig. S6C(ii)] and specific [fig. S6C(iii)] forces over a 3- to 6-s period. Together, these data showed that iMS cells had the capacity to respond appropriately to the injured environment and contribute to tissue-specific regeneration without impeding function.

We have optimized a protocol, originally designed for mouse osteocytes, to convert human primary adipocytes into iMS cells. We show that these long-term repopulating cells regenerate tissues in vivo in a context-dependent manner without generating ectopic tissues or teratomas.

PDGF-AB, AZA, and serum are indispensable ingredients in reprograming media, but the underlying reasons for their cooperativity and the observed dose-response variability between patients are not known. PDGF-AB is reported to bind and signal via PDGFR- and PDGFR- but not PDGFR- subunits (21). Mouse osteocytes and human adipocytes lack PDGFR, although surface expression was detectable as cells transition during reprogramming [mouse; day 2 of 8 (14) and human day 21 of 25]. However, these cells express PDGFR (14). Given that PDGFR inhibition attenuates iMS cell production in both mice (14) and humans, a degree of facilitated binding of PDGF-AB to PDGF- subunits or signaling through a noncanonical receptor is likely to occur, at least at the start of reprogramming. PDGF-Bcontaining homo- and heterodimers are potent mitogens that increase the pool of undifferentiated fibroblasts and preosteoblasts with rhPDGF-BB used in the clinic to promote healing of chronic ulcers and bone regeneration (34). However, the unique characteristics of PDGF-AB but not PDGF-BB or PDGF-AA that facilitate reversal and plasticity of cell identity in combination with AZA and serum (14) remain unknown.

PDGF-AB was replenished in culture throughout the reprogramming period, but AZA treatment was limited to the first 2 days for both mouse osteocyte and human adipocyte cultures. DNA replication is required for incorporation of AZA into DNA (35) and hence DNA demethylation is unlikely to be an initiating event in the conversion of terminally differentiated nonproliferating cells such as osteocytes and mature adipocytes. However, the majority of intracellular AZA is incorporated into RNA, which could directly affect the cellular transcriptome and proteome as an early event (36, 37). It is feasible that subsequent redistribution of AZA from RNA to DNA occurs when cells replicate resulting in DNA hypomethylation as a later event (38).

In the absence of serum, we could neither convert primary human adipocytes into iMS cells nor perpetuate these cells long term in culture. The efficiency of conversion and expansion was significantly higher with human versus FCS and highest with AS. The precise serum factor(s) that are required for cell conversion in conjunction with PDGF-AB and AZA are not known. The volumes of blood (~50 ml 2) and subcutaneous fat (5 g) that we harvested from donors were not limiting to generate sufficient numbers of P2 iMS cells (~10 106) for in vivo implantation and are in the range of cell numbers used in prospective clinical trials using mesenchymal precursor cells for chronic discogenic lumbar back pain (NCT02412735; 6 106) and hypoplastic left heart syndrome (NCT03079401; 20 106).

Our motivation was to optimize a protocol that could be applied to primary uncultured and easily accessible cells for downstream therapeutic applications, and adipose tissue satisfied these criteria. We have not surveyed other human cell types for their suitability for cell conversion using this protocol. It would be particularly interesting to establish whether tissue-regenerative properties of allogeneic mesenchymal precursor populations that are currently in clinical trials could be boosted by exposure to PDGF-AB/AZA. However, given that iMS cells and MSCs share stromal cell characteristics, identifying a unique set of cell surface markers that can distinguish the former is a priority that would assist in future protocol development and functional assessment of iMS cells.

Producing clinical-grade autologous cells for cell therapy is expensive and challenging requiring suitable quality control measures and certification. However, the advent of chimeric antigen receptor T cell therapy into clinical practice (39) has shown that production of a commercially viable, engineered autologous cellular product is feasible where a need exists. Although there were no apparent genotoxic events in iMS cells at P2, ex vivo expansion of cells could risk accumulation of such events and long-term follow-up of ongoing and recently concluded clinical trials using allogeneic expanded mesenchymal progenitor cells will be instructive with regard to their teratogenic potential. The biological significance of the observed expression of pluripotency-associated transcription factors in 2 to 3% of murine and human iMS cells is unknown and requires further investigation. However, their presence did not confer teratogenic potential in teratoma assays or at 12-month follow-up despite persistence of cells at the site of implantation. However, this risk cannot be completely discounted, and the clinical indications for iMS or any cell therapy require careful evaluation of need.

In regenerating muscle fibers, it was noteworthy that iMS cells appeared to follow canonical developmental pathways in generating muscle satellite cells that were retained and primed to regenerate muscle following a second muscle-specific injury. Although iMS cells were generated from adipocytes, there was no evidence of any adipose tissue generation. This supports the notion that these cells have lost their native differentiation trajectory and adopted an epigenetic state that favored response to local differentiation cues. The superior in vivo differentiation potential of iMS cells vis--vis matched AdMSCs was consistent with our data showing that despite the relatively minor transcriptomic differences between these cell types, the epigenetic state of iMS cells was better primed for systems development. Another clear distinction between iMS cells and AdMSCs was the ability of the former to produce CD31+ endothelial tube-like structures that were enveloped by PDGFR+ pericytes. An obvious therapeutic application for iMS cells in this context is vascular regeneration in the setting of critical limb ischemia to restore tissue perfusion, an area of clear unmet need (40).

An alternative to ex vivo iMS cell production and expansion is the prospect of in situ reprogramming by local subcutaneous administration of the relevant factors to directly convert subcutaneous adipocytes into iMS cells, thereby eliminating the need for ex vivo cell production. AZA is used in clinical practice and administered as a daily subcutaneous injection for up to 7 days in a 28-day cycle, with responders occasionally remaining on treatment for decades (41). Having determined the optimal dose of AZA required to convert human adipocytes into iMS cells in vitro (2 days, 5 M), the bridge to ascertaining the comparable in vivo dose would be to first measure levels of AZA incorporation in RNA/DNA following in vitro administration and match the dose of AZA to achieve comparable tissue levels in vivo. A mass spectrometrybased assay was developed to measure in vivo incorporation of AZA metabolites (AZA-MS) in RNA/DNA and is ideally suited to this application (38). The duration of AZA administration for adipocyte conversion was relatively short (i.e., 2 days), but PDGF-AB levels were maintained for 25 days. One mechanism of potentially maintaining local tissue concentrations would be to engineer growth factors to bind extra cellular matrices and be retained at the site of injection. Vascular endothelial growth factor A (VEGF-A) and PDGF-BB have recently been engineered with enhanced syndecan binding and shown to promote tissue healing (42). A comparable approach could help retain PDGF-AB at the site of injection and maintain local concentrations at the required dose. While our current data show that human adipocytederived iMS cells regenerate tissues in a context-dependent manner without ectopic or neoplastic growth, these approaches are worth considering as an alternative to an ex vivo expanded cell source in the future.

Extended methods for cell growth and differentiation assays and animal models are available in the Supplementary Materials, and antibodies used are detailed in the relevant sections.

The primary objective of this study was to optimize conditions that were free of animal products for the generation of human iMS cells from primary adipocytes and to characterize their molecular landscape and function. To this end, we harvested subcutaneous fat from donors across a broad age spectrum and used multiple dose combinations of a recombinant human growth factors and a hypomethylating agent used in the clinic and various serum types. We were particularly keen to demonstrate cell conversion and did so by live imaging and periodic flow cytometry for single-cell quantification of lipid loss and gain of stromal markers. Using our previous report generating mouse iMS cells from osteocytes and adipocytes as a reference, we first characterized the in vitro properties of human iMS cells including (i) long-term growth, (ii) colony-forming potential, (iii) in vitro differentiation, and (iv) molecular landscape. Consistent with their comparative morphology, cell surface markers, and behavioral properties, the transcriptomes (RNA sequencing) were broadly comparable between iMS cells and matched AdMSCs, leading to investigation of epigenetic differences [Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) histone chromatin immunoprecipitation sequencing (ChIP-seq), and RRBS for DNA methylation differences] that might explain properties that were unique to iMS cells (expression of pluripotency factors, generation of endothelial tubes in vitro with pericyte envelopes, and in vivo regenerative potential). Context-dependent in vivo plasticity was assessed using a tissue injury model that was designed to promote bone/cartilage/muscle/blood vessel contributions from donor cells and simultaneously assess the absence of ectopic/malignant tissue formation by these cells (labeled and tracked in vivo using a bioluminescence/fluorescence marker). Tissue-specific regeneration and the deployment of canonical developmental pathways were assessed using a specific muscle injury model, and donor cell contributions in all injury models were performed on multiple serial tissue sections in multiple mice with robust statistical analyses (see below). Power calculations were not used, samples were not excluded, and investigators were not blinded. Experiments were repeated multiple times or assessments were performed at multiple time points. Cytogenetic and Copy Number Variation (CNV) analyses were performed on iMS and AdMSCs pretransplant, and their teratogenic potential was assessed both by specific teratoma assays and long-term implantation studies.

Subcutaneous fat and blood were harvested from patients undergoing surgery at the Prince of Wales Hospital, Sydney. Patient tissue was collected in accordance with National Health and Medical Research Council (NHMRC) National Statement on Ethical Conduct in Human Research (2007) and with approval from the South Eastern Sydney Local Health District Human Research Ethics Committee (HREC 14/119). Adipocytes were harvested as described (43). Briefly, adipose tissue was minced and digested with 0.2% collagenase type 1 (Sigma-Aldrich) at 37C for 40 min and the homogenized suspension passed through a 70-m filter, inactivated with AS, and centrifuged. Primary adipocytes from the uppermost fatty layer were cultured using the ceiling culture method (44) for 8 to 10 days. AdMSCs from the stromal vascular pellet were cultured in MEM + 20% AS + penicillin (100 g/ml) and streptomycin (250 ng/ml), and 200 mM l-glutamine (complete medium).

Adherent mature adipocytes were cultured in complete medium supplemented with AZA (R&D systems; 5, 10, and 20 M; 2 days) and rhPDGF-AB (Miltenyi Biotec; 100, 200, and 400 ng/ml; 25 days) with medium changes every 3 to 4 days. For inhibitor experiments, AG1296 was added for the duration of the culture. Live imaging was performed using an IncuCyte S3 [10 0.25numerical aperture (NA) objective] or a Nikon Eclipse Ti-E (20 0.45-NA objective). Images were captured every 30min for a period of 8 days starting from day 15. Twelve-bit images were acquired with a 1280 1024 pixel array and analyzed using ImageJ software. In vitro plasticity was determined by inducing the cells to undergo differentiation into various cell types using differentiation protocols adapted from a previous report (45).

Animals were housed and bred with approval from the Animal Care and Ethics Committee, University of New South Wales (UNSW; 17/30B, 18/122B, and 18/134B). NSG (NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ) and SCID/Beige (C.B-Igh-1b/GbmsTac-Prkdcscid-Lystbg N, sourced from Charles River) strains were used as indicated. The IVIS Spectrum CT (Perkin Elmer) was used to capture bioluminescence. Briefly, 15 min after intraperitoneal injection of d-luciferin (150 mg/kg), images were acquired for 5 min and radiance (photon s1 cm2 sr1) was used for subsequent data analysis. The scanned images were analyzed using the Living Image 5.0 software (Perkin Elmer).

Teratoma assays (46) were performed on 3- to 4-month-old female NSG mice. Lentiviral-tagged cells (5 105) in 20 l of phosphate-buffered saline containing 80% Matrigel were injected under the right kidney capsule using a fine needle (26 gauges) and followed weekly by BLI until sacrifice at week 8. Both kidneys were collected, fixed in 4% paraformaldehyde (PFA) for 48 hours, embedded in optimal cutting temperature compound (OCT), cryosectioned, and imaged for GFP.

Posterior-lateral intervertebral disc injury model (29). Lentiviral-tagged (28) AdMSCs (1 106) or iMS cells were loaded onto Helistat collagen sponges and implanted into the postero-lateral gutters in the L4/5 lumbar spine region of anesthetized NSG mice following decortication of the transverse processes. Animals were imaged periodically for bioluminescence to track the presence of transplanted cells. At 3, 6, or 12 months, mice were euthanized, and spines from the thoracic to caudal vertebral region, including the pelvis, were removed whole. The specimens were fixed in 4% PFA for 48 hours, decalcified in 14% (w/v) EDTA, and embedded in OCT.

Muscle injury model (47). The left TA and EDL muscles of 3- to 4-month-old female SCID/Beige mice were injured by injection with 15 l of 10 M CTX (Latoxan). Confocal images of three to four serial sections (TA) per mouse were captured by Zen core/AxioVision (Carl Zeiss) and visualized by ImageJ with the colocalization and cell counter plugins [National Institutes of Health; (48)]. Tetanic force contractions were performed on EDL muscles (49).

Total RNA was extracted using the miRNeasy Mini Kit (Qiagen) according to manufacturers instructions, and 200 ng of total RNA was used for Illumina TruSeq library construction. Library construction and sequencing was performed by Novogene (HK) Co. Ltd. Raw paired-end reads were aligned to the reference genome (hg19) using STAR (https://github.com/alexdobin/STAR), and HTSeq (50) was used to quantify the transcriptomes using the reference refFlat database from the UCSC Table Browser (51). The resulting gene expression matrix was normalized and subjected to differential gene expression using DeSeq2 (52). Normalized gene expression was used to compute and plot two-dimensional principal components analysis, using the Python modules sklearn (v0.19.1; https://scikit-learn.org/stable/) and Matplotlib (v2.2.2; https://matplotlib.org/), respectively. Differentially expressed genes (log2 fold change |1|, adjusted P < 0.05) were the input to produce an unsupervised hierarchical clustering heat map in Partek Genomics Suite software (version 7.0) (Partek Inc., St. Louis, MO, USA). Raw data are available using accession GSE150720.

ChIP was performed as previously described (53) using antibodies against H3K27Ac (5 g per IP; Abcam, ab4729), H3K4Me3 (5 g per IP; Abcam ab8580), and H3K27Me3 (5 g per IP; Diagenode, C15410195). Library construction and sequencing were performed by Novogene (HK) Co. Ltd. Paired-end reads were aligned to the hg38 genome build using Burrows Wheeler Aligner (BWA) (54) duplicate reads removed using Picard (http://broadinstitute.github.io/picard/), and tracks were generated using DeepTools bamCoverage (https://deeptools.readthedocs.io/en/develop/). Peaks were called using MACS2 (55) with the parameter (P = 1 109). Differentially bound regions between the AdMSC and iMS were calculated using DiffBind (http://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf) and regions annotated using ChIPseeker (56). Raw data are available using accession GSE151527. Adipocyte ChIP data were downloaded from Gene Expression Omnibus (GEO); accession numbers are as follows for the three histone marks: GSM916066, GSM670041, and GSM772771.

Total genomic DNA was extracted using the DNA MiniPrep Kit (Qiagen), and RRBS library construction and sequencing were performed by Novogene (HK) Co. Ltd. Raw RRBS data in fastq format were quality and adapter trimmed using trim_galore (0.6.4) with rrbs parameter (www.bioinformatics.babraham.ac.uk/projects/trim_galore). The trimmed fastq files were then aligned to a bisulfite-converted genome (Ensembl GRCh38) using Bismark (2.3.5), and methylation status at each CpG loci was extracted (57). The cytosine coverage files were converted to BigWig format for visualization. Differentially methylated cytosines (DMCs) and DMRs were identified using methylKit (1.10) and edmr (0.6.4.1) packages in R (3.6.1) (58, 59). DMCs and DMRs were annotated using ChIPseeker (56), and pathway enrichment was performed as detailed below. Raw data are available using accession number GSE151527. Adipocyte RRBS data were downloaded from GEO: GSM2342293 and GSM2342392.

IPA (Qiagen) was used to investigate enrichment in molecular and cellular functions, systems development and function, and canonical pathways.

Statistical analysis was performed in SAS. For the dose-optimization experiments (Fig. 1), a linear mixed model with participant-level random effects was used to estimate maximum time by dose level and age group. A linear mixed model with participant-level random effects was used to analyze statistical differences in lineage contribution outcomes between treatment groups (Fig. 3) and at different time points posttransplant, to estimate the percentage of cells by treatment and lineage. For the in vivo regeneration experiment (Fig. 4), a linear model was used to model the percent of cells over time for each group. Quadratic time terms were added to account for the observed increase from 1 to 2 weeks and decrease from 2 to 4 weeks. In the muscle regeneration experiment, a linear model was applied to cohort A and cohort B, to estimate and compare percent cells by treatment and source. Statistical modeling data are included in table S2.

Acknowledgments: We are indebted to the patients who donated tissue to this project. We thank E. Cook (Prince of Wales Private Hospital), B. Lee (Mark Wainwright Analytical Centre, UNSW Sydney), and technicians at the UNSW BRC Facility for assistance with sample and data collection and animal care; Y. Huang for technical assistance; and A. Unnikrishnan and C. Jolly for helpful discussions and critical reading of the manuscript. We acknowledge the facilities and scientific and technical assistance of the National Imaging Facility, a National Collaborative Research Infrastructure Strategy (NCRIS) capability, at the BRIL (UNSW). The STRO-1 antibody was a gift from S. Gronthos, University of Adelaide, Australia. Funding: We acknowledge the following funding support: A.Y. was supported by an Endeavour International Postgraduate Research scholarship from the Australian Government. S.S. is supported by an International Postgraduate Student scholarship from UNSW and the Prince of Wales Clinical School. P.S. is supported by an International Postgraduate Student scholarship from UNSW. M.L.T. and D.D.M. acknowledge funding from St. Vincents Clinic Foundation and Arrow BMT Foundation. K.A.K. acknowledges funding from Australian Research Council (FT180100417). J.M. is supported, in part, by the Olivia Lambert Foundation. M.K. is supported by a NHMRC Program Grant (APP1091261) and NHMRC Principal Research Fellowship (APP1119152). L.B.H. acknowledges funding from MTPConnect MedTech and Pharma Growth Centre (PRJ2017-55 and BMTH06) as part of the Australian Governmentfunded Industry Growth Centres Initiative Programme and The Kinghorn Foundation. D.B. is supported by a Peter Doherty Fellowship from the National Health and Medical Research Council of Australia, a Cancer Institute NSW Early Career Fellowship, the Anthony Rothe Memorial Trust, and Gilead Sciences. R.M. acknowledges funding from Jasper Medical Innovations (Sydney, Australia). J.E.P., V.C., and E.C.H. acknowledge funding from the National Health and Medical Research Council of Australia (APP1139811). Author contributions: The project was conceived by V.C. and J.E.P., and the study design and experiments were planned by A.Y., V.C., and J.E.P. Most of the experiments and data analyses were performed by A.Y., guided and supervised by V.C. and J.E.P. S.S., R.A.O., C.A.L., D.C., F.Y., M.L.T., P.S., T.H., J.R.P., P.H., W.R.W., and V.C. performed additional experiments and data analyses, with further supervision from R.M., C.P., J.A.I.T., D.C., J.W.H.W., L.B.H., D.B., and E.C.H. Statistical analyses were performed by J.O. R.M., D.D.M., J.M., K.A.K., and M.K. provided critical reagents. The manuscript was written by A.Y., J.A.I.T., V.C., and J.E.P., and reviewed and agreed to by all coauthors. Competing interests: V.C. and J.E.P. are named inventors on a patent A method of generating cells with multi-lineage potential (US 9982232, AUS 2013362880). All other authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a treatment process for restoring normal hematopoietic and immune functions. In this method, patients undergo high-dose radiotherapy and chemotherapy, and immunosuppressive pre-treatment is done to eliminate abnormal hematopoietic and immune systems. The patient is then transfused with allogeneic hematopoietic stem cells. This strategy is an effective cure for blood diseases, bone marrow failure syndrome, and immune deficiency.1,2 However, neutrophil deficiency, impaired mucosal barrier, and weakened immune function typically occur after transplantation, which increases the risk of infection after HSCT.3

Bloodstream infections (BSI) are a severe type of systemic infectious disease caused by the invasion of the circulatory system by pathogenic microorganisms. Notably, BSI is a common complication in the early stages of allo-HSCT and has an incidence rate of 13.6%38.9%.47 According to literature, the occurrence of bloodstream infections is a huge risk factor to early deaths after HSCT.810 The occurrence of BSI after HSCT is exacerbated by the widespread use of antibiotics and the resultant antibacterial resistance, especially multi-drug-resistant bacteria (MDR) that seriously affects the survival of transplant patients.1113 Thus, evaluation of the distribution and prevalence of drug-resistant pathogens of the bloodstream in allo-HSCT patients and the study of the BSI risk factors could guide the course of clinical treatment for BSI prevention and control. This study retrospectively analyzed the BSI risk factors in patients with allo-HSCT in the First Affiliated Hospital of Zhengzhou University from 2013 to 2017. The detection rate, distribution, and drug sensitivity of pathogenic bacteria after allo-HSCT was also evaluated.

From January 2013 to December 2017, 397 patients who received allogeneic HSCT for the treatment of hematological diseases in the First Affiliated Hospital of Zhengzhou University were selected. The patients included 242 males and 155 females, with a median age of 21 (162) years. Of these, 115 cases had acute myeloid leukemia (AML), 110 with severe aplastic anemia (SAA), 102 with acute lymphocytic leukemia (ALL), and 70 patients with other conditions.

According to the difference in the histocompatible typing and relationship, allo-HSCT is divided into matched sibling transplantation, partially matched related transplantation and matched unrelated transplantation. Among the 397 cases of allo-HSCT, 177 were matched sibling transplantation, 165 were partially matched related transplantation, and 55 were matched unrelated transplantation. According to the stem cell source, there were 333 cases of peripheral hematopoietic origin, 55 from peripheral blood combined with bone marrow transplantation, and nine involved cord blood transplantation.

Central vein catheterization was performed for all patients before transplantation conditioning. Modified busulfan/cyclophosphamide (Bu/Cy) and total body irradiation/cyclophosphamide (TBI/Cy) conditioning regimens were used for patients with acute leukemia, myelodysplastic syndrome, and lymphoma. Meanwhile, cyclophosphamide + anti-thymocyte globulin (Cy-ATG) and FluCy-ATG pre-treatment regimens were used for severe aplastic anemia. The GVHD prevention program used cyclosporine combined with mycophenolate mofetil and methotrexate, of which 272 cases were also treated with ATG to prevent GVHD.

All HSCT patients were admitted to the laminar flow purification ward after a medicated bath, and were given a sterile diet, and received oral, eye, nose, and perianal care. Take a 1:2000 chlorhexidine liquid medicinal bath for 20 minutes; routinely gargle with saline and cermetium chloride before and after three meals a day, add metronidazole solution if necessary; use 1% chloramphenicol, 0.5% Rifampicin eye drops alternate eye drops, 4 times/d; alternate nose drops with houttuynia cordata and streptomycin nasal drops, 4 times/d; rinse the perineum with warm water after each bowel movement, 3% boric acid solution for a bath for 20 Minutes, mupirocin is applied to the perianal area. Itraconazole, berberine, and compound sulfamethoxazole were administered orally for intestinal disinfection two weeks before transplantation. If the body temperature of patients got to 38.00C during transplantation or shivering occurred, 10 mL of blood from the peripheral vein was collected using standard. The blood was drawn twice in a row for separate cultivation of aerobic and anaerobic bacteria. For positive cases, broad-spectrum antibiotics were administered intravenously, and the treatment efficacy was evaluated 48 hours after the initial treatment. Treatment efficacy was empirically assessed based on blood culture results, WBC, C-reactive protein, and procalcitonin levels, after which ineffective treatment strategies were adjusted.

Agranulocytosis refers to the absolute value of neutrophils <0.5 109/L,14 while granulocyte reconstitution refers to neutrophils 0.5 109/L for three consecutive days after transplantation.

Fever is a single measurement of oral temperature 38.3C (axillary temperature 38.0C) or 38.0C (axillary temperature 37.7C) for more than 1 hour.

The pathogenic diagnosis of BSI was made after the isolation of pathogenic microorganisms from blood culture. If the same patient isolates the same bacteria, if the drug sensitivity is the same, it is 1 BSI. BSI-related mortality was defined as death occurring within 30 days after the diagnosis of BSI. Pre-engraftment BSI is defined as the infection that arises from the onset of the pre-treatment regimen to the time before granulocyte implantation.

VersaTREK automatic blood culture instrument (Thermo Fisher, USA), VITEK MS IVD 3.0 mass spectrometer identification instrument and VITEK2 Compact automatic microbial identification, and drug sensitivity analysis system for bacterial culture, identification, and drug sensitivity detection, spread through paper (K-B) method and E-Test were used in in vitro susceptibility tests and review of abnormal susceptibility results. The results were interpreted according to the standards issued by the United States Committee for Clinical and Laboratory Standardization (CLSI).15

The SPSS21.0 software was used for statistical analysis, and descriptive statistics were used to summarize clinical features. The univariate analysis used a chi-square test, while logistic regression was applied for multivariate analysis. A P-value of 0.05 was used as the level of significance; thus, P<0.05 indicated statistically significant differences.

Among the 397 HSCT patients, 294 had agranulocytosis fever, out of which 52 were microbiologically confirmed as BSIs. Therefore, the incidence of BSI was 17.7% (52/294), accounting for 13.1% (52/397) of all transplant patients. The implantation time of neutrophils is 13 days (11,15), and the time from agranulocytosis to BSI is 12 days (7,30). For 294 patients, we did 607 blood cultures, among which 60 were positive (9.9% positive blood culture rate). Out of the 294 patients, six had two or more pathogenic bacteria.

Sixty pathogens were detected in 52 patients, including 43 Gram-negative bacteria (71.67%), 10 Gram-positive bacteria (16.67%), and 7 fungi (11.67%). We found that Gram-negative bacteria accounted for most BSIs, followed by Gram-positive bacteria, and fungal infections were the least. The numbers and proportions of different strains of pathogenic bacteria are shown in Figure 1. In terms of drug resistance, the extended-spectrum -lactamase (ESBL) detection rates of E. coli and K. pneumoniae were 46.7% (7/15) and 30% (3/10), respectively. Carbapenem-resistant Enterobacteriaceae (CRE) accounted for 17.9% (5/28). The recorded patterns for Gram-negative bacteria drug susceptibility are shown in Table 1. The two staphylococci detected in Gram-positive bacteria were all methicillin-resistant, and all the three enterococci were sensitive to vancomycin, teicoplanin, and linezolid. The detected fungi belong to the genus Candida, and the resistance rates to itraconazole and voriconazole were 57.1% and 28.6%, respectively.

Table 1 Resistance Rate of Major Gram-Negative Bacteria to Common Antibacterial Drugs

Figure 1 Distribution of 60 isolated pathogenic bacteria pathogen.

Out of the 52 BSI patients, 33 improved after treatment, while 19 died after treatment failed (36.5%). Among the 19, 13 had Gram-negative bacteria infection, three were Candida infections, while another three were mixed Gram-negative and Gram-positive bacterial infection. Six of the seven patients who were resistant to carbapenems died.

We divided the 294 patients with agranular fever into two groups: BSI-free (242) and BSI (52). Univariate and multivariate analyses were applied for the study of BSI risk factors, including patients age, gender, disease type, stem cell source, pre-treatment application of ATG, combined diarrhea, oral ulcers, and presence of granules. Univariate analysis results demonstrated that the occurrence of BSI was correlated to the transplantation method, pre-treatment application of ATG, agranulocytosis time (21 days), and stem cell source (Table 2). Meanwhile, multivariate analysis showed that pre-treatment application of ATG, agranulocytosis time (21 days), and stem cell source were risk factors for BSI (Table 3).

Table 2 Univariate Analysis of Risk Factors for BSI

Table 3 Multivariate Analysis of Risk Factors for BSI

Allo-HSCT patients undergo prolonged agranulocytosis and develop an impaired mucosal barrier. Besides, the long-term use of immunosuppressive agents increases the incidence of bloodstream infections.47 In the present study, the incidence of bloodstream infections was 13.1% in all patients, and 17.7% in patients with febrile neutropenia. A previous study conducted in China reported that the incidence of bloodstream infections in patients with febrile neutropenia was 17.0%.16 Thus, our findings are consistent with earlier results of other studies. The mortality rate of allo-HSCT bloodstream infections in our center was 36.5%, which is higher than the 26.9% reported by Mikulska et al17 and the 31.1% reported by Stoma et al.18 In addition, studies by Stoma et al also found that the application of fluoroquinolones can reduce the incidence of bloodstream infections by affecting the colonization of intestinal bacteria, while insufficient empirical antibacterial treatment is associated with increased mortality.18,19 This disparity suggests that we should pay attention to the prevention and treatment of bloodstream infections in transplant patients and formulate anti-infection strategies based on the distribution of pathogens and drug resistance patterns to improve transplantation and survival rates.

This study detected 60 pathogens in BSIs, of which gram-negative bacteria (71.67%) were the main ones, followed by gram-positive bacteria (16.67%), and fungi were the least (11.67%) (Figure 1). Gram-negative bacteria were mainly of the Enterobacteriaceae family, particularly E. coli and K. pneumoniae. The non-fermenting bacteria P. aeruginosa was also detected. A 25-year study in Spain showed that BSIs after HSCT were mainly caused by gram-positive bacteria, with a downward trend in positive bacteria and an increasing trend in gram-negative bacteria.20 Blennow et al also reported similar conclusions.21 However, many transplant centers in China have reported that BSIs after HSCT are mainly caused by gram-negative bacteria, followed by gram-positive bacteria, while fungi make up the least proportion. Thus, the epidemiology of BSIs in our center conforms to the distribution pattern reported in other centers in China.22,23

In this study, the common Enterobacteriaceae (E. coli and K. pneumoniae) had ESBL detection rates of 46.7% and 30%, respectively, and carbapenem resistance rates of the two bacteria were 6.7% and 30%, respectively (Table 1). Thus, we found that E. coli is highly sensitive to carbapenem drugs, suggesting that these drugs can be used for empiric antibacterial treatment. The ESBL positivity rate and carbapenem resistance rate of K. pneumoniae were both 30% (Table 1), indicating that its clinical treatment can be a combination of tigecycline, polymyxin, and other drugs. Notably, research shows that combination therapy with antibacterial medications such as cyclin and polymyxin can reduce the mortality of patients.24,25 In the present study, the resistance rate of P. aeruginosa to carbapenems was 28.6%, while its resistance rate to both aminoglycosides and quinolones was 14.3% (Table 1). Thus, a combination of carbapenems, aminoglycosides, and quinolones can be used for clinical treatment. Multi-center research in China reported carbapenem resistance rates of 3.6% and 18.9% for E. coli and K. pneumoniae, respectively.26 Similarly, this study revealed high resistance of E. coli and K. pneumoniae to carbapenem. The high rate of mycene resistance could be attributed to the repeated use of broad-spectrum antibiotics in transplant patients and the continuous increase in multi-drug-resistant bacteria in recent years.27 In response to the rise in multi-drug-resistant bacteria, our center uses perianal swabs to regularly screen intestine colonizing bacteria in transplant patients. As such, pathogenic bacteria are identified early, and treatment strategies are adjusted based on drug sensitivity results. The sensitivity of Gram-positive bacteria to the glycopeptides vancomycin, linezolid, and teicoplanin was 100.0%, suggesting that Gram-positive bacteria BSIs can be completely treated in clinical practice. Thus, glycopeptide or azole drugs can be the first choice for the treatment of Gram-positive bacteria BSIs.

All the seven fungi in this study were Candida, and Candida tropicalis was the predominant species. The resistance rates to itraconazole and voriconazole were 57.1% and 28.6%, respectively. The mortality rate of candidiasis was high, which significantly threatened the survival of transplant patients. According to previous studies, caspofungin should form the first choice fungal treatment after allo-HSCT in clinical practice, combined with antifungal treatment if necessary.28,29

The single-factor and multi-factor analysis results showed that pre-treatment application of ATG, agranulocytosis time (21 days), and stem cell source were risk factors for BSI. The removal of T-lymphocytes from the body of ATG-pretreated patients significantly delays immune reconstitution,30 and the continued lack of granulocytes causes immunodeficiency in transplant patients, thus increasing the risk to BSIs. Peripheral blood combined with bone marrow transplantation, hematopoietic implantation is relatively fast, which may be the reason for the lower incidence of BSIs in this group of patients, relative to peripheral blood and cord blood transplantation.3133

The results of this study show that BSI is a common complication of allo-HSCT patients with agranulocytosis. Gram-negative bacteria were the most prevalent pathogen in BSIs, and drug resistance to carbapenem drugs was relatively high. The use of ATG in pre-treatment, agranulocytosis time (21 days), and stem cell source are risk factors for BSI. The high mortality rate of BSI substantially affects the prognosis of transplant patients, and attention should be paid on the distribution of pathogenic bacteria and drug resistance in the bloodstream of transplant patients. Besides, the treatment plan should be adjusted based on the specific bacteria and drug resistance patterns.

The patient consent was waived, since the research involves no more than minimal risk to the subjects because the review of subjects medical records is for limited information. The information is not sensitive in nature, and the data are derived from clinically indicated procedures. The precautions taken to limit the record review to specified data and the coding of the data further minimize the primary risk, which is a breach of confidentiality. This study has been approved by the ethics review committee of the research project of the First Affiliated Hospital of Zhengzhou University, and has obtained relevant certificates.

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work. This study complies with the Declaration of Helsinki.

This project was supported by the Key Scientific Research Project Plan of Higher Education Institutions in Henan Province (18A320040).

The authors report no conflicts of interest in this work.

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[Full text] Clinical Analysis of Bloodstream Infections During Agranulocytosis Aft | IDR - Dove Medical Press

Recommendation and review posted by Bethany Smith

Introduction

Recent literature reviews suggest that bisphosphonates (BPs) may contribute to the growing number of cases of osteonecrosis involving the maxilla and mandible that are associated with the pathogenesis of BP-related osteonecrosis of the jaw (BRONJ).1 In the discussion concerning BRONJ, a distinction must be made between diseases featuring reduced osseous mineral content, which may be counteracted by BPs (such as those occurring during menopause or in cases of osteoporosis), and cases that present with indications for BPs (such as tumors). BPs have been used in the treatment of multiple myeloma, breast cancer, prostate cancer, and other tumors. In patients with metastatic breast cancer, the bones are affected in around two-thirds of cases. To protect patients from bone fractures and to reduce pain, patients are often prescribed BPs or a special antibody that prevents the breakdown of, and subsequently stabilizes, affected bone. BRONJ is a newly emerging problem that is recognized as a serious complication of BP therapy, primarily following intravenous (IV) administration.2

The concern is that BPs affect the natural remodeling of bone tissues and delay the breakdown of older bone structures. BPs are potent inhibitors of bone resorption and have a chronic effect over a half-life of at least 5 years, possibly exerting their effects for more than 10 years. BRONJ is a seemingly growing epidemic associated with osteonecrosis of the jawbone (ONJ).35 The long-term effects of oncological-related BP treatment on alveolar bone quality include the impact on BP-induced overexpression of alveolar bone remodeling. There are increased osteosclerotic properties in the alveolar bone that are associated with significantly greater bone volume and higher bone density.6,7 The risk of BP therapy is divided into two categories: local and systemic risk factors; thus, a distinction must be made between oral and IV administration. Local oral risk factors for BRONJ in cancer patients include dentoalveolar surgery, dental extraction, and dental implant insertion.8 Periodontal infections also significantly increase the risk of BRONJ in cancer patients.9 In addition, there is a significant correlation between the use of removable prostheses, the administration of high-dose IV BPs, and an increased risk of BRONJ.10 In patients receiving oral BP therapy for the treatment of osteoporosis, the prevalence of BRONJ only increased 0.21% from close to 0%. Systemically, however, there is a much higher risk associated with the IV injection of BPs. This is closely related to the frequent use of BPs in cancer patients who receive a significantly higher total dose over a longer duration.11 The mean and minimum time for the development of ONJ is 1.8 years and 10 months, respectively.12 The risk of BRONJ in cancer patients exposed to BP therapy is from 50100 times higher than in cancer patients treated with a placebo. The BRONJ risk for the RANKL inhibitor denosumab was between 0.7% and 1.9%.13,14 The risk of ONJ in cancer patients treated with high doses of IV BPs appears to be significantly higher: in the range of 110 per 100 patients (depending on therapy duration).15 A recent review reported a wide-ranging BRONJ incidence of 027.5% that was associated with the IV administration of BPs, with an average incidence of 7%.16 The cumulative frequency varied from 0.812.0% and was estimated to be up to 30.0% in some reports.17,18 Despite numerous publications on the subject, the overall pathogenesis of BRONJ does not yet appear to be fully understood. In particular, the reasons why only a subset of patients (<30%) receiving IV BPs develop BRONJ remain unclear. Although most patients that develop BRONJ have a history of tooth extraction or injury, these factors do not fully explain the occurrence of BRONJ.8 The development of BRONJ in edentulous areas in patients with no apparent history of injury suggests that pre-existing conditions, such as subclinical infections or potentially necrotic areas of the jawbone, may contribute to the conditions that lead to the development of BRONJ.

Why does BRONJ develop in up to 30% of individuals following IV BP therapy and not the remaining 70%? This review raises the question of whether little-known or difficult-to-identify, pre-existing, impaired bone remodeling, such as that occurring in aseptic-ischemic osteonecrosis of the jaw (AIOJ), bone marrow defects (BMD), or fatty-degenerative osteonecrosis of the jawbone (FDOJ), represents a local risk factor in the development of BRONJ.

There is still a limited scientific understanding of the relationship between ONJ and BPs.19 In order to clarify the research question and present the background and specific common characteristics of AIOJ/BMD/FDOJ and BRONJ, an extensive literature search was carried out in PubMed Central. In the literature, the terms aseptic-ischemic osteonecrosis of the jaw (AIOJ), bone marrow defects (BMD), and fatty-degenerative osteonecrosis of the jawbone (FDOJ) are used to describe an intramedullary phenomenon with the same pathogenesis, morphology, and pathohistology.

The American Association of Oral and Maxillofacial Surgeons published four staging criteria (at risk, Stage 03).20 Stage 0 is of particular interest in our research as it refers to patients with no clinical evidence of exposed bone, but presence of non-specific symptoms or clinical and/or radiographic abnormalities. The discussion concerning BRONJ is complicated by the fact that there are two clinical forms of BRONJ. The first presents as exposed bone in the maxillofacial region with clinically recognizable necrotic bone that is visibly exposed through the oral mucosa or facial skin, and present for more than 8 weeks, which is referred to as so-called exposed BRONJ.15 The second form of BRONJ is particularly interesting for our investigation; it was recently emphasized that BRONJ does not always appear with necrotic bone visible through a breech in the oral mucosa.21 This form is referred to as non-exposed BRONJ (NE-BRONJ). In the absence of exposed bone, it is characterized by clinical features associated with the jaw, such as unexplained jawbone pain, fistulas/sinus tracts, loose teeth, and swelling.22,23 Diagnosing NE-BRONJ is difficult, as other common jawbone diseases, such as odontogenic infections, may cause similar symptoms and must be excluded. The non-exposed variant may comprise up to one third of all BRONJ cases and is thus not uncommon;24 however, this previously underestimated NE-BRONJ is difficult to accurately diagnose. Recently published papers emphasize that NE-BRONJ has received little attention so far and does not fulfill the current definition of BRONJ.25 Nevertheless, NE-BRONJ belongs to the same disease as exposed BRONJ and should be identified as part of the full spectrum of BRONJ (see the section titled, Case descriptions of AIOJ/BMD/FDOJ, non-exposed BRONJ, and Actinomyces colonization).26

Our investigation requires the identification of the basic immune mechanisms associated with BP administration. Specifically, which mechanism is behind the anti-tumor activity of BPs in cancer patients?

Various studies postulate that BPs change the bone microenvironment around cancer cells, which may prevent cancer cell survival and disease recurrence.27 BPs may also reduce the appearance of disseminated tumor cells. The formation of metastases is complex; mesenchymal stem cells (MSCs) are predominantly found in the bone marrow.28 MSCs may contribute to the formation of metastases through various mechanisms: (1) MSCs are recruited to develop breast tumors where they can enhance the metastatic potential of weakly tumorigenic breast cancer cells;29 (2) MSCs and other bone marrow cells may form a pre-metastatic niche within the specific tissues to which tumor cells metastasize;30 and (3) MSCs are able to maintain the growth and survival of cancer cells in the bone microenvironment where they may contribute to the formation of niches for dormant micrometastases that can later form distant metastases. BPs significantly reduce the ability of MSCs to migrate, thereby reducing the growth and survival of cancer cells.31 Thus, the effects of BPs on MSCs in the bone marrow microenvironment contribute to anti-tumor activity by affecting the ability of MSCs to migrate and develop tumors in pre-metastatic niches. BPs disrupt the interaction between MSCs and breast cancer cells within the bone microenvironment, where BPs may also directly inhibit breast cancer cell growth.

The antiangiogenic effect of BP administration in tumor patients also plays a role in therapy.32 When administered systemically, BPs effectively inhibit angiogenesis. The pronounced antiangiogenic properties of BPs enhance their effectiveness in the treatment of malignant bone diseases. In addition to suppressing RANTES/CCL5 (R/C) expression in MSCs, BP administration plays a role in the treatment of tumor patients.33 Similar to exogenous glucocorticoids and estrogen,34 BPs are ischemic and hypoxia-related stressors of bone health that alter jawbone metabolism, thus leading to osteonecrosis. While tumor-associated BP therapy is currently the heavy weight for bone health, it may accelerate existing, chronic pathophysiological events within the microcirculation of bone marrow compartments in the jaw. BRONJ development is often characterized by a slow start and usually presents with infarcts and thrombosis of small vascular sections of the supplying artery within the medullary canal; these features also correspond to AIOJ/BMD/FDOJ. Myeloid elements (including fat marrow) liquefy and cancellous trabeculae are resorbed, so that individual bone spaces merge and gradually create larger cavities.

If we compare the findings in the sections titled, Bisphosphonates and mesenchymal stem cells and Bisphosphonates and antiangiogenesis to pre-existing AIOJ/BMD/FDOJ, several strikingly common characteristics shared by BRONJ and AIOJ/BMD/FDOJ can be observed that help to answer our research question. In the sections following Bisphosphonates and antitumor therapy, we present the foundations for the development of AIOJ/BMD/FDOJ and draw similarities with the development of BRONJ.

The key function of proinflammatory chemokines R/C in the formation of breast cancer and its metastasis, as well as a possible connection with the intramedullary signaling of R/C overexpression from AIOJ/BMD/FDOJ areas, has been pointed out in previous studies.35,36 The conspicuous overexpression of R/C in little-known BMDs, as found in AIOJ/BMD/FDOJ, has been reported.37,38 R/C overexpression is a regulator of healthy bone metabolism in bone needing repair. The starting point for a typical AIOJ/BMD/FDOJ BMDs is the expression of R/C and its chemokine receptors (CCR5) in both osteoblasts (OBs) and osteoclasts (OCs). Ligands (CCL5) and receptors (CCR5) simultaneously activate autocrine and paracrine mechanisms in the bone.39 One study examined the effects of BPs on human primary OBs and was able to show that the overexpression of proinflammatory R/C from BP-treated OBs also occurs in areas affected by BRONJ.40 The secretion of proinflammatory cytokines interleukin (IL)-8 and R/C increased after 14 days of treatment with the highest dose of BPs.40 The complexity of cytokine control becomes clear at this point. In contrast to the tumor, where BPs in the MSCs reduce R/C expression to such an extent that metastasis is prevented, R/C expression is increased by BPs in OBs. If AIOJ/BMD/FDOJ is already present, it may be assumed that the associated increased R/C secretion is thus further increased by BPs. Specifically, NE-BRONJ may develop as BPs increase the expression of IL-8 and R/C.41 Other researchers have confirmed increases in the secretion of proinflammatory IL-8 and R/C from BP-treated OBs.42 Combined with the lower proliferation rate of OBs and a decrease in their differentiation, higher doses or accumulations of BPs cause undesirable local changes in the bone by increasing the secretion of IL-8 and R/C from OBs. If these findings are applied to BP administration in the context of a chronic, pre-existing AIOJ/BMD/FDOJ area, then such areas may be expected to exhibit increased R/C secretion in response to BPs. This increase may result from the inhibition of OC activity, leading to the development of BRONJ. Figure 1 summarizes the effects of BP administration on the pre-existing physiological derailments associated with tumor and osteoporosis development.

Figure 1 Comparison of the effects of BP administration (+BP) in the context of a tumor (upper part of Figure 1) and pre-existing osteoporosis (lower part of Figure 1). Legend: The red arrows indicate overactivity; the green arrows show reversal following BP administration.

In the literature, the vascular composition of AIOJ/BMD/FDOJ is characterized by the fact that blood flow in the medullary canal is impaired by micro-infarcts, which leads to chronic marrow ischemia.43 BRONJ also shows reduced vascularization in the medullary canal.44 Several publications have shown that ischemic bone diseases such as AIOJ/BMD/FDOJ and BRONJ are of multifactorial origin and emphasize the multiple stroke model as the cause of ischemic bone diseases.45,46 In the orthopedic literature, intensive research conducted on the development of ischemic bone disease in the early stages of the disease process is presented.47 Our aim here is to apply this knowledge not only to extreme forms of the disease, such as osteoradionecrosis and BRONJ, but also to chronic, subclinical, and ischemic forms such as bone marrow edema and AIOJ/BMD/FDOJ, which often progress asymptomatically. Many of these forms are manifestations of both local and systemic risk factors that compromise circulation in the bone marrow, and may also impact on the homeostasis of bone resorption and formation, in addition to BP therapy. The importance of this multifactorial exposure to risk factors for ischemia and the associated causal genetics that are very similar to those in cases of AIOJ/BMD/FDOJ is shown by observing how bone that is exposed to BPs demonstrates minimal OC activity, followed by the deposition of newly formed, thicker bone with reduced vascular supply.48 The resulting mosaic-like pattern of bone remodeling is strikingly similar to that found in Pagets disease, which tends to be associated with the development of osteomyelitis.49 Similar to AIOJ/BMD/FDOJ, the remodeling induced by BPs leaves cavities, otherwise known as cavitations, which leads to both necrosis and unlike that which is found in AIOJ/BMD/FDOJ subsequent infection by colonizing bacteria. Many patients with AIOJ/BMD/FDOJ have inherited prothrombotic tendencies, which is comparable to what is found in patients with idiopathic osteonecrosis of the femoral head (Pagets disease) and includes thrombophilia and hypofibrinolysis.5052 Although a consensus has been reached that ischemic marrow edema is not part of the pathogenesis of BRONJ,53 it is regarded as a typical characteristic of AIOJ/BMD/FDOJ, serving as a precursor to BRONJ development. Systemic antibiotic therapy has limited access to these avascular zones and surgical debridement is usually necessary.

The initial OB situation found in AIOJ/BMD/FDOJ is highly characteristic; under pathological conditions, OBs express R/C chemokines in a non-physiological manner.54,55 The increasing frequency of ONJ and its possible association with high cumulative doses of BPs was investigated in one study, which concluded that high doses of BPs had both OC and OB effects, and thus bone remodeling was inhibited in vivo.56 Other researchers have examined the proliferation, viability, expression, and secretion of bone markers and cytokines/chemokines from primary OBs following exposure to BPs.42 Increased concentrations of proinflammatory cytokines were found in response to BPs. Similarly, increased R/C expression is present in AIOJ/BMD/FDOJ. Following treatment with the highest dose of BPs, the secretions of proinflammatory cytokines IL-8 (P<0.001) and R/C (P<0.001) were significantly increased after 14 days. In addition, the secretion of proinflammatory R/C from OBs exposed to BPs increased. It has also been determined that R/C plays a role in the etiology of the osteolytic changes that are present in AIOJ/BMD/FDOJ.37,57 The aim of another study was to investigate the effect of BPs on human OBs in vitro, while considering RANKL and osteoprotegerin (OPG), both of which mediate OC differentiation.40 OPG increased significantly in the group that received BPs at a dose of 10 M, while RANKL expression decreased significantly with different concentrations of BPs. In summary, exposure to various BP concentrations had a positive effect on OB differentiation, but did not affect proliferation. In contrast, the BP-associated changes in RANKL and OPG production contributed to the suppression of osteoclastic bone resorption. Excess R/C leads to OC inhibition which, in our model, also leads to a disturbance in RANK/RANKL homeostasis (see Figure 2). The chain of reactions that arise from pre-existing AIOJ/BMD/FDOJ and BP administration result in the development of BRONJ in response to the subsequent OB depression; it also leads to increased OC apoptosis. In addition, bone densification takes place following BP administration as a result of increased OB activity. As such, osteonecrosis occurs in the jawbone when BPs are used parenterally. The reasons for these different reactions to BPs have not yet been clarified.

Figure 2 The effects of BP administration and the characteristics of AIOJ/BMD/FDOJ both include depressed alkaline phosphatase (AP) activity with subsequent R/C overexpression. On the one hand, this leads to OC inhibition and, on the other, to RANK/RANKL deactivation, which subsequently causes increased OC apoptosis and depressed OB activity resulting in BRONJ development. Legend: The red arrows indicate deactivation; the green arrows show a reversal of the effect following BP administration.

The first step in tumor necrosis factor alpha (TNF-a)-induced OC genesis occurs in the bone marrow.58 Although mature OCs erode the resorption of the bone as a focal point over the course of months to years, the lifespan of individual OCs is only a few weeks. Thus, mature OCs must be constantly replaced. With respect to OC formation, TNF-a directly stimulates the formation of mature OCs,59,60 and supports and promotes the survival of mature OCs.61 TNF-a increases the survival time of OCs to extend the duration of bone resorption. In the early stages of AIOJ/BMD/FDOJ, the situation for OCs is highly contradictory: the extremely low TNF-a values found in areas of AIOJ/BMD/FDOJ as compared to the values in healthy jawbone samples (as documented in our previous studies) indicate that any inflammatory erosion due to TNF-a supported OC formation is unlikely. Due to reduced TNF-a activation, OC formation in AIOJ/BMD/FDOJ is inhibited, which results in a fatty-degenerative morphology.62

In the same way, BPs inhibit the ability of OCs to resorb bone. They do so by suppressing farnesyl diphosphate synthetase activity, which inhibits OC recruitment and impacts the life expectancy of OCs through increased apoptosis. Where the OC function is excessively inhibited, dying OCs will not be replaced, and the capillary network of the bone will not be maintained, which leads to BRONJ.19 The ability of BPs to regulate bone turnover by suppressing OC activity has led to its widespread use in the treatment of osteoporosis, Pagets disease, humoral hypercalcemia, and in tumors metastasizing to bone.17,63 Several studies have shown the effectiveness of BPs in suppressing OC activity in arthritic bone erosions, which was comparable to the effects of OPG injections.64

The initial alkaline phosphatase (AP) situation in AIOJ/BMD/FDOJ is as follows: AP has an optimum pH in the alkaline range. The pH level of AIOJ/BMD/FDOJ areas, however, is reduced as a consequence of the proinflammatory characteristics of R/C overexpression, resulting in a chronic inflammatory state. AP activity is thus inhibited within the increasingly acidic environment of such areas. Furthermore, BPs increase R/C secretion from OBs, and the acidity of areas affected by AIOJ/BMD/FDOJ, together with an excess of R/C, leads to OC inhibition.65 At the same time, there is also reduced osteogenesis due to the suppression of AP activity,66 as well as the overexpression of R/C that is present in AIOJ/BMD/FDOJ areas and also caused by BP administration. In our model, these two factors led to OC inhibition via disturbed RANK//RANKL homeostasis. In addition, depressed OB activity and increased OC apoptosis result in BRONJ development. While the skeletal bone consolidation that results from BP administration occurs in response to increased OB activity, BRONJ develops in the jawbone when BP is administered parenterally. The reasons for these different responses to BPs have not yet been clarified. If we apply these considerations to an existing AIOJ/BMD/FDOJ area (as shown in Figure 2), then BRONJ and AIOJ/BMD/FDOJ both show suppressed AP activity with subsequent R/C overexpression.67 This leads to OC inhibition and RANK/RANKL deactivation and, subsequently, increased OC apoptosis. Decreased OB activity may ultimately lead to the development of exposed BRONJ.

Despite the similarities detailed in the section titled Osteoimmunological parameters of AIOJ/BMD/FDOJ and BRONJ with the same impact in response to BPs, BRONJ and AIOJ/BMD/FDOJ present two very different clinical pictures; different reactions to BP administration are also likely to occur.

The initial involvement of RANKL in AIOJ/BMD/FDOJ has been described in the literature as follows: pathological increases in levels of R/C and MCP-3 from activated OBs stimulate chemotactic recruitment and RANKL formation of resorptive OCs and aggravate local osteolysis. However, BP administration indirectly inhibits OC maturation by increasing OPG protein secretion and decreases transmembrane RANKL expression in human OBs. Several studies have shown that although BPs do not significantly affect RANKL gene expression, they reduce transmembrane RANKL protein expression in OBs.68,69 This shows that BPs, in addition to directly inhibiting mature OCs, prevent OC recruitment and differentiation by splitting transmembrane RANKL into OBs. OC activation and RANKL activation in areas of AIOJ/BMD/FDOJ, and OC inhibition and RANKL inhibition in BRONJ distinguish these two forms of derailed bone metabolism and thus yield different clinical results. Specifically, imperceptible fatty osteolysis of the marrow structures in AIOJ/BMD/FDOJ and painful BRONJ sequestrum arise as a result. BPs have been shown to downregulate the expression of RANKL, the OC-differentiating factor produced by OBs.70

The initial involvement of OPG in AIOJ/BMD/FDOJ is described in the literature. Since the TNF-a level found in AIOJ/BMD/FDOJ represents only 50% of the TNF-a level in healthy jawbone,36,37 the OPG enzyme that belongs to the TNF family is deactivated. In the resulting osteolysis found in areas of AIOJ/BMD/FDOJ, this leads to reduced RANKL binding and thus results in OC activation. In conclusion, data from previously published studies have suggested that BPs modulate the production of OPG by normal OBs, which may contribute to the inhibition of OC bone resorption.71 As the production of OPG increases with OB maturation, the amplification of OPG by BPs may be linked to OB differentiation via stimulatory BP effects. BPs have been shown to increase the gene expression for the decoy receptor, OPG, in human OBs.71 OPG balance is disturbed in both AIOJ/BMD/FDOJ and BRONJ, albeit in opposite ways. However, the prior imbalance of OPG activity in AIOJ/BMD/FDOJ may increase the effects associated with BP administration.

With respect to the exposed variant of BRONJ, radiographic procedures are required in order to determine the extent to which the degree of ossification has increased.72 However, the existence of this variant of BRONJ is clinically evident. In contrast, the non-exposed BRONJ variant and AIOJ/BMD/FDOJ are associated with very similar problems in terms of diagnostic imaging. As with AIOJ/BMD/FDOJ, the prevalence of this variant of BRONJ is largely underestimated as the disease is often underdiagnosed and under-reported.73 Studies have shown that almost a quarter of patients with BRONJ remain undiagnosed.74

The initial histopathological presentation of AIOJ/BMD/FDOJ found in the literature is as follows: Bouquot describes these bone modeling disorders as ischemic osteonecrosis, which is a bone disease characterized by the degeneration and death of marrow and bone due to a slow or abrupt decrease in marrow blood flow.75 Clumps of coalesced, liquefied fat (oil cysts) may be seen. Bone death is represented by a focal loss of OCs. Dark masses of calcific necrotic detritus may often be present.75 The histopathological features of AIOJ/BMD/FDOJ include necrotic adipocytes and fibrosis, but an almost complete absence of inflammatory cells.76 Additional research has shown the role of aseptic necrosis following injury or drug therapy in the pathophysiology of BRONJ. Aseptic bone necrosis, as found in AIOJ/BMD/FDOJ, has been reported as a manifestation of selected systemic diseases and also documented following operations, trauma, and immunosuppressive therapy at the site of BRONJ.77,78 The development of aseptic necrosis has been documented in the upper and lower jaw, particularly following osteotomies.79,80 Researchers have observed a relationship between oral BP use and non-specific aseptic osteonecrosis among a cohort of older cardiovascular patients.81 Other researchers have identified necrotic liquefaction, which often extend to large areas of the jaw, especially within BRONJ lesions of cancer patients, as shown using digital volume tomography (DVT)/cone beam computed tomography (CBCT).82 Research has been published on BRONJ samples that were characterized by low to moderate inflammation.83 This is in accordance with other reports of histopathological analyses of BRONJ samples.48,78,8486 Bone samples from BRONJ patients were investigated by microscopy and the presence of inflammatory infiltrates in the bone tissues was not observed.87 These studies have demonstrated that aseptic necrosis, a lack of inflammatory reactions, and empty OC lacunae are common histopathological features of AIOJ/BMD/FDOJ and BRONJ.

The diagnostic difficulties associated with BRONJ and AIOJ/BMD/FDOJ present another common feature. In order to diagnose BRONJ with imaging procedures, the Task Force Report of the American Society for Bone and Mineral Research highlights that the differential diagnosis of BRONJ should exclude other common intraoral diseases such as periodontitis, gingivitis, infectious osteomyelitis, osteoradionecrosis, neuralgia-inducing cavitational osteonecrosis (NICO), bone tumors, and metastases.15 The authors of the report thus rule out an etiological equation for diagnosing NICO and BRONJ. The current review is focused on the potential role of imaging techniques in the diagnosis of the early stages of BRONJ. A combination of clinical and radiological symptoms suggest that, while not specific to BRONJ, they may collectively be more comprehensive and representative of the bone disease process.2 The American Association of Maxillofacial Surgery accepts the use of imaging techniques when detecting BRONJ during presurgical evaluation.72 It is important for the BRONJ patient that various imaging methods be examined critically prior to being adopted for the early detection and diagnosis of BRONJ.

Figure 3 Left panel shows jawbone area 18; hematoxylin and eosin staining, magnification 200. The lower half of the image illustrates eosinophilic bone substance with empty osteocyte cavities corresponding to devitalized bone sequestrum. Middle part of the left panel: Highly irregular trabecular surfaces with a wide edging comprised of Actinomyces colonies surrounded by a wall of leukocytes. Upper part of left panel: Fibrin particles and individual lymphocytes. Right panel: Actinomyces granules visualized in a PAS reaction; the red color represents a broad band of granules in the middle. The lower edge of the right panel images once again shows a bone sequestrum and typically empty osteocyte lacunae. Diagnosis: Aseptic bone necrosis with Actinomyces colonization.

The histopathological changes in necrotic bone may be visualized with MRI scans, as with CBCT/DVT. The images detect progressive cell death and the repair response (ie, edema). As the fat cells in normal bone marrow provide high signal intensity, it may be assumed that signal changes evident in the marrow are related to the death of fat cells. Necrotic adipocytes are a morphological characteristic of AIOJ/BMD/FDOJ.76 Following the application of a contrast agent, areas of ischemia may be identified as non-enhancing regions. Cases in which fibrosis and sclerosis of the bone occur may also result in lower signal intensity. Nevertheless, the currently available data on MRI results for BRONJ are limited,96 as are those related to AIOJ/BMD/FDOJ. Studies showed positron-emission tomography (PET) as a sensitive method for diagnosis of BRONJ. Thus, PET could be useful for evaluating the severity of BRONJ.97

2D-OPG is used to identify osteopathies of the jawbone. However, this imaging technique fails to show AIOJ/BMD/FDJ areas, thus generating false-negative findings. As a result, AIOJ/BMD/FDOJ have been highly neglected in dentistry and medicine.98 Therefore, transalveolar ultrasound sonography (TAU) appears to be necessary as an additional imaging technique in order to improve the diagnosis of AIOJ/BMD/FDOJ.99,100 A newly developed TAU device (TAU-n) measures sound velocity attenuation when the bone marrow has been penetrated. An ultrasound transmitter is placed over the jaw area and a thumbnail-sized receiver is placed inside the mouth. To obtain reproducible results when measuring bone density, the transmitter and receiver are arranged in a coplanar and fixed position. The parts of the receiving unit are placed inside a patients mouth, the acoustic coupling between those parts and the alveolar ridge is performed with the aid of a semi-solid gel (Figure 3). With the receiver containing 91 piezoelectric fields, sound waves are registered and converted into a color graph of the corresponding areas of bone density (Figure 4).On the graphic visualization, green indicates healthy, dense, and solid bone, yellow indicates the presence of ischemic metabolism, and orange and red highlight areas of AIOJ/BMD/FDOJ presence.101

Figure 4 Left panel shows positioning of transmitter (outside) and receiver (enoral) in the lower jaw; the red band marks the cheek. Right panel shows the transmitter (in blue at the right) and receiver (in green at the left) in a fixed coplanar position (blue bar connecting the transmitter and receiver); semi-solid gel pads between the transmitter and the cheek on the outside of the mouth and between receiver and the alveolar ridge in the enoral position; trans-alveolar ultrasonic impulse from the transmitter to receiver (arrows in blue).

Figure 5 Inconspicuous 2D-OPG findings (left panel); suspected osteolytic processes in areas 1719 in the sagittal section of the image using DVT (right panel). Lower panel: TAU measurement from region 17 to retromolar region 19. Legend: Green areas indicate normal bone density; yellow, orange, and red areas show decreasing bone density until complete osteolysis is reached.

A clinical case of a 55-year-old patient with prostate carcinoma who was treated with parenteral BPs received an X-ray diagnosis of non-exposed BRONJ with normal intraoral findings in the right upper jawbone from area 17 to retromolar area 19. While 2D-OPG of area 18/19 showed no suspicious findings, the CBCT/DVT image demonstrated ossification irregularities and partial cavities that resembled AIOJ/BMD/FDOJ. The development and progression of BRONJ could not be reliably determined by reference to these images and it was not possible to make a differential diagnosis. In contrast, TAU-n images clearly indicated osteolysis (see Figure 4, below). The postoperative light microscopy findings from area 18/19 showed marrow with adipose tissue, significant fibrillar and myxoid degeneration of adipocytes, individual lymphocytes, and mast cells; however, no florid inflammation was observed. These are the typical histological features of AIOJ/BMD/FDOJ.76 It is worth noting, however, that there was a large bone sequestrum with empty OC cavities, highly irregular trabecular surfaces, and empty marrow spaces, with Actinomyces colonization (Figure 3).

Several reviews have indicated that light microscopy examinations were able to detect that 68.8% of BRONJ cases featured Actinomyces colonization.32 Anaerobic Actinomyces has long been associated with necrotic bone findings in BRONJ lesions.102 Actinomyces colonization is thus a top priority as a possible pathological trigger with respect to BRONJ. Since we have not identified bacterial colonization in areas of AIOJ/BMD/FDOJ in our own studies,103 an accompanying secondary Actinomyces colonization seems to be an additional prerequisite for the development of BRONJ from an area of AIOJ/BMD/FDOJ in response to BP administration.

Table 1 displays all studies and their impact on the research question based on the inclusion and exclusion criteria in literature review.

Table 1 The Table Displays the Criteria for Inclusion of Specific Manuscripts in Our Research. Exclusion Criteria Were Unspecific Reviews Concentrating on Exposed BRONJ Only

Can hitherto little-known, yet according to our clinical experience37,76 epidemiologically widespread AIOJ/BMD/FDOJ represent cofactors in the development of BRONJ? The development of biological processes takes place in different stages and during various phases of transition. This also seems to be the case for BRONJ, as the exposed form found in the maxillofacial region represents the final, late-stage form of the NE-BRONJ variant. The focus of our study is thus on the early stage of BRONJ (Stage 0) without exposed bone, as based on the recommendations of the American Association of Oral and Maxillofacial Surgeons.5,20,104 Our hypothesis considers the NE-BRONJ variant as one stage of development featuring an unrecognized BMD that is characteristic of AIOJ/BMD/FDOJ and amplified by BP administration. The cumulative effects of BPs on pre-existing AIOJ/BMD/FDOJ support this premise. The relationship between AIOJ/BMD/FDOJ and the administration of BPs (as shown in Figure 6) leads, etiologically, to the non-exposed BRONJ variant, which is less clearly described in the literature than the late-stage form of BRONJ, and also results in considerable oral impairment.

Figure 6 Overview of the individual osteoimmunological signal cascades present in AIOJ/BMD/FDOJ and their conversion or amplification following BP administration, resulting in the development of BRONJ. Legend: A pair of arrows, one red and one green, indicates the reinforcement or, in one instance, the reversal of the typical overexpression or inhibition found in AIOJ/BMD/FDOJ following BP administration.

As BPs and AIOJ/BMD/FDOJ exert the same effects, resulting in the hyperfunctioning of R/C expression, OB activity, hypoxia/ischemia, and the inhibition of OC activity, vascularization, and AP activity, AIOJ/BMD/FDOJ may be regarded as a prerequisite to the formation of BRONJ. Changes in silent AIOJ/BMD/FDOJ processes, including strongly inhibited OC production, reduced RANKL activity, and increased OPG activity, appear to induce the occurrence of BRONJ. Figure 7 presents a hypothetical three-step model detailing the basic stages for the development of BRONJ at AIOJ/BMD/FDOJ areas. Regions with fatty-degenerative changes may be the focal point for the subsequent development of BRONJ, as such changes may constitute an additional risk factor. This is consistent with the hypothesis described in the literature, whereby bone necrosis precedes clinically evident ONJ that is exposed through the oral mucosa.78,105 Regions featuring subclinical changes and necrotic bone may represent significant risk factors in the development of BRONJ.104 Further, it is known that patients at each stage exhibit a very different bone composition.104

Figure 7 Three-step model for the development of BRONJ beginning with undetected AIOJ/BMD/FDOJ followed by the development of the NE-BRONJ variant, and finally by BRONJ.Notes: Exposed bone BNOJ (left panel). Bony sequestrum BRONJ (right panel). Figure courtesy of Professor J Bouquot.

The prevention of BRONJ is of paramount importance and has been repeatedly emphasized.106108 Thus, BPs should not be regarded as the sole cause of osteonecrosis. The results of this study indicate that unresolved areas of wound healing at extraction sites especially in former wisdom tooth areas may directly contribute to the pathogenesis of BRONJ. Other research has already described the involvement of the jaw in BRONJ as opposed to other bone sites.109 This may be because BPs are preferentially deposited in bones with high turnover rates such as the jawbone. The jawbone also presents with hidden conditions that according to our hypothesis share common characteristics with those found in AIOJ/BMD/FDOJ. Under the influence of BPs, areas of AIOJ/BMD/FDOJ may develop the pathological features of BRONJ. Efforts to prevent BRONJ, therefore, should not ignore the fact that BRONJ and AIOJ/BMD/FDOJ share similar osteoimmunological characteristics with respect to amplifying or reversing derailed signal cascades. Since AIOJ/BMD/FDOJ represent chronic, subclinical states, the sudden formation of BRONJ may be interpreted as a subsequent acute event. The early detection of BRONJ (as well as AIOJ/BMD/FDOJ) using X-ray techniques appears to be difficult. A new risk-benefit analysis should be considered: Patients should be screened for hidden oral risk factors, such as AIOJ/BMD/FDOJ. Thus, TAU may be used to measure bone density and fill this diagnostic gap. When parenteral BP therapy is administered, periodontal prophylaxis and tooth restoration should take precedence;110,111 furthermore, AIOJ/BMD/FDOJ should be diagnosed first, preferably (and accurately) with TAU-n, and then surgically eliminated. The formation of difficult-to-treat BRONJ could be avoided in certain cases if the exacerbation of pre-existing areas of AIOJ/BMD/FDOJ is prevented before initiating anti-tumorigenic BP therapy. Surgical opening of the cortex, removal of ischemic marrow, and accompanying wound care represent the only way to address cases of AIOJ/BMD/FDOJ.112 Consultation with an oncologist is mandatory, as the oncologist may insist on radiation therapy and the prevention of osteoradionecrosis of the jawbones via tooth restoration. To the best of our knowledge, we have highlighted, for the first time, the possible impact chains flowing from AIOJ/BMD/FDOJ and leading to the development of NE-BRONJ and further to exposed BRONJ. We also support the hypothesis presented herein with scientific data from the available literature. Due to the lack of clinical studies investigating these impact chains, multiple studies are necessary to elucidate the hypothesized relationships.

AIOJ, aseptic-ischemic osteonecrosis of the jawbone; BMD, bone marrow defects; BRONJ, bisphosphonate (BP)-related osteonecrosis of the jaw; CBCT, cone beam computed tomography; CCL5, chemokine (C-C motif) ligand 5; DVT, digital volume tomography; FDOJ, fatty-degenerative osteonecrosis/osteolysis of the jawbone; HU, hounsfield units; OPG, orthopantomogram; R/C, RANTES/CCL5; RANTES, regulated on activation, normal T cell expressed and secreted; TAU, transalveolar ultrasonography; TAU-n, new transalveolar ultrasonography device.

Hereby we confirm that written informed consent has been provided by the patient to have the case details and any accompanying images published. The data were collected as part of the normal everyday medical care of the patients and evaluated retrospectively. Institutional approval was not required to publish the case details.

English language editing of this manuscript was provided by Journal Prep Services. Additional English language editing was provided by Natasha Gabriel.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The corresponding author, Johann Lechner, is the holder of a patent used in the TAU-n apparatus and its associated software and reports a patent CaviTAU licensed to Dr. Johann Lechner. Bernd Zimmermann is an employee of QINNO. The authors report no other potential conflicts of interest for this work.

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Originally posted here:
[Full text] Osteonecrosis of the Jaw Beyond Bisphosphonates: Are There Any Unknown | CCIDE - Dove Medical Press

Recommendation and review posted by Bethany Smith

A SHIPYARD worker has potentially saved a stranger's life after donating his stem cells to a person in desperate need.

Brad Lawson, from Walney, first signed up to be a stem cell donor six years ago after an event at his college.

Stem cells are cells with the potential to develop into many different types of cells in the body.

Every 14 minutes, someone is diagnosed with blood cancer such as leukaemia.

For many, a bone marrow or blood stem cell transplant is their only chance.

They need cells from a healthy person with the same tissue type to replace and repair their own damaged cells.

About 30 per cent of people in need can find a suitable donor in their family but the other 70 per cent rely on a stranger to save their lives.

This is what prompted Mr Lawson to travel hundreds of miles to London to give his much-needed donation.

The 23-year-old said: "I first signed onto the register six years ago and hadn't thought much about it since.

"Then I was shocked to get a phone call the other week to say they'd matched a patient with my stem cells.

"It's quite rare to match with someone - it's only one in 800 people so I knew I had to help."

Mr Lawson travelled down to London where he underwent peripheral blood stem cell collection.

The process involves having a course of injections prior to collection to stimulate the bone marrow and increase the number of stem cells and white blood cells in the blood.

He said: "I had no hesitation about going down there when I got the call. When you sign up, you need to be fully committed if you do get a call.

"This could be someone's chance of survival and I would never pull out of something like that.

"The process was actually really easy. It takes about five hours and isn't painful at all.

"I absolutely hate needles and didn't find it painful at all."

Mr Lawson said it felt 'rewarding' to know his donation could have possibly saved a stranger's life.

"You could potentially give someone the chance to survive by signing up," he said.

"It's an amazing thing to do which could seriously make a difference.

"I may be in that position one day where I desperately need stem cells and would like to think someone out there would help me.

"Donations literally saves lives. It's a really rewarding thing to do to be able to help someone in this way."

Mr Lawson is urging the public to sign up to the register.

"Only about two per cent of people in the UK are actually on the register," he said.

"I'm telling everyone to sign up and raise awareness of stem cell donation.

"The more people we can get to sign up, and save lives, the better."

To register, visit: http://www.dkms.org.uk/en/register-now.

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Shipyard worker Brad Lawson from Walney may have saved a stranger's life with his stem cell donation - NW Evening Mail

Recommendation and review posted by Bethany Smith

Bone marrow aspiration and trephine biopsy are usually performed on the back of the hipbone, or posterior iliac crest. An aspirate can also be obtained from the sternum (breastbone). For the sternal aspirate, the patient lies on their back, with a pillow under the shoulder to raise the chest. A trephine biopsy should never be performed on the sternum, due to the risk of injury to blood vessels, lungs or the heart.

The need to selectively isolate and concentrate selective cells, such as mononuclear cells, allogeneic cancer cells, T cells and others, is driving the market. Over 30,000 bone marrow transplants occur every year. The explosive growth of stem cells therapies represents the largest growth opportunity for bone marrow processing systems.

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Europe and North America spearheaded the market as of 2018, by contributing over 74.0% to the overall revenue. Majority of stem cell transplants are conducted in Europe, and it is one of the major factors contributing to the lucrative share in the cell harvesting system market.

In 2018, North America dominated the research landscape as more than 54.0% of stem cell clinical trials were conducted in this region. The region also accounts for the second largest number of stem cell transplantation, which is further driving the demand for harvesting in the region.

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Asia Pacific is anticipated to witness lucrative growth over the forecast period, owing to rising incidence of chronic diseases and increasing demand for stem cell transplantation along with stem cell-based therapy. Japan and China are the biggest markets for harvesting systems in Asia Pacific. Emerging countries such as Mexico, South Korea, and South Africa are also expected to report lucrative growth over the forecast period. Growing investment by government bodies on stem cell-based research and increase in aging population can be attributed to the increasing demand for these therapies in these countries.

Major players operating in the global bone marrow processing systems market are ThermoGenesis (Cesca Therapeutics inc.), RegenMed Systems Inc., MK Alliance Inc., Fresenius Kabi AG, Harvest Technologies (Terumo BCT), Arthrex, Inc. and others.

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Bone Marrow Processing Systems Market- Global Research Analysis, Trends, Competitive Share and Forecasts 2018 2025 - NeighborWebSJ

Recommendation and review posted by Bethany Smith

The Global Adipose Derived Stem Cell Therapy Market report provides a holistic evaluation of the market for the forecast period (20192025). The report comprises various segments as well as an analysis of the trends and factors that are playing a substantial role in the market. These factors; the market dynamics involve the drivers, restraints, opportunities and challenges through which the impact of these factors in the market are outlined. The drivers and restraints are intrinsic factors whereas opportunities and challenges are extrinsic factors of the market. The Global Adipose Derived Stem Cell Therapy Market study provides an outlook on the development of the market in terms of revenue throughout the prognosis period.

In order to present an executive-level model of the market and its future perspectives, the Adipose Derived Stem Cell Therapy Market report presents a clear segmentation based on different parameters. The factors that affect these segments are also discussed in detail in the report.

Adipose derived stem cells (ADSCs) are stem cells derived from adipocytes, and can differentiate into variety of cell types. ADSCs have multipotency similar to bone marrow mesenchymal stem cells, thus ADSCs substitute for bone marrow as a source of stem cells. Numerous manual and automatic stem cell separation procedures are adopted in order to separate adipose stem cells (ASCs) from adipose tissue. Flow cytometry can also be used to isolate ADSCs from other stem cells within a cell solution.

Major Players included in this report are as follows BioRestorative Therapies, Inc., Celltex Therapeutics Corporation, Antria, Inc., Cytori Therapeutics Inc., Intrexon Corporation, Mesoblast Ltd., iXCells Biotechnologies, Pluristem Therapeutics, Inc., Thermo Fisher Scientific, Inc., Tissue Genesis, Inc., Cyagen US Inc., Celprogen, Inc., and Lonza Group, among others.

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Adipose Derived Stem Cell Therapy Market 2018: Production, Sales, Supply, Demand, Analysis and Forecast To 2026 | BioRestorative Therapies, Inc.,...

Recommendation and review posted by Bethany Smith

Bone marrow aspiration and trephine biopsy are usually performed on the back of the hipbone, or posterior iliac crest. An aspirate can also be obtained from the sternum (breastbone). For the sternal aspirate, the patient lies on their back, with a pillow under the shoulder to raise the chest. A trephine biopsy should never be performed on the sternum, due to the risk of injury to blood vessels, lungs or the heart.

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The need to selectively isolate and concentrate selective cells, such as mononuclear cells, allogeneic cancer cells, T cells and others, is driving the market. Over 30,000 bone marrow transplants occur every year. The explosive growth of stem cells therapies represents the largest growth opportunity for bone marrow processing systems.Europe and North America spearheaded the market as of 2016, by contributing over 74.0% to the overall revenue. Majority of stem cell transplants are conducted in Europe, and it is one of the major factors contributing to the lucrative share in the cell harvesting system market.

In 2016, North America dominated the research landscape as more than 54.0% of stem cell clinical trials were conducted in this region. The region also accounts for the second largest number of stem cell transplantation, which is further driving the demand for harvesting in the region.Asia Pacific is anticipated to witness lucrative growth over the forecast period, owing to rising incidence of chronic diseases and increasing demand for stem cell transplantation along with stem cell-based therapy.

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Japan and China are the biggest markets for harvesting systems in Asia Pacific. Emerging countries such as Mexico, South Korea, and South Africa are also expected to report lucrative growth over the forecast period. Growing investment by government bodies on stem cell-based research and increase in aging population can be attributed to the increasing demand for these therapies in these countries.

Major players operating in the global bone marrow processing systems market are ThermoGenesis (Cesca Therapeutics inc.), RegenMed Systems Inc., MK Alliance Inc., Fresenius Kabi AG, Harvest Technologies (Terumo BCT), Arthrex, Inc. and others

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Bone Marrow Processing System Market: Segmental Highlights and Table of Content 2025 - NeighborWebSJ

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DBMR has added a new report titled Global stem cell banking market with analysis provides the insights which bring marketplace clearly into the focus and thus help organizations make better decisions. This market research report contains fundamental, secondary and advanced information related to the global status and trend, market size, sales volume, market share, growth, future trends analysis, segment and forecasts from 2020 2027. This Global stem cell banking market report helps businesses to define their own strategies for the up gradation in the existing product, possible modifications required in the future product, sales, marketing promotion and distribution of the product in the existing and the new market. In this report, a thorough investment analysis is offered which forecasts imminent opportunities for the market players and develops the strategies to grow return on investment (ROI).

Global stem cell banking market is set to witness a substantial CAGR of 11.03% in the forecast period of 2019- 2026. The report contains data of the base year 2018 and historic year 2017. The increased market growth can be identified by the increasing procedures of hematopoietic stem cell transplantation (HSCT), emerging technologies for stem cell processing, storage and preservation. Increasing birth rates, awareness of stem cell therapies and higher treatment done viva stem cell technology.

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Competitive Analysis:

Global stem cell banking market is highly fragmented and the major players have used various strategies such as new product launches, expansions, agreements, joint ventures, partnerships, acquisitions, and others to increase their footprints in this market. The report includes market shares of inflammatory disease drug delivery market for Global, Europe, North America, Asia-Pacific, South America and Middle East & Africa.

Key Market Competitors:

Few of the major competitors currently working in global inflammatory disease drug delivery market are: NSPERITE N.V, Caladrius, ViaCord, CBR Systems, Inc, SMART CELLS PLUS, LifeCell International, Global Cord Blood Corporation, Cryo-Cell International, Inc., StemCyte India Therapeutics Pvt. Ltd, Cordvida, ViaCord, Cryoviva India, Vita34 AG, CryoHoldco, PromoCell GmbH, Celgene Corporation, BIOTIME, Inc., BrainStorm Cell Therapeutics and others

Market Definition:Global Stem Cell Banking Market

Stem cells are cells which have self-renewing abilities and segregation into numerous cell lineages. Stem cells are found in all human beings from an early stage to the end stage. The stem cell banking process includes the storage of stem cells from different sources and they are being used for research and clinical purposes. The goal of stem cell banking is that if any persons tissue is badly damaged the stem cell therapy is the cure for that. Skin transplants, brain cell transplantations are some of the treatments which are cured by stem cell technique.

Cord Stem Cell Banking MarketDevelopment and Acquisitions in 2019

Cord Stem Cell Banking MarketScope

Cord Stem Cell Banking Marketis segmented on the basis of countries into U.S., Canada and Mexico in North America, Germany, France, U.K., Netherlands, Switzerland, Belgium, Russia, Italy, Spain, Turkey, Rest of Europe in Europe, China, Japan, India, South Korea, Singapore, Malaysia, Australia, Thailand, Indonesia, Philippines, Rest of Asia-Pacific (APAC) in the Asia-Pacific (APAC), Saudi Arabia, U.A.E, South Africa, Egypt, Israel, Rest of Middle East and Africa (MEA) as a part of Middle East and Africa (MEA), Brazil, Argentina and Rest of South America as part of South America.

All country based analysis of the cord stem cell banking marketis further analyzed based on maximum granularity into further segmentation. On the basis of storage type, the market is segmented into private banking, public banking. On the basis of product type, the market is bifurcated into cord blood, cord blood & cord tissue. On the basis of services type, the market is segmented into collection & transportation, processing, analysis, storage. On the basis of source, market is bifurcated into umbilical cord blood, bone marrow, peripheral blood stem, menstrual blood. On the basis of indication, the market is fragmented into cerebral palsy, thalassemia, leukemia, diabetes, autism.

Cord stem cell trading is nothing but the banking of the vinculum plasma cell enclosed in the placenta and umbilical muscle of an infant. This ligament plasma comprises the stem blocks which can be employed in the forthcoming time to tackle illnesses such as autoimmune diseases, leukemia, inherited metabolic disorders, and thalassemia and many others.

Market Drivers

Market Restraint

Key Pointers Covered in the Cord Stem Cell Banking MarketIndustry Trends and Forecast to 2026

Key Developments in the Market:

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Global stem cell banking market Analysis With Key Players, Applications, Trends And Forecasts 2028 - The Courier

Recommendation and review posted by Bethany Smith

Little Evie's mum confessed it's "torture" and the "worst bit for a parent" waiting to find out results of her daughter's life-saving transplant.

Evie Hodgson, eight, was given a vital transplant from a stem cell donor after she was diagnosed with the rare blood disorder, aplastic anaemia.

The mother and daughter spoke to Holly Willoughby and Phillip Schofield on This Morning via video link from hospital in Newcastle on Tuesday.

It was at Great North Childrens Hospital where the little girl had the six-hour operation for the transplant.

Tina, 37, of Whitby, North Yorkshire, admitted it was "torturous" waiting to find out whether her daughter's body accepts the new bone marrow.

Their journey isn't over yet as the family won't find out for three to four weeks.

However, the mother expressed her relief was "phenomenal" now Evie had the life-saving transplant.

She said: "This is the worst bit for a parent. We got the donor, we had been searching for the transplant. It went ahead. The relief was phenomenal.

"We're waiting for her body to accept new stem cells. They've said three to four weeks. It's torture."

She added: "Absolutely, hopefully we will have some good news then."

Little Evie told This Morning presents that she feels "great" right now.

The brave girl gushed about the "kind" nurses and doctors who have been looking after her.

She said: "Yes, all the nurses and doctors are very kind to me. They're always getting me chocolate milk."

The mother and daughter duo will join Holly and Phil once again in three to four weeks' time once the results are in.

Evie was diagnosed with aplastic anaemia in May last year.

Aplastic anaemia is a disease where the body has a deficiency of all blood cells types.

Last year, Evie was unable to undergo vital surgery because her original donor dropped out in September.

At the time, the original donor was the only match available in the world before the new match was found.

The Mirrors Change the Law for Life crusade saw a new opt out' system introduced in England last May.

Named Max and Keiras Law in honour of our poster boy Max Johnson, 13, of Winsford, Cheshire, and his heart donor Keira Ball, nine, who died in a car accident near her home in Barnstaple, Devon, it means everyone is understood to be a donor when they die.

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Mum of girl, 8, with rare illness is in 'torture' waiting for results of vital operation - Irish Mirror

Recommendation and review posted by Bethany Smith

Gosselies, Belgium and Modena, Italy, 14January 2021, 7am CET BONE THERAPEUTICS (Euronext Brussels and Paris: BOTHE), the cell therapy company addressing unmet medical needs in orthopedics and other diseases, and Rigenerand SRL, the biotech company that both develops and manufactures medicinal products for cell therapy applications, primarily for regenerative medicine and oncology, today announce the signing of a first agreement for a process development partnership.

Allogeneic mesenchymal stem cell (MSC) therapies are currently being developed at an incredible pace and are evaluated in numerous clinical studies covering diverse therapeutic areas such as bone and cartilage conditions, liver, cardiovascular and autoimmune diseases in which MSCs could have a significant positive effect. Advances in process development to scale up these therapies could have major impacts for both their approval and commercial viability. This will be essential to bring these therapies to market to benefit patients as quickly as possible, said Miguel Forte, CEO, Bone Therapeutics. Hence, whilst Bone Therapeutics is driving on its existing clinical development programs, we have signed a first formal agreement with Rigenerand as a fellow MSC-based organization. This will result in both companies sharing extensive expertise in the process development and manufacturing of MSCs and cell and gene therapy medicinal products. Bone Therapeutics also selected Rigenerand to partner with for their additional experience with wider process development of advanced therapy medicinal products (ATMPs), including the conditioning and editing of MSCs. Rigenerand was founded by Massimo Dominici, a world opinion leader in the cell therapy with an unparalleled MSC expertise and knowledge.

The scope of collaborations between Bone Therapeutics and Rigenerand aims to focus on different aspects of product and process development for Bone Therapeutics expanding therapeutic portfolio. Rigenerand will contribute to improving the processes involved in the development and manufacture of Bone Therapeutics MSC based allogeneic differentiated cell therapy products as they advance towards patients. The first collaboration between the two organizations will initially focus on augmented professional bone-forming cells cells that are differentiated and programmed for a specific task. There is also potential for Bone Therapeutics to broaden its therapeutic targets and explore new mechanisms of action with potential gene modifications for its therapeutic portfolio.

In addition to Rigenerands MSC expertise, Bone Therapeutics also selected Rigenerand as a partner for Rigenerands GMP manufacturing facility. This facility, situated in Modena, Italy, has been designed to host a number of types of development processes for ATMPs. These include somatic, tissue engineered and gene therapy processes. These multiple areas of Rigenerand capabilities enable critical development of new processes and implementation of the gene modification of existing processes. In addition, Rigenerand has built considerable experience in cGMP manufacturing of MSC-based medicinal products, including those that are genetically modified.

Process development and manufacturing is a key part of the development for ATMPs internationally. Navigating these therapies through the clinical development phase and into the market requires a carefully considered process development pathway, said Massimo Dominici, scientific founder, Rigenerand, professor of medical oncology, and former President of the International Society for Cell & Gene Therapy (ISCT). This pathway needs to be flexible, as both the market and materials of these therapies continues to evolve alongside an improved clinical efficacy.

Rigenerand will offer considerable input from its experience of MSC-based therapies to enable Bone Therapeutics to keep and further accelerate the pace in development of the product processes of its MSC based allogeneic differentiated cell therapy as they advance towards patients, said Giorgio Mari, CEO, Rigenerand. We will continue to use our MSC expertise in the development of Rigenerands own products, as well as in process development and manufacturing cell and gene therapies for partner organizations across the globe.

About Bone Therapeutics

Bone Therapeutics is a leading biotech company focused on the development of innovative products to address high unmet needs in orthopedics and other diseases. The Company has a, diversified portfolio of cell and biologic therapies at different stages ranging from pre-clinical programs in immunomodulation to mid-to-late stage clinical development for orthopedic conditions, targeting markets with large unmet medical needs and limited innovation.

Bone Therapeutics is developing an off-the-shelf next-generation improved viscosupplement, JTA-004, which is currently in Phase III development for the treatment of pain in knee osteoarthritis. Consisting of a unique combination of plasma proteins, hyaluronic acid - a natural component of knee synovial fluid, and a fast-acting analgesic, JTA-004 intends to provide added lubrication and protection to the cartilage of the arthritic joint and to alleviate osteoarthritic pain and inflammation. Positive Phase IIb efficacy results in patients with knee osteoarthritis showed a statistically significant improvement in pain relief compared to a leading viscosupplement.

Bone Therapeutics core technology is based on its cutting-edge allogeneic cell therapy platform with differentiated bone marrow sourced Mesenchymal Stromal Cells (MSCs) which can be stored at the point of use in the hospital. Currently in pre-clinical development, BT-20, the most recent product candidate from this technology, targets inflammatory conditions, while the leading investigational medicinal product, ALLOB, represents a unique, proprietary approach to bone regeneration, which turns undifferentiated stromal cells from healthy donors into bone-forming cells. These cells are produced via the Bone Therapeutics scalable manufacturing process. Following the CTA approval by regulatory authorities in Europe, the Company has initiated patient recruitment for the Phase IIb clinical trial with ALLOB in patients with difficult tibial fractures, using its optimized production process. ALLOB continues to be evaluated for other orthopedic indications including spinal fusion, osteotomy, maxillofacial and dental.

Bone Therapeutics cell therapy products are manufactured to the highest GMP (Good Manufacturing Practices) standards and are protected by a broad IP (Intellectual Property) portfolio covering ten patent families as well as knowhow. The Company is based in the BioPark in Gosselies, Belgium. Further information is available at http://www.bonetherapeutics.com.

About Rigenerand

Rigenerand SRL is a biotech company that both develops and manufactures medicinal products for cell therapy applications, primarily for regenerative medicine and oncology and 3D bioreactors as alternative to animal testing for pre-clinical investigations.

Rigenerand operates through three divisions:

Rigenerand is developing RR001, a proprietary ATMP gene therapy medicinal product for the treatment of pancreatic ductal adenocarcinoma (PDAC). RR001 has been granted an Orphan Drug Designation (ODD) by US-FDA and from the European Medicine Agency. The Clinical trial is expected to start in Q2 2021.

Rigenerand is headquartered in Medolla, Modena, Italy, with more than 1,200 square metres of offices, R&D and quality control laboratories and a cell factory of 450 square metres of sterile cleanroom (EuGMP Grade-B) with BSL2/BSL3 suites for cell and gene therapies manufacturing. It combines leaders and academics from biopharma and medical device manufacturing sectors.

For further information, please contact:

Bone Therapeutics SAMiguel Forte, MD, PhD, Chief Executive OfficerJean-Luc Vandebroek, Chief Financial OfficerTel: +32 (0)71 12 10 00investorrelations@bonetherapeutics.com

For Belgian Media and Investor Enquiries:BepublicCatherine HaquenneTel: +32 (0)497 75 63 56catherine@bepublic.be

International Media Enquiries:Image Box CommunicationsNeil Hunter / Michelle BoxallTel: +44 (0)20 8943 4685neil.hunter@ibcomms.agency / michelle@ibcomms.agency

For French Media and Investor Enquiries:NewCap Investor Relations & Financial CommunicationsPierre Laurent, Louis-Victor Delouvrier and Arthur RouillTel: +33 (0)1 44 71 94 94bone@newcap.eu

Certain statements, beliefs and opinions in this press release are forward-looking, which reflect the Company or, as appropriate, the Company directors current expectations and projections about future events. By their nature, forward-looking statements involve a number of risks, uncertainties and assumptions that could cause actual results or events to differ materially from those expressed or implied by the forward-looking statements. These risks, uncertainties and assumptions could adversely affect the outcome and financial effects of the plans and events described herein. A multitude of factors including, but not limited to, changes in demand, competition and technology, can cause actual events, performance or results to differ significantly from any anticipated development. Forward looking statements contained in this press release regarding past trends or activities should not be taken as a representation that such trends or activities will continue in the future. As a result, the Company expressly disclaims any obligation or undertaking to release any update or revisions to any forward-looking statements in this press release as a result of any change in expectations or any change in events, conditions, assumptions or circumstances on which these forward-looking statements are based. Neither the Company nor its advisers or representatives nor any of its subsidiary undertakings or any such persons officers or employees guarantees that the assumptions underlying such forward-looking statements are free from errors nor does either accept any responsibility for the future accuracy of the forward-looking statements contained in this press release or the actual occurrence of the forecasted developments. You should not place undue reliance on forward-looking statements, which speak only as of the date of this press release.

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Bone Therapeutics and Rigenerand sign partnership for cell therapy process development - GlobeNewswire

Recommendation and review posted by Bethany Smith