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

By daniellenierenberg

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: Regional analysis includes:

The study will also feature the key companies operating in the industry, their product/business portfolio, market share, financial status, regional share, segment revenue, SWOT analysis, key strategies including mergers & acquisitions, product developments, joint ventures & partnerships an expansions among others, and their latest news as well. The study will also provide a list of emerging players in the Adipose Derived Stem Cell Therapy Market.

Adipose Derived Stem Cell Therapy Market scope

A basic summary of the competitive landscape A detailed breakdown of the regional expanse A short overview of the segmentation

Furthermore, this study will help our clients solve the following issues:

Cyclical dynamics We foresee dynamics of industries by using core analytical and unconventional market research approaches. Our clients use insights provided by us to maneuver themselves through market uncertainties and disruptions.

Identifying key cannibalizes Strong substitute of a product or service is the most prominent threat. Our clients can identify key cannibalizes of a market, by procuring our research. This helps them in aligning their new product development/launch strategies in advance.

Spotting emerging trends Our Ecosystem offering helps the client to spot upcoming hot market trends. We also track possible impact and disruptions which a market would witness by a particular emerging trend. Our proactive analysis helps clients to have an early mover advantage.

Interrelated opportunities This report will allow clients to make decisions based on data, thereby increasing the chances that the strategies will perform better if not best in the real world.

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Some of the Major Highlights of TOC covers:

Adipose Derived Stem Cell Therapy Regional Market Analysis

Adipose Derived Stem Cell Therapy Production by Regions Global Adipose Derived Stem Cell Therapy Production by Regions Global Adipose Derived Stem Cell Therapy Revenue by Regions Adipose Derived Stem Cell Therapy Consumption by Regions

Adipose Derived Stem Cell Therapy Segment Market Analysis (by Type)

Global Adipose Derived Stem Cell Therapy Production by Type Global Adipose Derived Stem Cell Therapy Revenue by Type Adipose Derived Stem Cell Therapy Price by Type

Adipose Derived Stem Cell Therapy Segment Market Analysis (by Application)

Global Adipose Derived Stem Cell Therapy Consumption by Application Global Adipose Derived Stem Cell Therapy Consumption Market Share by Application (2014-2019)

Adipose Derived Stem Cell Therapy Major Manufacturers Analysis

Adipose Derived Stem Cell Therapy Production Sites and Area Served Product Introduction, Application and Specification Adipose Derived Stem Cell Therapy Production, Revenue, Ex-factory Price and Gross Margin (2014-2019)Main Business and Markets Served

Key questions answered in the report:

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Key Benefits

Major countries in each region are mapped according to individual market revenue. Comprehensive analysis of factors that drive and restrict market growth is provided. The report includes an in-depth analysis of current research and clinical developments within the market. Key players and their key developments in recent years are listed.And More.

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

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Global Cell Isolation Market SWOT Analysis, Key Indicators, Forecast 2027 : Becton, Dickinson, and Company, Thermo Fisher Scientific KSU | The…

By daniellenierenberg

The market research report titled Cell Isolation Market by Product (Instruments and Consumables), by Cell Type (Animal and Human), by Cell Source (Adipose Tissue, Embryonic/Cord Blood Stem Cells, and Bone Marrow), by Technique (Surface Marker-Based Cell Isolation, Centrifugation-Based Cell Isolation, and Filtration-Based Cell Isolation), by Application (Cancer Research, Biomolecule Isolation, Tissue Regeneration & Regenerative Medicine, Stem Cell Research, In Vitro Diagnostics, and Others), and By End-User (Hospitals & Diagnostic Laboratories, Research Laboratories & Institutes, Biotechnology & Biopharmaceutical Companies, and Others): Global Industry Perspective, Comprehensive Analysis, and Forecast, 20182025 published by Zion Market Research provides an insightful comprehension about the growth aspects, dynamics, and working of the globalCell IsolationMarket. The report entails details about the market with data collected over the years with its wide-ranging analysis. It also comprises the competitive landscape within the market together with a detailed evaluation of the leading players within the global Cell Isolation Market. In addition, it sheds light on the profiles of the key vendors/manufacturers comprising thorough assessment of the market share, production technology, market entry strategies, revenue forecasts, and so on. Further, the report will encompass the fundamental strategic activities such as product developments, mergers & acquisitions, launches, events, partnerships, collaborations, and so on. Apart from this, it will also present the new entrants contributing their part in the market growth.

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Global Cell Isolation Market: Competitive Players

Becton, Dickinson, and Company, Thermo Fisher Scientific, Inc., Merck KGaA, Beckman Coulter Inc., Terumo BCT, Bio-Rad Laboratories, Inc.

The Cell Isolation Market report also entails exhaustive examination of the key factors likely to propel or restrict the expansion of the global Cell Isolation Market during the forecast period in addition to the most recent and promising future trends in the market. Moreover, the report uses SWOT analysis and other methodologies to analyze the numerous segments [Product, Applications, End-Users, and Major Regions] of the global Cell Isolation Market. Furthermore, it comprises valuable understanding about the segments like their growth potential, market share, and developments. It also evaluates the market on the basis of its major geographical regions [Latin America, North America, Asia Pacific, Middle & East Africa, and Europe]. It entails quantitative and qualitative facets of the market in association to each country and region enlisted in the report.

Promising Regions & Countries Mentioned In The Cell Isolation Market Report:

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The Cell Isolation Market report also stipulates the computed expected CAGR of the market estimated on the basis of the existing and previous records concerning the global Cell Isolation Market. The report analyzes the market with the aim of being capable to get a clear picture of prevailing and anticipated growth patterns of the market. Furthermore, it entails the impact of numerous federal policies and rules on the growth and dynamics of the market during the forecast period. The thorough assessment put forth by our analysts assist to get more profound acquaintance of global markets and related industries. In addition, the report encompasses various tactics to discover the weaknesses, opportunities, risks, and strengths having the potential to impact the global market expansion.

Impact of COVID-19 (Coronavirus):The report will also entail a dedicated section assessing the influence of COVID-19 on the expansion of the global Cell Isolation Market during the coming period.

Report Customization:Clients can request for customization of report as per their need for additional data.

Apart from this, our team will be accessible around the clock to our clients for any kind of assistance.

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Global Cell Isolation Market SWOT Analysis, Key Indicators, Forecast 2027 : Becton, Dickinson, and Company, Thermo Fisher Scientific KSU | The...

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‘I was excited to help somebody’: Montana Western’s Dylan Pope reflects on donating bone marrow – MontanaSports

By daniellenierenberg

DILLON Dylan Pope concurs that 2020 was, by and large, not a great year. But he still found a way to make the most of it.

"It was a pretty tough year but having this to look forward to and reflect on has been something pretty big for me," he said.

Pope, a Montana Western defensive back, made the decision to donate bone marrow in December.

"I was nervous, but I was excited to help somebody," Pope said.

At the encouragement of his sister, Mariah, Pope registered with a non-profit called Be The Match in March, shortly after coronavirus knocked the world off kilter.

According to the organization's website, only one out of every 430 registered members will actually go on to donate bone marrow. Pope's sister has been registered for years without a match.

So, Pope was understandably taken aback when, after a little more than three months, he received a call telling him that he had been deemed a suitable donor for an anonymous recipient to receive his blood stem cells, which are derived from bone marrow.

"At first I thought it was fake," Pope said. "I didn't think there was any way it was going to happen after just three months."

With a donation date set in December -- because of confidentiality policies, Pope can't disclose what state or hospital the procedure took place at -- the next months were what one would expect: a lot of paperwork and a lot of blood tests.

The week before the donation, he began receiving daily injections to increase his stem cell count. He then made the trip with his younger brother, Brayton.

The process took eight hours and required only local anesthesia. A needle in his right arm drew blood, ran it through a machine that extracted stem cells and then a needle in his left arm injected blood back into his body.

"It's really not nearly as scary when you get there as you think it's going to be," Pope said.

It'll be a year before Pope learns the identity of who received his bone marrow. He's certain it'll be a moving, powerful experience.

"I bet it'll be pretty emotional thing for both of us, because it was pretty cool to be able to help them," Pope said.

Ryan Nourse, Montana Western's head football coach, said he wasn't surprised by Pope's willingness to donate bone marrow and said he and the program supported him the entire way.

"I think that's a really brave thing, courageous thing for Dylan to go do," Nourse said. "I think that selflessness will shine through to the other guys knowing that maybe I could help somebody in a similar position someday."

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Global Bone Marrow-Derived Stem Cells (BMSCS) Market 2021 Overview, Trends, Growth Factors and Leading Players With Detailed Analysis of Industry…

By daniellenierenberg

By providing an absolute overview of the market, Global Bone Marrow-Derived Stem Cells (BMSCS) Market report covers various aspects of market analysis, product definition, market segmentation, key developments, and the existing vendor landscape. Such market insights can be accomplished with this comprehensive Global Bone Marrow-Derived Stem Cells (BMSCS) Market research report which takes into account all the aspects of current and future market. The report provides wide-ranging analysis of the market structure along with the estimations of the various segments and sub-segments of the market. This Global Bone Marrow-Derived Stem Cells (BMSCS) Market research report delivers an analytical measurement of the main challenges faced bythe business currently and in the upcoming years.

Bone marrow-derivedstem cells(BMSCS) market is expected to gain market growth in the forecast period of 2020 to 2027. Data Bridge Market Research analyses the market to growing at a CAGR of 10.4% in the above-mentioned forecast period. Increasing awareness regarding the benefits associates with the preservation of bone marrow derived stem cells will boost the growth of the market.

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The major players covered in the bone marrow-derived stem cells (BMSCS) market report are CBR Systems, Inc, Cordlife Sciences India Pvt. Ltd., Cryo-Cell International, Inc.ESPERITE N.V., LifeCell International Pvt. Ltd., StemCyte India Therapeutics Pvt. Ltd, PerkinElmer Inc, Global Cord Blood Corporation., Smart Cells International Ltd., Vita 34 among other domestic and global players.

Some of the factors such as introduction of novel technologies for the preservation of stem cells and their storage, surging investment that will help in research activities leading to stem cells benefits, adoption of hemotopoietic stem cell transplantation system will accelerate the growth of the bone marrow-derived stem cells (BMSCS) market in the forecast period of 2020-2027. Various factors that will create opportunities in the bone marrow-derived stem cells (BMSCS) market are increasing occurrences of various diseases along with rising applications in emerging economies.

Large cost of operation and strict regulatory framework will restrict the growth of bone marrow-derived stem cells (BMSCS) market in the above mentioned forecast period. Ethical concern leading to stem cells will become the biggest challenge in the market growth.

Global Bone Marrow-Derived Stem Cells (BMSCS) Market By Service Type (Sample Preservation and Storage, Sample Analysis, Sample Processing, Sample Collection and Transportation), Application (Personalized Banking Applications, Research Applications, Clinical Applications), Country (U.S., Canada, Mexico, Germany, Italy, U.K., France, Spain, Netherland, Belgium, Switzerland, Turkey, Russia, Rest of Europe, Japan, China, India, South Korea, Australia, Singapore, Malaysia, Thailand, Indonesia, Philippines, Rest of Asia- Pacific, Brazil, Argentina, Rest of South America, South Africa, Saudi Arabia, UAE, Egypt, Israel, Rest of Middle East & Africa), Market Trends and Forecast to 2027

Global Bone Marrow-Derived Stem Cells (BMSCS) Market Scope and Market Size

Bone marrow-derivedstem cells(BMSCS) market is segmented on the basis of service type and application. The growth amongst these segments will help you analyse meagre growth segments in the industries, and provide the users with valuable market overview and market insights to help them in making strategic decisions for identification of core market applications.

Thisbonemarrow-derived stem cells (BMSCS) market report provides details of new recent developments, trade regulations, import export analysis, production analysis, value chain optimization, market share, impact of domestic and localised market players, analyses opportunities in terms of emerging revenue pockets, changes in market regulations, strategic market growth analysis, market size, category market growths, application niches and dominance, product approvals, product launches, geographic expansions, technological innovations in the market. To gain more info on bone marrow-derived stem cells (BMSCS) market contactData Bridge Market Researchfor anAnalyst Brief, our team will help you take an informed market decision to achieve market growth.

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Bone Marrow-Derived Stem Cells (BMSCS) Market Country Level Analysis

Bone marrow-derivedstem cells(BMSCS) market is analysed and market size insights and trends are provided by country, service type and application as referenced above.

The country section of the bone marrow-derivedstem cells(BMSCS) market report also provides individual market impacting factors and changes in regulation in the market domestically that impacts the current and future trends of the market. Data points such as consumption volumes, production sites and volumes, import export analysis, price trend analysis, cost of raw materials, down-stream and upstream value chain analysis are some of the major pointers used to forecast the market scenario for individual countries. Also, presence and availability of global brands and their challenges faced due to large or scarce competition from local and domestic brands, impact of domestic tariffs and trade routes are considered while providing forecast analysis of the country data.

Healthcare Infrastructure Growth Installed Base and New Technology Penetration

Bone marrow-derived stem cells (BMSCS) market also provides you with detailed market analysis for every country growth in healthcare expenditure for capital equipments, installed base of different kind of products for bone marrow-derived stem cells (BMSCS) market, impact of technology using life line curves and changes in healthcare regulatory scenarios and their impact on the bone marrow-derived stem cells (BMSCS) market. The data is available for historic period 2010 to 2018.

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Key Highlights of Report

Competitive Landscape and Bone Marrow-Derived Stem Cells (BMSCS) Market Share Analysis

Bone marrow-derived stem cells (BMSCS) market competitive landscape provides details by competitor. Details included are company overview, company financials, revenue generated, market potential, investment in research and development, new market initiatives, global presence, production sites and facilities, production capacities, company strengths and weaknesses, product launch, product width and breadth, application dominance. The above data points provided are only related to the companies focus related to bone marrow-derived stem cells (BMSCS) market.

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Global Bone Marrow-Derived Stem Cells (BMSCS) Market 2021 Overview, Trends, Growth Factors and Leading Players With Detailed Analysis of Industry...

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Brave Evie Hodgson from Sleights finally has bone marrow transplant after one last ‘twist in the tale’ – Yorkshire Live

By daniellenierenberg

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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Mesoblast Limited: Is Stemcell Therapy Ready For Prime Time? – Sick Economics

By daniellenierenberg

Mesoblast, MESO, is an Australian based biopharmaceutical company that has been a market favorite, even though the companys ups and downs have confused many investors.

The MESO share price has been inconsistent lately. This has prompted many investors to ask why. Analyzed carefully, MESO has done better than many stem cell businesses. Most stem cell businesses fail to ever make a profit and fail to even get a product to market. This can cause long-term problems with the stock price of any company.

ByMichael A. Mannen, MS

Mesoblast as a company is committed to offering groundbreaking cellular therapies for the treatment of many severe diseases using Mesenchymal Stem Cells. They are dedicated to cellular medicines and leveraging their stem cell technology. There are not many successful companies in this niche.

Adult stem cells are undifferentiated cells that divide and rebuild the damaged tissue. Mesenchymal Stem Cells are a type of adult stem cells generated from some of the adult tissues present in the body.

Stem cells have been found by scientists to have two properties: self-renewal and the potential to divide into specialized cell types. Multi-potent, mesenchymal stem cells are found to be present in many adult tissues. The bone marrow is considered by many scientists to be the most usable reservoir of adult human stem cells.

For several disorders, such as heart failure, the capacity to rebuild tissue may be groundbreaking for treatment. And this has been the inspiration for many companies exploring stem cell therapies.

However, what differentiates Mesoblast from other stem cell companies is its approach to treating inflammatory diseases. Their products have the potential to make breakthroughs a reality for many diseases.

The company has developed and manufactured its own patented mesenchymal lineage cells to be used for a range of ailments. These have a potential for the regeneration of tissues. These cells, however, secrete a number of biomolecules which can help the body heal more than just tissue damage. They may be important to supporting immune responses needed for recovery in many diseases.

Possible rejection of the patients immune system is the biggest problem with the use of stem cell therapies in heart diseases and other diseases. This can worsen many illnesses.

MESO does appear committed to the quality of its product. For MESO it is a question of the effectiveness and safety of their products. Its a long and winding road to provide adequate scientific proof when presenting breakthrough treatments to regulators. Many less reputable organizations have touted stem cells without doing the necessary scientific investigation or seeking the necessary regulatory approval. Mesoblast is trying to do things the right way. Committing to doing science the right way leads to a lot of inevitable ups and downs. This raises financial speculation and can lead to wild fluctuations in the stock price of any company.

A further significant advantage of some of Mesoblasts products is that they apparently can be administered to patients without needing donor matching. This increases their viability. Moreover, it allows for a wide spectrum of patients to be treated from their products. This gives them an advantage in comparison with other firms and should potentially allow them to increasingly gain a larger market share.

Of great interest to investors include the many clinical trial phase 3 products that Mesoblast has in its pipeline. These include MPC-06-ID, Remestemcel-L, and REVASCOR.

Remestemcel-L is a Mesoblast therapy that may theoretically have properties to help with the treatment of ventilator-dependent patients with COVID-19 patients. However, a clinical trial reported some concerns with the therapy meeting its primary endpoint. And it sent the stock down in December 2020. Obviously, there is a large demand for the treatment of complications linked to Covid-19, so this bad news disappointed investors.

However, another therapy has shown promise in the DREAM-HF Phase 3 for patients with chronic heart failure. Although the Revasacor did not stop heart failure, it did seem to deliver dramatic reductions in heart attacks and other negative cardiovascular events that plague heart failure patients.

Heart failure is a pathology that involves ones heart having trouble pumping. The condition impacts millions of people worldwide. In order to feed and maintain it working, the heart muscle depends on a continuous supply of oxygen rich blood. Having stem cell therapies is highly desirable to treat cardiovascular diseases. Hopefully, many Cardiovascular disorders can be treated with stem cell therapies in the future.

Other conditions such as hypertension and Coronary artery disease can help lead to heart failure. According to the Mayo Clinic, heart failure can cause significant health complications and lead to Liver and Kidney damage in patients.

Some scientists believe that Mesenchymal Stem Cells when used to treat cardiovascular diseases can preserve the myocardium by reducing the intensity of inflammation and supporting angiogenesis. Angiogenesis is a mechanism used by the body to create new blood vessels. Their low immunogenicity once more makes them a perfect treatment. This helps ensure that the immune system of the patient does not produce a negative response to the therapy. This theoretically can give stem cell therapies an advantage over some protein-based treatments that are easily recognized by the patients immune system.

This product could be a major development for Mesoblast moving forward, although further analysis and testing is still needed.

Stem cell therapies are not without experimental and medical challenges. For example, there are concerns with the ability of stem cell migration to tissues that require regeneration. There may also be cases whereby stem cells are divided into unintended cells. There may also be difficulties with the manufacturing and culturing of stem cells. Identification of Mesenchymal stem cells in cell populations can be problematic. From a scientific point of view, bone marrow derived Mesenchymal Stem Cells are known to be the best source for obtaining these cells in the human body.

Mesoblast has a wide range of advanced research programs related to different stem cell therapies. MPC-06-ID could potentially be a viable therapy for treating chronic low back pain attributable to degenerative disc disease.

These are products that consumers should be thrilled about.

The company has solid financials for a stem cell company and has a lot of cash on hand. The stock had a market cap of over 2 billion on 9/30/2020 and a 52-week high of 21.28. Lately the news surrounding the companys clinical trials has been a potpourri of both good and bad, so the share price has settled at around $9. It has a float of 93.7 million shares.

Mesoblast is a really exciting healthcare business. The business has made a commitment for the future. And it should be a stock that investors continue to follow.

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Mesoblast Limited: Is Stemcell Therapy Ready For Prime Time? - Sick Economics

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[Full text] Identification and Targeting of ThomsenFriedenreich and IL1RAP | OTT – Dove Medical Press

By daniellenierenberg

Introduction

Chronic myeloid leukemia (CML) is a hematological malignancy that develops when the 9;22 translocation in a single hematopoietic stem cell (HSC) results in the expression of BCR-ABL1 tyrosine kinase fusion protein. If left untreated, CML progresses over approximately 5 years, from relatively benign chronic phase to accelerated phase, and then to fatal blast crisis. The introduction of tyrosine kinase inhibitors (TKIs) specifically targeting the BCR-ABL1 fusion protein was a breakthrough in the management of CML, leading to a significant reduction in mortality and improved 5-year survival rates. However, despite the high annual acquisition costs of all the TKIs; first-, second-, and-third line TKIs1 induce only transient responses in the 10% to 15% of CML patients diagnosed in advanced phase, suboptimal responses in approximately 30% of CML patients during chronic phase (CP) cases that experience disease progression each year during, and only 1020% chance of successful treatment discontinuation due to disease persistence.2 Among the causes of disease persistence, studies have shown that CML leukemia stem cells (LSC) play a major role in inducing therapeutic resistance and disease progression because they are able to self-renew.3,4 These LSC a rare subset of immature cells residing in the bone marrow niche are protected from the action of TKI5 because these cells are normally quiescent and the TKIs are designed to target malignant blast cells that proliferate. That is why current strategies are not able to effectively eliminate the LSC or the disease.3 In CML, LSC are primitive cells expressing CD34+ CD38- with the 9;22 translocations, or the Philadelphia chromosome (Ph).6 However, these markers cannot distinguish the cancer hematopoietic cells from normal ones. Additionally, the BCR-ABL fusion gene encodes for an intracellular tyrosine kinase protein rather than a surface protein, calling for the need to identify unique surface biomarkers for efficient targeting of this cell population with subsequent eradication of the root of the disease.

In 2010, a single biomarker, Interleukin 1 receptor accessory protein (IL1RAP), was found to be up-regulated on the cell surface of BCR-ABL+ LSC. They were able to distinguish Ph+ from Ph- LSCs using IL1RAP.7 A polyclonal anti-human IL1RAP was generated that not only targeted the LSC population but also killed normal peripheral blood mononuclear cells, indicating that this marker was not specific to the LSC.7 Another characteristic cell surface marker has been investigated; ThomsenFriedenreich antigen (TF, or CD176) a tumor-associated carbohydrate epitope. The CD176 antigen was found to be expressed on the surface of various cancer-initiating cells, such as breast carcinomas,8 colorectal carcinomas,9 several leukemias,10 and other types of cancer, but was absent from almost all normal adult cell types.11 CD176 was also found to be expressed on the surface of CD34+ hematopoietic stem cells of the K562 erythroblastic leukemia cell line; a cell line derived from a CML patient. Being strongly expressed on the surface of cancer cells and virtually absent from normal tissues, CD176 was evaluated as a suitable target for cancer biotherapy8 with the development of an anti-CD176 antibody that induced apoptosis of leukemic cells.12

Using monoclonal antibodies (mAb) as a tool for cancer therapy still has its limitations. Patients who receive mAb therapy may develop drug resistance or fail to respond to treatment owing to the multiple signaling pathways involved in the pathogenesis of cancer and other diseases.13 Targeting more than one molecule has proven to circumvent the regulation of parallel pathways and avoid resistance to the treatment.14 Bi-specific antibodies (Bis-Ab) are antibodies that can recognize two different epitopes. They can redirect specific immune cells to the tumor cells to enhance tumor eradication, enable the simultaneous blocking of two different targets that have common signaling pathways, or interact with two different cell-surface antigens instead of one with subsequent boosting of the binding specificity.13 Thus, the identification of two surface markers specific to the cancer stem cells would be useful in characterizing and targeting CML stem cells, without affecting other blood cells.

In this study, we evaluated co-expression of IL1RAP, linked to BCR-ABL+ expression, and the CD176 antigen, carried on the hematopoietic stem cell marker CD34 molecule, in CML patients. We identified PBMCs co-expressing CD34, IL1RAP, and CD176 antigens using flow cytometry, a finding that allowed for subsequent separation and targeting of such cells from normal HSCs. A bi-specific antibody (TF/RAP), was generated in order to target the IL1RAP+ and CD176+ cell population among PBMCs in patients with CML. We used a flow-cytometry assay as a cell-based assay to measure the antibody binding capability of the TF/RAP Bis-Ab to the cell surface antigens. Our TF/RAP Bis-Ab, increased targeting of the IL1RAP+ and CD176+ cell population among CML PBMCs but not corresponding normal cells, using complement-dependent cytotoxicity assay (CDC). This novel TF/RAP Bis-Ab may provide a novel strategy for the eradication of CML stem cells.

Deidentified samples of peripheral blood from healthy volunteers were obtained from Gulf Coast Regional Blood Bank (Houston, TX, USA) after signing informed consent and used as reference samples. Deidentified samples of peripheral blood mononuclear cells (PBMCs) from consented patients with CML were obtained from Oncology Research Gundersen BioBank (https://www.gundersenhealth.org/research/biobank/, La Crosse, WI, USA). While the samples were de-identified, necessary CML patient characteristics were collected (Table 1). The collection and dissemination protocols for the samples are approved by The Gundersen Human Subjects Committee/Institutional Review Board (IRB) and are in full compliance with National Cancer Institute Best Practices for Biospecimen Resources. Because the de-identified samples were received through Biobanks and not through direct intervention/interaction with a research subject, the Tulane University Human Research Protection Office was notified and this study was classified by the IRB as exempt as the study did not meet the definition of human subjects research according to US Federal policy (HHS regulations, 45 CFR part 46, subpart A, also known as the Common Rule). The study was conducted in accordance with the Declaration of Helsinki.

Table 1 CML Patients Characteristics

HEK 293FT cell line (Invitrogen # R70007) was cultured in DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, 100 g/mL streptomycin sulfate, and 4.0 mM L-glutamine (Gibco BRL products, Gaithersburg, MD), at 37C in a humidified 5% CO2 incubator. The KG1 cell line (ATCC #CCL-246) and transduced derivative cells were cultured in Iscoves Modified Dulbeccos Medium (Life technologies) supplemented with 20% FBS at 37C in a humidified 5% CO2 incubator. K562 cell line (ATCC# CCL-243) was maintained in RPMI-1640 (Life technologies) supplemented with 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin sulfate at 37C in a humidified 5% CO2 incubator.

The IL1RAP cDNA was PCR amplified from an expression plasmid containing Human IL-1RAcP/IL-1R3 Gene ORF cDNA (Sino biological Inc., HG10121-CM) using Clone Amp HiFi PCR Premix (Takara Bio USA, Inc.), and primers that included either a BamHI or an XhoI site (F-IL1RAP: acgggatccccaccaagcttggtaccatgac; R-IL1RAP: acgctcgagttatacatttttcaaagatg). The PCR fragment was gel extracted as above, sub-cloned into BamHI and XhoI sites in the pHRST-MPSV vector according to standard protocols and confirmed by restriction mapping and sequencing.

Transient production of lentiviral particles in adherent HEK293T was modified from previously described.15 Briefly, HEK293T cells were seeded in a T-75 flask, where we used 4.0 g of envelope plasmid pMPSV-VSV-G, 10.0 g packaging plasmid psPAX2, and 26 g transfer plasmid that has the gene of interest. In our case, the transfer plasmid is either the antibody plasmid or the control. The plasmids were mixed into 500 L 0.25 M CaCl2 (Sigma Aldrich, St. Louis, MO) and incubated at room temperature for 5 minutes, and then mixed with 500 L 2xHBS and briefly vortexed. The mixed transfection cocktail was then incubated for 3 minutes at room temperature, and added into the medium of the cells, and mixed gently to make an even distribution. After 16 hours of incubation, the medium was replaced with fresh medium and collected every 24 hours for 3 days. The conditioned medium that contained the vector virus was then pelleted for 10 minutes at 1500 g and passed through a 0.45-m filter to remove the cell debris, and then frozen at 80C for long-term storage, or used for the transduction of target cells.

Lentiviral transduction was done as previously described.1618 In brief, lentiviral supernatant was added to KG1 cells cultured in complete IMEM. After overnight incubation, the lentiviral vector was removed, and fresh media was added. After 48 hours, IL1RAP expression was demonstrated by flow cytometry using anti-Human IL-1 RAcP/IL-1 R3 PE-conjugated antibody (#FAB676P, R&D Systems, Minneapolis, MN).

The CH and CL constant domains in the pLM219 plasmids were amplified with 0.5 nM overlapping mutant primers (Table S1), Deep Vent Polymerase (New England Biolabs), and reaction buffer for forty cycles at 94C for 10 seconds, 60C for 45 seconds, and 72C for 2 minutes. Initial fragments were purified, combined, and used to amplify the entire heavy or light domains (Table S2). The mutated fragments were then gel purified and sub-cloned into their corresponding vectors using restriction enzymes according to standard protocols (Table S2). Sequences were then verified by restriction digestion and sequencing.

For antibody sequences towards CD176 (TF) and IL1RAP, the VH and VL domains from two clones with the most conserved amino acid sequences (TF Clone 1 and Clone 2 called TF1 and TF2 for CD176; Clone 4B6 and Clone 4G9 called RAPa and RAPb for IL1RAP, respectively) were chosen from published sequences.20,21 IL1RAP antibody was designed to target the extracellular membrane anchor-proximal region that comprises an amino acid primary sequence VPAPRYTVELAC within 10 to 15 amino acids of amino acid 361 of human ILR1AP (Gene bank accession Q9NPH3) while the TF antibody was designed to target the same Gal(13)GalNAc disaccharide epitope20 as the Bis-Ab. Variable domains (VD) were codon-optimized and synthesized (Gene Art, Invitrogen) to be compatible with 15 base pairs of homologous sequences on both the 3 and 5 ends of pLM2 recipient plasmid flanking the EcoRI restriction enzyme site.

The pLM2 expression vector was digested with EcoRI to generate a double-stranded break. An In-Fusion HD cloning kit (Clontech, Inc) was used to clone the VD regions of the antibodies between the leader and constant regions of the pLM2 vectors. The correct clones were identified by PCR and restriction mapping and then verified by sequencing.

Adherent HEK cells were transfected as above. A total of 14 g high-quality plasmid-DNA, 10% GFP plasmid for assessment of transfection efficiency, while the rest was heavy and light chain plasmid DNA combined at a ratio of 1:1. Six to 8 hours later, cells were gently washed once with PBS and fresh growth medium added. Sixteen hours post-transfection, the medium was replaced with DMEM supplemented with 5% FCS and incubated at 5% CO2 for 24 hours prior to the initial collection of antibody supernatant. A second collection was made after a further 24 hours.

Flow antibodies used were as follows: anti-TF/CD176 mAb mouse IgM (Glycotope, Berlin, Germany) targeting Gal1-3GalNAc epitope; FITC-conjugated anti-mouse IgM secondary antibody (-chain specific, #F9259; Sigma); PE-conjugated mouse anti-human IL-1 RAcP/IL-1 R3 monoclonal IgG1 antibody, epitope Ser21-Glu359 (#FAB676P, R&D Systems); APC-conjugated mouse anti-human CD34 monoclonal IgG1 antibody (#QBEnd10, FAB7227A-025, R&D Systems); APC-conjugated mouse antihuman IgG monoclonal antibody (Clone G18-145, mouse IgG1 , #550,931, BD Pharmingen).

LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (#L34957, Invitrogen); Vibrio Cholera Neuraminidase (VCN; Sigma Aldrich Inc), an enzyme used to expose the CD176 on the surface of expressing cells. Flow cytometric analyses were performed in a BD LSR Fortessa (BD Biosciences, USA) and flow cytometric cell sorting was done in a FACSAriaII (P0010) cell sorter (BD Biosciences, USA). The amount of bi-specific antibody bound to the receptors was calculated from the frequency of total IgG bound receptors.

Sorted cells were received in RPMI media and then fixed using the standard 3:1 methanol: acetic acid fixative. Standard procedures were used for FISH hybridization and washing.22 The BCR/ABL1 Plus translocation, dual fusion probe set (Cytocell Inc., Tarrytown, NY) was used. Slides were analyzed using Leica Biosystems Cyto Vision. FISH nomenclature was described according to the ISCN 2016.23

CD34+CD176+IL1RAP+ and CD34+CD176+IL1RAP- cells were sorted from PBMC samples derived from patients with CML. Cells (1 x 103) were plated in Metho Cult Express (#04437, Stem Cell Technologies, Vancouver, Canada) semi-solid media containing recombinant human IL-3, IL-6, G-CSF, GM-CSF, SCF, TPO and cultured for 2 weeks in a humidified atmosphere at 37C with 5% CO2. Fourteen days after plating, the number of colonies was counted by microscopy.24,25

The capacity to induce CDC was assessed essentially as has been described.2628 Briefly, target cells (1105 cells) were pre-incubated at 37C for 60 min with diluted antibodies. Human serum from human male AB (Sigma Aldrich) (20% v/v) was added to the cells as a source of complement and incubated at 37C for an additional 45 min. Cells were then put on ice and viability was determined by staining with LIVE/DEAD staining and detected using a FORTESSA flow cytometer (BD Biosciences). CDC activity was expressed as a percentage of lyses as determined from the increase in the percentage of cells stained positive with the LIVE/DEAD marker compared to the control samples. Cycloviolacin O2 (CyO2, 0.05nM), a pore-forming peptide, was used as a positive control because it kills cells with the similar mechanisms as CDC by causing pores in the cell membrane.

The capacity to induce CDC was assessed essentially as has been described.2628 Briefly, target cells (1105 cells) were pre-incubated at 37C for 60 min with diluted antibodies. Human serum from human male AB (Sigma Aldrich) (20% (v/v)) was added to the cells as a source of complement and incubated at 37C for an additional 45 min. Cells were then put on ice and viability was determined by staining with LIVE/DEAD staining and detected using a FORTESSA flow cytometer (BD Biosciences). CDC activity was expressed as a percentage of lyses as determined from the increase in the percentage of cells stained positive with the LIVE/DEAD marker compared to the control samples. Cycloviolacin O2 (CyO2, 0.05nM), a pore-forming peptide, was used as a positive control because it kills cells with the similar mechanisms as CDC by causing pores in the cell membrane.

We measured the production of the Bis-Ab by ELISA. Plates were initially coated with goat anti-Human IgG heavy chain antibody (Axell) and blocked with PBS containing 0.5% Tween 20 (Fisher), 10% FBS (FetalPlex Animal Serum Complex, GeminiBio, Cat#100-602), 4% whey protein (BiPRO, AGROPUR). Undiluted or diluted supernatant was added, including the standard curve samples (human IgG MAb 1.7B, kindly provided by Dr. James Robinson), and negative blocking buffer. After incubating at 37C for 60 min, the plates were washed. Then, goat anti-Human lambda antibody conjugated to HRP (Southern Biotech, Cat# 207005) was added at 1:300 in blocking buffer for 60 min and washed five times. A mixture of 0.1M Na Acetate (pH 6), peroxide, and TMB substrate were added. The reaction was terminated by adding 1M phosphoric acid, and the absorbance of each well was measured at 450 nm using a Synergy H1 microplate reader (BioTek).

For each experiment, more than three independent replicates were conducted, and the results were expressed as average standard deviation. Comparison of multiple groups was conducted using ANOVA-based Test and p< 0.05 (*) represented significances with statistical meaning. Calculation of the Kd was done using the equation % RO = [Ab]/([Ab]+Kd) 100%, where RO is the receptor occupancy, Ab is the concentration of antibody and Kd is the equilibrium dissociation constant.

In order to analyze the co-expression of CD176 and IL1RAP antigens on CD34+ cells, peripheral blood mononuclear cells from a normal volunteer (NPBMCs), patients with CML, and K562 cells were isolated and stained with anti-CD34, anti-CD176, and anti-IL1RAP monoclonal antibodies and analyzed by flow cytometry (Figure 1A). It has been previously established that these markers were not expressed on normal PBMCs nor on stem cells7,10 CD34+ cell expression ranged from an average 938% in CML samples versus 83.7% in K562 cells (Figure 1A, upper panel). Within the CD34+ cell population, CD176 and IL1RAP antigens were variably expressed in CML samples, ranging from 1.35% in CML-4 to over 50% in CML-1 (Figure 1A, lower panel), while CD176+ IL1RAP+ was detected in 78% of CD34 cells in K562 cells. Surprisingly, surface co-expression of CD176 and IL1RAP was not only detectable on CD34+ cells in patients with BCR-ABL positive CML but was also demonstrable in cells from a treated patient who was BCR-ABL negative (CML-2) (Figure 1B). In Figure 1C, CD34+ cells revealed higher frequency of CD176+ IL1RAP+ in CML group compared to control sample (17.5% versus 3.4%, p<0.001).

Figure 1 CD176 and IL1RAP antigens are co-expressed on CD34+ Leukemia stem cells. Peripheral blood mononuclear cells from patients with CML and healthy volunteers were isolated and stained for flow-cytometry analysis. (A) FACS Dot Blot showing expression of CD34 (top row) and co-expression of CD176 and IL1RAP antigens on the CD34+ cells (bottom row) in PBMCs from patients with CML compared to NPBMCs. (B) Bar graphs showing the BCR-ABL status relative to the percentage of IL1RAP and CD176 co-expression in the CD34+ subsets from patients with CML as compared to the normal control and the positive control (K562 cells). The BCR-ABL status is indicated below the sample. The error bars represent the variation in two independent experiments. (C) Average percentage of CD34+ and CD34+ CD176+ IL1RAP+ subsets in normal versus CML patients respectively. (D) Bar graphs showing the average count of colony-forming units (CFU) per 1000 CD34+CD176+IL1RAP- cells (open bar) or CD34+CD176+IL1RAP+ cells (solid bar) obtained from CML-2 and CML-4 samples. **p< 0.01, n.s represents that there is no significant difference between groups.

In order to analyze the progenitor activity of the various subpopulations, CML-2 and CML-4 were flow-sorted for CD34+CD176+IL1RAP+ and CD34+CD176+IL1RAP- then plated in media t support hematopoietic colony formation. The number of colonies, or colony-forming units (CFU), in CD34+CD176+IL1RAP+ pool represented 6% of the sorted cells with a significant difference between both populations, p<0.01 (Figure 1D and Figure S1).

To facilitate correct interaction of the VH and VL domains, site-directed mutagenesis was used to generate knob-in-hole mutations in the heavy and light chains of the constant domains (Figure 2A) via polymerase chain reaction overlap extension (Figures S2 and 3). Two PCR reactions were performed to generate two amplicons with the specific mutations included in the overlapping primers. The two fragments were then combined in a subsequent fusion reaction, in which the overlapping ends anneal, allowing the 3 overlap of each strand to serve as a primer for the 3 extension of the complementary strand. The resulting fusion product served as a template for amplification of the entire constant domain. In order to circumvent the light chain mismatching, an Orthogonal Fab interface was generated. In one Fab, complementary mutation was introduced and verified at the heavy chain constant domain (CH1_H172A_ F174G) and at the light chain constant domain (CL_L135Y_S176W), respectively (Figures S46). For the heavy chain heterodimerization, we used the Knob-in-Hole strategy, where we inserted the CH3 mutations (S354C and T366W) into different heavy chains (Figures S7 and 8). The VH and VL sequences were synthesized and cloned into the new pLM2-CH and -CL plasmids (Figure 2A) where CD176 was represented by TF1 (VH1 and VL1) and TF2 (VH2 and VL2) while IL1RAP was represented by Clone 4B6 (VHa and VLa) and Clone 4G9 (VHb and VLb). Then, we generated the four different bi-specific antibody mixtures (TF1RAPa, TF1RAPb, TF2RAPa, and TF2RAPb) to evaluate the most effective Bis-Ab (Figure 2B). The bispecific antibody was quantified by ELISA at 283 ng/mL. Since ELISA used the human IgG heavy chain antibody as the primary antibody and a goat anti-human lambda antibody conjugated to HRP as the secondary antibody, these data also confirm the correct association of the heavy and light chains and ensure that monomers are excluded.

Figure 2 The bi-specific antibody arms. (A) Schematic diagram of the bi-specific antibody showing the mutant arms and the antigen-binding domains. Thomsen-Freidenrich or CD176 domains (TF); IL1RAP domains (RAP); variable domain-heavy chain (VH); variable domain-light chain (VL); L135Y and S176W mutations (Y-W) in constant domain-light chain; H172A and F174G mutations in CH1 domain (A-G); S354C (C) or T366W (W) mutations in CH3. (B) Antibody mixtures generated by transient transfection of HEK 293T cells. TF1 and TF2 was paired with RAPa and RAPb to generate four Bis-Ab mixtures. The bispecific antibody concentration was 283 ng/mL as measured with ELISA. The correct association of the human IgG heavy chain and the lambda light chain was confirm and monomers were excluded by using anti-IgG primary antibodies and anti-light chain secondary antibodies.

KG1 cell line is an acute myeloid leukemia cell line that is known to be a positive control for CD176. For optimizing the staining protocol of CD176, KG1 cells were pre-treated with VCN to expose CD176 antigens for better staining (Figure S9). In order to test the binding capability and functional potential of our bi-specific antibody, we generated a dual-positive cell line for expressing both IL1RAP and CD176 through lentiviral transduction (Figure S10A and B). IL1RAP expression was increased by 1.5 folds in KG1/RAP cells as verified by flow cytometry (Figure S10C and D).

CD176 antigen is a glycosylated antigen; a protein antigen bound to GAL-NAC moiety which makes the antigen displayed on the cell surface yet not easy to isolate.21 For this reason, a flow-cytometry assay was used to evaluate both the binding capability and toxicity of our Bis-Ab using the gating strategy in Figure S11. KG1 and KG1/RAP cell lines were treated with the various Bis-Ab mixtures. Binding percentage was calculated from the percentage of IgG positive cells, where the secondary IgG antibody is bound to the primary Bis-Ab. The TF1RAPa Bis-Ab showed the highest binding in KG1/RAP cells (Figure 3A) as compared to other mixtures (p<0.001). In contrast, the TF1RAPb antibody revealed slightly reduced binding in KG1/RAP cells. On treating KG1/RAP cells with increasing amounts of TF1RAPa, more binding to the dual-positive KG1/RAP cells was observed (Figure 3B). To demonstrate the specificity of the Bis-Ab, we measured the competition with the CD176 and the IL1RAP monoclonal antibodies. Increasing concentrations of the Bis-Ab specifically inhibited the binding of both the IL1RAP and CD176 mAbs (Figure S12). Then, our KG1/RAP cells were treated with the Bis-Ab TF1RAPa and complement prior to staining with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit, in order to evaluate whether CDC could be achieved using IL1RAP and CD176 as targets. Flow cytometric analysis revealed a significant increase in dead cells in the Bis-Ab treated CD176/IL1RAP dual-positive KG1/RAP population as antibody binding also increased (Figure 3C), p<0.001.

Figure 3 Validation of TF-RAP Bi-specific antibody in KG1 cell line and CML samples. (A) MFI for binding of different Bis-Ab mixtures in KG1/RAP (p <0.001). (B) Binding (%) of the Bis-Ab in KG1/RAP cell lines. (C) Shows live/dead (LD) staining (%) in KG1/RAP cell lines after treatment with the Bis-Ab and complement. (D) MFI for binding of different Bis-Ab mixtures p <0.001 in CML cells. (E) Binding of the Bis-Ab (%) in PBMCs from patients with CML. The binding affinity (Kd) of our bispecific antibody was 21ng/mL, calculated using the % RO = [Ab]/([Ab]+Kd) 100%, where RO is the receptor occupancy, Ab is the concentration of antibody, and Kd is the equilibrium dissociation constant. This Bis-Ab platform used in this study had the correct molecular weight (95 KDa) and assembled properly (93%) as revealed by SDS-PAGE analysis.38 (F) Live/dead (L/D) staining (%) from patients with CML after treatment with the Bis-Ab and complement. The red square were L/D positive cells treated with CyO2; the percent of L/D staining in normal PBMCs is shown in blue. Each point represents the mean increase in L/D staining SEM with three to four replicates. Data from normal samples were low for all doses (data not shown).

Binding of TF1RAPa, TF2RAPa, and TF2RAPb was also tested in PBMCs from patients with CML. Again, TF1RAPa showed the highest binding relative to other mixtures (p<0.001) (Figure 3D) and with increasing doses (Figure 3E). Based on the CML binding curve, the binding affinity (Kd) of our bispecific antibody was 21 ng/mL. Other therapeutic antibodies, such as ofatumumab directed against CD20, have shown significant CDC against peripheral blood cells obtained from CML patients in chronic phases26 and B cells in CLL,29 respectively. Thus, the TF1RAPa cocktail was used to generate the doseresponse curve and to evaluate whether CDC could be achieved using both IL1RAP and CD176 as targets. The ability of the TF1RAPa cocktail was compared to human anti-IL1RAP and anti-CD176 monoclonal antibodies to induce cell death in PBMCs from patients with CML. PBMCs from CML1-4 were tested in CDC assays in parallel to cells from healthy control samples. In CML cells, the binding of TF1RAPa mediated CDC at higher levels than in normal peripheral blood mononuclear control cells, correlating with the expression level of IL1RAP and CD176, particularly at lower antibody concentrations (Figure 3F). More strikingly, among peripheral blood cells, TF1RAPa did not induce CDC of normal cells, whereas a clear dose-dependent CDC effect was observed in CML cells (Figure S13A and B). To address the selectivity of IL1RAP/CD176-targeting antibodies, we also validated the bispecific antibody cytotoxicity on the various subpopulations in peripheral blood. The dual-positive CD176+IL1RAP+ cell populations showed the highest CDC activity as compared to CD176+IL1RAP-, CD176-IL1RAP+, and CD176-IL1RAP- populations (Figure 4 and S13CF, S14).

Figure 4 Dose-response curve of TF1RAPa Bis-Ab on CDC in CML samples. A dose-response curve showing the selective killing potential of CD176+IL1RAP+ subpopulation by the TF1RAPa Bis-Ab as compared to other subpopulations in PBMCs from patients with CML. Each point represents the mean SEM of the four samples.

Targeting molecules involved in multiple pathways is proving to be one of the most reliable strategies for eradicating cancer stem cells. In this report, we present a novel bi-specific antibody, TF/RAP, capable of targeting ThomsenFriedenreich (TF, CD176) and IL1RAP antigens on CD34+ HSCs in CML and on cell lines. TF is a glycoprotein that has many domains and motifs (eg, LGALS3, Gal(1,3)GalNAc, LGalS3BP), many related to signaling pathways. It is a known marker for ongoing tumorigenesis and metastasis, as it is expressed on various cancer-initiating cells.8 Interestingly, CD34 and LGALS3 were found to be co-expressed in myeloid cells.30,31 LGALS3 and ABL1 are involved in regulating RUNX1 and the transcription of genes involved in differentiation of hematopoietic stem cells,32 especially myeloid cells33 (Figure S15) IL1RAP, on the other hand, is a member of the Toll-like receptor superfamily and is a well-known co-receptor of IL1R1.34 IL1RAP plays a role in mediating the effect of the pro-inflammatory cytokine IL-1 and is also involved in activating T cells and mast cells after mediating the signal of IL-1 cytokine.35 It has previously been characterized as a tightly related marker for BCR-ABL positive cells.7 Together, both TF and IL1RAP were related to apoptotic pathways; IL1RAP up-regulation was associated with decreased apoptosis in AML,36 and anti-CD176 antibody induced apoptosis of CD176-positive leukemic cells through multiple pathways.12 Although we did not find a direct link between IL1RAP, CD176 and leukemogenesis, previous studies have shown that each of them is separately expressed on CD34+ cells in leukemia cell lines8,10,12 and patients with CML7

Therefore, we conducted this pilot study, in order to assess the co-expression of IL1RAP and ThomsenFriedenreich (CD176) antigens on CD34+ HSCs in peripheral blood of patients with CML, using FACS gene expression analyses. Flow-drop FISH and CFU assays were used for the separation of CD34+CD176 BCR-ABL+ and BCR-ABL CML stem cells, based on IL1RAP expression.7 CFU numbers were significantly lower in CD34+CD176+IL1RAP- cells than in CD34+CD176+IL1RAP+ cells, obtained from CML-2 and CML-4 samples (Figure 1D), particularly CML-2 sample which was obtained from a patient in remission (BCR-ABL-). We found that the frequency of clonogenic hematopoietic progenitor cells was increased in the CD34+ CD176+IL1RAP+ cells in these samples. Testing the stem-cell characteristics of these two cell populations in immune-deficient mice would have been advantageous. Yet, the low numbers of sorted CML cells acquired from the CD34+CD176+ IL1RAP and IL1RAP+ cell subpopulations, alongwith the general low engrafting efficiency of chronic phase CML cells in these mice7 prevented us from successfully performing such experiments. Importantly, as IL1RAP expression was correlated with changes from chronic phase (CP) into accelerated phase (AP) and blast phase (BP)37, we also found that the level of IL1RAP/CD176 co-expressionwas increased, in our patient samples, as the disease progressed, independent of the treatment status(Table S3).

To target both TF and IL1RAP simultaneously, we developed a Bis-Ab specific for both antigens. Because antibodies are normally heterodimers of two heavy and two light chains, we modified the constant domains in the Bis-Ab to maximize the correct interactions of the four immunoglobulin chains within single cells. Here, we used the orthogonal Fab design; CH1_H172A_F174G and CL_L135Y_S176W38 to facilitate selective assembly of the Fab arms for correct dimerization of the antigen-binding domains.39 Therefore, we mutated CH1 and CL binding sites to restrict the assembly of the Fab with the correct VD pairs. The RAP VDs were cloned with the wild type Fab; and the TF VD was linked to the mutant orthogonal Fab design. Published data have shown that the component proteins of this Bis-Ab platform proper assembly were detected at 93% and the complex had a molecular weight of 95 KDa, as revealed by SDS-PAGE analysis.38 Additionally, the CH3 for each Fab was mutated with previously described knob-into-hole mutations40,41 to facilitate hetero-dimerization between the TF and the RAP heavy chains. In our study, we used ELISA to demonstrate that both the VD and Fc were properly paired. Here, because the primary antibody was anti-human VL and the secondary antibody was anti-human IgG, quantifying the Bis-Ab also demonstrated the VD-Fc interactions.

To efficiently validate the specific binding of our Bis-Ab, we generated a dual-positive cell line; KG1/RAP. KG1 cell line expresses CD176+, but IL1RAP is low or absent. Therefore, we induced IL1RAP expression in KG1 cells by lentiviral mediated-gene transfer, as previously usedin both immune42 and leukemic cells.43 In the competitive binding assay, increasing concentrations of the Bis-Ab blocked the binding of CD176 and IL1-RAP monoclonal antibodies to the KG1/RAP and KG1 parental cells, demonstrating the specific binding of the Bis-Ab. The level of CD176 expression in KG1 cell line was detected before and after VCN treatment. Increased staining of the KG1/RAP cells compared to the parental KG1 cells indicated that expression of the IL1RAP facilitates the interaction of the Bis-Ab with the target cell. This increased binding of the Bis-Ab to the KG1/RAP cells also increased their susceptibility to complement-dependent cytotoxicity (CDC). We also observed increased binding and increased CDC in the CD176+ IL1RAP+ population of the peripheralblood from patients with CML. As a pilot study and given that on average, 50% of the cells within the CD34+ subpopulation in the patients tested were dual positive for CD176 and IL1RAP antigens, in addition to the almost undetectable CDC in CD34+ cells in normal controls, our data strongly support the idea that the bi-specific antibody (TF/RAP) indeed induces CDC preferentially in CD176+ IL1RAP+ CML CD34+ cells. In generating a bi-specific antibody that targets CD176 and IL1RAP, we are unique in providing proof of concept that CML CD34+CD176+ IL1RAP+ cells can be targeted while preserving corresponding normal cells. The potential to target multiple antigens is supported by studies that demonstrated increased or synergistic CDC activity by non-cross blocking CD20 antibody combinations.44

Therapeutic antibodies are commonly administered intravenously, yet selectivity and specificity are a major concern for reduced toxicity. CD176/IL1RAP co-expression was not present in monocytes unlike the reported weak but present IL1RAP expression in monocytes.7 Both antigens were low or absent in most types of normal bone-marrow progenitor and mature cell types, suggesting that CD176/IL1RAP dual targeting antibodies are expected to show low toxicity on normal hematopoietic cells. Being strongly expressed on the surface of cancer cells and virtually absent from normal tissues, CD176 was evaluated as a potential target for cancer biotherapy with the development of anti-CD176 antibody that induced apoptosis of leukemic cells.8 Added to this, antibodies against IL1RAP were found to be capable of blocking IL-1 signaling as well as inhibiting tumor cells' growth in AML,34 CML,7 breast cancer,45 prostate cancer, breast cancer, lung cancer, colorectal cancer, melanomas, bladder cancer, brain/CNS cancer, cervical cancer, esophageal cancer, gastric cancer, head/neck cancer, kidney cancer, liver cancer, lymphomas, ovarian cancer, pancreatic cancer, and sarcomas46 especially in cancer stem cells, or (CSCs) and progenitor cells, which are responsible, directly or indirectly, for the development of a solid tumor.47 Thus, it may be thatour Bis-Ab will not only eradicate the CD176+IL1RAP+ drug-resistantCML stem cells but also may have universal therapeutic potential for preventing relapses in both solid and hematological cancers.Given that the mode of action in CDC is having the antibody direct the complement pathway to target cell killing, we suggest that this therapeutic strategy would be independent of known mechanisms of TKI resistance in CML. Thus, the concept of complement-mediated killing of IL1RAP/CD176 expressing cells may also have the potential to eradicate such cells in patients, either alone or in combination with current regimens, in order to increase their therapeutic effectiveness. And finally, expanded studies need to be performed in order to confirm the co-expression of both markers, especially in resistant and relapsed cancer patients as well as in patient-derived xenografts (PDX).

The experimental research was mostly supported by a fellowship to REE from the Egyptian Ministry of Higher Education, Cultural, and Missions Section (JS 3577). The lentiviral vectorHRST-cmvGFPand the packaging plasmids were akind gift from Richard C.Mulligan in the Harvard Gene Therapy Institute. The human IgG heavy and light chain constant genes were provided by JE Robinson (Tulane University). C Wu and SEB were supported by AI110158 and/or OD01104-51; EUA and SEB were supported by the Applied Stem Cell Laboratory.

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. All authors have given approval of the final version of the article; and have agreed to be accountable for all aspects of the work.

The abstract of this paper was presented at the AACR annual Meeting 2019; March 29 April3, 2019; Atlanta, GA, as a poster presentation with interim findings. The posters abstract was published in Poster Abstracts in the AACR meeting proceedings and as a supplement in the AACR Cancer Research Journal [https://cancerres.aacrjournals.org/content/79/13_Supplement/1222A].

Raghda Eldesouki reports grants from Egyptian Ministry of Higher Education. Stephen EBraun reports grants from Egyptian Ministry of Education, Alliance for Cardiovascular Research, NIAID OD01104, and Braun/McGroarty Charitable Fund, during the conduct of the study. In addition, Dr Raghda Eldesouki, Dr Stephen Braun, Dr Fouad Badr and Dr Eman Abdel-Moemen Mohammedhave apatent, PCT/EG2019/000014, pending. The authors report no other conflicts of interest in this work.

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[Full text] Identification and Targeting of ThomsenFriedenreich and IL1RAP | OTT - Dove Medical Press

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

By daniellenierenberg

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.

1. Barriga F, Ramirez P, Wietstruck A, Rojas N. Hematopoietic stem cell transplantation: clinical use and perspectives. Biol Res. 2012;45(3):307316. doi:10.4067/S0716-97602012000300012

2. Passweg JR, Baldomero H, Bader P, et al. Use of haploidentical stem cell transplantation continues to increase: the 2015 European Society for Blood and Marrow Transplant activity survey report. Bone Marrow Transplant. 2017;52(6):811817. doi:10.1038/bmt.2017.34

3. Gudiol C, Garcia-Vidal C, Arnan M, et al. Etiology, clinical features and outcomes of pre-engraftment and post-engraftment bloodstream infection in hematopoietic SCT recipients. Bone Marrow Transplant. 2014;49(6):824830.

4. Kikuchi M, Akahoshi Y, Nakano H, et al. Risk factors for pre- and post-engraftment bloodstream infections after allogeneic hematopoietic stem cell transplantation. Transpl Infect Dis. 2015;17(1):5665.

5. Mori Y, Yoshimoto G, Nishida R, et al. Gastrointestinal Graft-versus-Host Disease Is a Risk Factor for Postengraftment Bloodstream Infection in Allogeneic Hematopoietic Stem Cell Transplant Recipients. Biol Blood Marrow Transplant. 2018;24(11):23022309.

6. Mikulska M, Raiola AM, Galaverna F, et al. Pre-Engraftment Bloodstream Infections after Allogeneic Hematopoietic Cell Transplantation: impact of T Cell-Replete Transplantation from a Haploidentical Donor. Biol Blood Marrow Transplant. 2018;24(1):109118.

7. Weisser M, Theilacker C, Tschudin Sutter S, et al. Secular trends of bloodstream infections during neutropenia in 15 181 haematopoietic stem cell transplants: 13-year results from a European multicentre surveillance study (ONKO-KISS). Clin Microbiol Infect. 2017;23(11):854859.

8. Poutsiaka DD, Munson D, Price LL, Chan GW, Snydman DR. Blood stream infection (BSI) and acute GVHD after hematopoietic SCT (HSCT) are associated. Bone Marrow Transplant. 2011;46(2):300307.

9. Youssef A, Hafez H, Madney Y, et al. Incidence, risk factors, and outcome of blood stream infections during the first 100 days post-pediatric allogeneic and autologous hematopoietic stem cell transplantations. Pediatr Transplant. 2020;24(1):e13610.

10. Wang CH, Chang FY, Chao TY, et al. Characteristics comparisons of bacteremia in allogeneic and autologous hematopoietic stem cell-transplant recipients with levofloxacin prophylaxis and influence on resistant bacteria emergence. J Microbiol Immunol Infect. 2018;51(1):123131. doi:10.1016/j.jmii.2016.02.003

11. Forcina A, Lorentino F, Marasco V, et al. Clinical Impact of Pretransplant Multidrug-Resistant Gram-Negative Colonization in Autologous and Allogeneic Hematopoietic Stem Cell Transplantation. Biol Blood Marrow Transplant. 2018;24(7):14761482. doi:10.1016/j.bbmt.2018.02.021

12. Averbuch D, Tridello G, Hoek J, et al. Antimicrobial Resistance in Gram-Negative Rods Causing Bacteremia in Hematopoietic Stem Cell Transplant Recipients: intercontinental Prospective Study of the Infectious Diseases Working Party of the European Bone Marrow Transplantation Group. Clin Infect Dis. 2017;65(11):18191828. doi:10.1093/cid/cix646

13. Girmenia C, Rossolini GM, Piciocchi A, et al. Infections by carbapenem-resistant Klebsiella pneumoniae in SCT recipients: a nationwide retrospective survey from Italy. Bone Marrow Transplant. 2015;50(2):282288. doi:10.1038/bmt.2014.231

14. Freifeld AG, Bow EJ, Sepkowitz KA, et al. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the infectious diseases society of america. Clin Infect Dis. 2011;52(4):e5693.

15. Arendrup MC, Prakash A, Meletiadis J, Sharma C, Chowdhary A. Comparison of EUCAST and CLSI Reference Microdilution MICs of Eight Antifungal Compounds for Candida auris and Associated Tentative Epidemiological Cutoff Values. Antimicrob Agents Chemother. 2017;61(6):6. doi:10.1128/AAC.00485-17

16. Han TT, Huang XJ, Liu KY, et al. [Blood stream infections during agranulocytosis period after hematopoietic stem cell transplantation in one single center]. Zhonghua Nei Ke Za Zhi. 2011;50(8):654658.

17. Mikulska M, Del Bono V, Bruzzi P, et al. Mortality after bloodstream infections in allogeneic haematopoietic stem cell transplant (HSCT) recipients. Infection. 2012;40(3):271278. doi:10.1007/s15010-011-0229-y

18. Stoma I, Karpov I, Milanovich N, Uss A, Iskrov I. Risk factors for mortality in patients with bloodstream infections during the pre-engraftment period after hematopoietic stem cell transplantation. Blood Res. 2016;51(2):102106. doi:10.5045/br.2016.51.2.102

19. Stoma I, Littmann ER, Peled JU, et al. Compositional flux within the intestinal microbiota and risk for bloodstream infection with gram-negative bacteria. Clin Infect Dis. 2020. doi:10.1093/cid/ciaa068

20. Puerta-Alcalde P, Cardozo C, Marco F, et al. Changing epidemiology of bloodstream infection in a 25-years hematopoietic stem cell transplant program: current challenges and pitfalls on empiric antibiotic treatment impacting outcomes. Bone Marrow Transplant. 2020;55(3):603612. doi:10.1038/s41409-019-0701-3

21. Blennow O, Ljungman P, Sparrelid E, Mattsson J, Remberger M. Incidence, risk factors, and outcome of bloodstream infections during the pre-engraftment phase in 521 allogeneic hematopoietic stem cell transplantations. Transpl Infect Dis. 2014;16(1):106114. doi:10.1111/tid.12175

22. Liu C-Y, Lai Y-C, Huang L-J, et al. Impact of bloodstream infections on outcome and the influence of prophylactic oral antibiotic regimens in allogeneic hematopoietic SCT recipients. Bone Marrow Transplantation. 2020;55(3):12311239. doi:10.1038/bmt.2010.286

23. Wang L, Wang Y, Fan X, Tang W, Hu J. Prevalence of Resistant Gram-Negative Bacilli in Bloodstream Infection in Febrile Neutropenia Patients Undergoing Hematopoietic Stem Cell Transplantation: A Single Center Retrospective Cohort Study. Medicine. 2014;16(1):e1931. doi:10.1097/MD.0000000000001931

24. Tumbarello M, Viale P, Viscoli C, et al. Predictors of mortality in bloodstream infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae: importance of combination therapy. Clin Infect Dis. 2012;55(7):943950. doi:10.1093/cid/cis588

25. Qureshi ZA, Paterson DL, Potoski BA, et al. Treatment outcome of bacteremia due to KPC-producing Klebsiella pneumoniae: superiority of combination antimicrobial regimens. Antimicrob Agents Chemother. 2012;56(4):21082113.

26. Zhang R, Liu L, Zhou H, et al. Nationwide Surveillance of Clinical Carbapenem-resistant Enterobacteriaceae (CRE) Strains in China. EBioMedicine. 2017;19:98106.

27. Taur Y, Xavier JB, Lipuma L, et al. Intestinal domination and the risk of bacteremia in patients undergoing allogeneic hematopoietic stem cell transplantation. Clin Infect Dis. 2012;55(7):905914.

28. Mousset S, Buchheidt D, Heinz W, et al. Treatment of invasive fungal infections in cancer patients-updated recommendations of the Infectious Diseases Working Party (AGIHO) of the German Society of Hematology and Oncology (DGHO). Ann Hematol. 2014;93(1):1332.

29. de Naurois J, Novitzky-Basso I, Gill MJ, et al. Management of febrile neutropenia: ESMO Clinical Practice Guidelines. Ann Oncol. 2010;21(Suppl 5):v252256.

30. Mackall C, Fry T, Gress R, et al. Background to hematopoietic cell transplantation, including post transplant immune recovery. Bone Marrow Transplant. 2009;44(8):457462.

31. Ge J, Yang T, Zhang L, et al. The incidence, risk factors and outcomes of early bloodstream infection in patients with malignant hematologic disease after unrelated cord blood transplantation: a retrospective study. BMC Infect Dis. 2018;18(1):654.

32. Laughlin MJ, Eapen M, Rubinstein P, et al. Outcomes after transplantation of cord blood or bone marrow from unrelated donors in adults with leukemia. N Engl J Med. 2004;351(22):22652275.

33. Rocha V, Labopin M, Sanz G, et al. Transplants of umbilical-cord blood or bone marrow from unrelated donors in adults with acute leukemia. N Engl J Med. 2004;351(22):22762285.

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

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BrainStorm Announces the Publication of Preclinical Data Highlighting the Potential of a NurOwn Derived Exosome-Based Treatment for COVID-19 ARDS -…

By daniellenierenberg

NEW YORK, Jan. 20, 2021 /PRNewswire/ --BrainStorm Cell Therapeutics Inc. (NASDAQ: BCLI), a leading developer of adult stem cell therapies for neurodegenerative diseases, announced today the peer-reviewed publication of a preclinical study in the journal Stem Cell and Research Therapy. The study, entitled "MSC-NTF (NurOwn) exosomes: a novel therapeutic modality in the mouse LPS-induced ARDS model," evaluated the use of NurOwn (MSC-NTF cell) derived exosomes in a mouse model of acute respiratory distress syndrome (ARDS).

ARDS is a type of respiratory failure that is frequently associated with COVID-19 and mediated by dysregulated cytokine production. While there are currently no effective therapies to prevent or reverse ARDS, mesenchymal stem cell (MSC)-derived exosomes have been suggested as a potential novel treatment option due to their ability to penetrate deep into tissues and efficiently deliver immunomodulatory molecules.

Results from the recently published study showed that intratracheal administration of NurOwn derived exosomes led to a statistically significant reduction in lung disease severity score (p < 0.05; based on criteria set forth by the American Thoracic Society Documents: Matute-Bello et al., Am J Respir Cell Mol Biol 44;725-738, 2011) and improvements in several additional clinically relevant lipopolysaccharide (LPS)-induced ARDS markers such as lung function, fibrin presence, neutrophil accumulation, cytokine expression, and blood oxygenation levels. Notably, these improvements were significantly superior to those observed following administration of nave MSC-derived exosomes.

"These exciting preclinical data suggest that NurOwn derived exosomes have the potential to treat COVID-19-induced ARDS or other severe respiratory complications, and that they are more effective than exosomes isolated from nave MSCs at combatting the various symptoms of the syndrome," said Dr. Revital Aricha, Vice President of Research & Development at BrainStorm. "This publication in a highly regarded journal provides important validation for the scientific advances and significance of BrainStorm's preclinical research programs, including on our exosome-based technology platform."

Chaim Lebovits, Brainstorm's Chief Executive Officer added, "While our primary focus is on advancing NurOwn towards regulatory approval in ALS, we continue to evaluate the potential of our exosome-based platform to address unmet medical needs. The publication of these proof-of-concept data highlights this potential, and we are now actively assessing next steps to determine how to best generate value. We are also actively discussing with possible partners several development opportunities for the exosome technology."

About NurOwn

The NurOwn technology platform (autologous MSC-NTF cells) represents a promising investigational therapeutic approach to targeting disease pathways important in neurodegenerative disorders. MSC-NTF cells are produced from autologous, bone marrow-derived mesenchymal stem cells (MSCs) that have been expanded and differentiated ex vivo. MSCs are converted into MSC-NTF cells by growing them under patented conditions that induce the cells to secrete high levels of neurotrophic factors (NTFs). Autologous MSC-NTF cells can effectively deliver multiple NTFs and immunomodulatory cytokines directly to the site of damage to elicit a desired biological effect and ultimately slow or stabilize disease progression.

About BrainStorm Cell Therapeutics Inc.

BrainStorm Cell Therapeutics Inc. is a leading developer of innovative autologous adult stem cell therapeutics for debilitating neurodegenerative diseases. The Company holds the rights to clinical development and commercialization of the NurOwn technology platform used to produce autologous MSC-NTF cells through an exclusive, worldwide licensing agreement. Autologous MSC-NTF cells have received Orphan Drug status designation from the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of amyotrophic lateral sclerosis (ALS). BrainStorm has completed a phase 3 pivotal trial in ALS (NCT03280056); this trial investigated the safety and efficacy of repeat-administration of autologous MSC-NTF cells and was supported by a grant from the California Institute for Regenerative Medicine (CIRM CLIN2-0989). BrainStorm is in active discussions with the FDA to identify regulatory pathways that may support NurOwn's approval in ALS. BrainStorm is also conducting an FDA-approved phase 2 open-label multicenter trial in progressive multiple sclerosis (MS). The phase 2 study of autologous MSC-NTF cells in patients with progressive MS (NCT03799718) completed dosing inDecember 2020, and topline results are expected by the end of the first quarter 2021.

For more information, visit the company's website atwww.brainstorm-cell.com.

Safe-Harbor Statement

Statements in this announcement other than historical data and information, including statements regarding future clinical trial enrollment and data, constitute "forward-looking statements" and involve risks and uncertainties that could cause BrainStorm Cell Therapeutics Inc.'s actual results to differ materially from those stated or implied by such forward-looking statements. Terms and phrases such as "may," "should," "would," "could," "will," "expect,""likely," "believe," "plan," "estimate," "predict," "potential," and similar terms and phrases are intended to identify these forward-looking statements. The potential risks and uncertainties include, without limitation, BrainStorm's need to raise additional capital, BrainStorm's ability to continue as a going concern, regulatory approval of BrainStorm's NurOwn treatment candidate, the success of BrainStorm's product development programs and research, regulatory and personnel issues, development of a global market for our services, the ability to secure and maintain research institutions to conduct our clinical trials, the ability to generate significant revenue, the ability of BrainStorm's NurOwn treatment candidate to achieve broad acceptance as a treatment option for ALS or other neurodegenerative diseases, BrainStorm's ability to manufacture and commercialize the NurOwn treatment candidate, obtaining patents that provide meaningful protection, competition and market developments, BrainStorm's ability to protect our intellectual property from infringement by third parties, heath reform legislation, demand for our services, currency exchange rates and product liability claims and litigation; and other factors detailed in BrainStorm's annual report on Form 10-K and quarterly reports on Form 10-Q available athttp://www.sec.gov. These factors should be considered carefully, and readers should not place undue reliance on BrainStorm's forward-looking statements. The forward-looking statements contained in this press release are based on the beliefs, expectations and opinions of management as of the date of this press release. We do not assume any obligation to update forward-looking statements to reflect actual results or assumptions if circumstances or management's beliefs, expectations or opinions should change, unless otherwise required by law. Although we believe that the expectations reflected in the forward-looking statements are reasonable, we cannot guarantee future results, levels of activity, performance or achievements.

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CONTACTS

Investor Relations:Corey Davis, Ph.D.LifeSci Advisors, LLCPhone: +1-646-465-1138[emailprotected]

Media:Paul TyahlaSmithSolvePhone: +1-973-713-3768[emailprotected]

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BrainStorm Announces the Publication of Preclinical Data Highlighting the Potential of a NurOwn Derived Exosome-Based Treatment for COVID-19 ARDS -...

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Cancer requires more tutoring, with Meyer continuing to Teaching Cancer a lesson – News – vintontoday.com

By daniellenierenberg

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Click to view a previous story about Carly's battle.

October 30th brought a second challenge to Vinton-Shellsburg Kindergarten teacher, Carly Meyer. After battling her first round of leukemia, she suffered another relapse with a second diagnosis of leukemia.

"I thought I was done with these updates... but should have known 2020 wasnt done messing stuff up yet!" Carly shared. "For those of you who don't know, I was diagnosed with Acute Myeloid Leukemia in August 2019 and completed chemo treatments in December 2019, but unfortunately my lab results on October 30, showed some "blasts", which are the cancerous cells in my blood." She explained back in November that her lab results also showed that my WBC's the infection fighting cells, were very low.

At the beginning of November, she had another bone marrow biopsy which Wes, her husband believes is her 6th. She was then admitted to the University of Iowa Hospital for a month long stay.

Carly finished up her 5 days of chemotherapy on November 11th with only a couple of side effects (fatigue and loss of appetite) which are a couple of the more common side effects with chemotherapy treatments. Unfortunately, she suffered from dehydration as well and this caused her to pass out a couple of times, and one of the falls caused her to hit her head. This of course triggered a trip for a CT Scan just to make sure she was alright, fortunately, she didn't have any side effects from the fall.

"It is fairly common for leukemia patients to spike fevers and to get random bugs because we are neutropenic and our body cant fight off simple things they normally would," Carly explained. She did come down with an infection during this time but it was able to be pinpointed and treated right away. On Thanksgiving, she was able to return home 10 days earlier from her hospital stay than had been anticipated,

Her journey continues to beat cancer with a trip back to the hospital at the end of December, to begin preparation for her bone marrow transplant. "My hero of a brother started getting shots December 30 to prep and will be donating his Stem Cells on Monday, January 4th." Carly explained how the process works. Her brother Kyle was hooked up to a machine she said it is similar to donating blood/plasma and that the procedure lasts for about 5 hours. Fortunately, her brother Kyle was a 100% perfect match to be her donor.

The stem cells were then put into her IV Powerline over about 30 minutes while they closely monitored Carly for any side effects. "Then its just a waiting game after that," she said.

After the transplant, Carly's immune system was down to zero. Unfortunately, it is common for SCT patients to spike fevers and even get an infection after transplant.

New Year, New Me has never rang more true than this year Carly said.

She is hoping to be home at the end of the week. She said that this last stay has been "extremely exhausting mentally and physically." Developing mucositis, extreme sores and pain in her mouth, it has made it very hard to eat or drink anything. Mucositis is very common after receiving the strong chemo that she received just before her bone marrow transplant. She is slowly recovering from this.

She said that she is excited to be coming home with her husband and fur-baby Maverick if all goes well, by the end of the week.

"I am so lucky to have an amazing support system (especially my husband) to get me through this tough time," she said.

Please keep the couple in your prayers as Carly continues to heal.

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Cancer requires more tutoring, with Meyer continuing to Teaching Cancer a lesson - News - vintontoday.com

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[Full text] Effects of Caffeic Acid and Its Derivatives on Bone: A Systematic Revi | DDDT – Dove Medical Press

By daniellenierenberg

Introduction

Bone remodelling is a tightly coupled lifelong process, whereby old bone is removed by osteoclasts (bone resorption) and new bone is formed by osteoblasts (bone formation).1,2 Osteocytes, which act as mechanosensors/endocrine cells, and bone lining cells3 are also involved in bone remodelling.4 Myriad pathophysiological factors affecting bone remodelling have been observed in skeletal diseases such as osteoporosis, arthritis and periodontal disease.5 Oxidative stress is one of the pathophysiological factors affecting bone remodelling. Oxidative stress stimulates osteoclast differentiation, thereby enhancing bone resorption.6,7 Reactive oxygen species (ROS) stimulate the apoptosis of osteoblasts and osteocytes, thus affecting bone formation. ROS also activate mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERK1/2), c-Jun-N terminal kinase (JNK) and p38, and enhance osteoclastogenesis and bone resorption.811 These phenomena skew the bone remodelling process in favour of bone loss.

Antioxidants are compounds which reduce free radicals and oxidative stress.12 Antioxidants have been reported to promote differentiation of osteoblasts, bone formation and survival of osteocytes, as well as suppressing osteoclast differentiation and activity.8,1315 Some studies associate the age-related reduction in circulating antioxidants to osteoporosis in rats and women.1618 A decline in antioxidant levels has been reported to promote bone loss by triggering the tumour necrosis factor-alpha (TNF)-dependent signalling pathway,6 while administration of antioxidants, such as vitamin C, E, N-acetylcysteine and lipoic acid, have been reported to exert favourable effects in animal models of osteoporosis1921 and individuals with osteoporosis.2225

Caffeic acid (CA) is a metabolite of hydroxycinnamate and phenylpropanoid commonly synthesized by all plant species. It is a polyphenol present in many food sources like coffee, tea, wine, blueberries, apples, cider, honey and propolis.26 CA and its major derivatives including caffeic acid phenethyl ester (CAPE) and caffeic acid 3,4-dihydroxy-phenethyl ester (CADPE) are reported to possess potential antibacterial, antidiabetic, antioxidant, anti-inflammatory, antineoplastic and cardioprotective activities (reviewed in2729). As a potent antioxidant, CA has been demonstrated to decrease lipoperoxyl radicals (ROO) by donating a hydrogen atom to its corresponding hydroperoxide, which terminates the lipid peroxidation chain reaction. It also inhibits human low-density lipoprotein (LDL) oxidation induced by cupric ions.30 Furthermore, it interacts with other compounds, such as -tocopherol, chlorogenic and caftaric acids, to exert more potent antioxidant activity in a variety of different systems.3133 Therefore, the antioxidant activities of CA might protect against the negative effects of oxidative stress on bone cells and the skeletal system. This systematic review aims to summarise the effects of CA and its derivatives on bone cells and bone in literature.

A systematic literature search was conducted from July until November 2020 using PubMed, Scopus, Cochrane Library and Web of Science databases to identify studies on the effects of caffeic acid on bone and bone cells including osteoblasts, osteoclasts and osteocytes. The search string used was (1) caffeic acid AND (2) (bone OR osteoporosis OR osteoblasts OR osteoclasts OR osteocytes).

Studies with the following characteristics were included: (1) original research article with the primary objective of determining the effects of caffeic acid on bone and bone cells; (2) studies using cellular or animal models, or humans; (3) studies administering caffeic acid as a single compound but not in a mixture or food. Articles were excluded if they (1) do not contain original data; (2) use food rich in caffeic acid or mixtures containing caffeic acid. The bibliography of relevant review articles was traced for potential articles missed during database search. The search results were organised using EndNoteTM software (Clarivate Analytics, Philadelphia, USA). Duplicates were identified using EndNoteTM and confirmed by manual checking.

Two authors (S.O.E. and K.L.P.) searched the same databases using the search string mentioned and screened the search results. All the articles that did not match the selection criteria were excluded. Next, the articles which used caffeic acid in treating models other than bone-related diseases were removed. Finally, articles which used caffeic acid in combination with other compounds were also excluded. Any disagreement on the inclusion or exclusion of articles was resolved through discussion among the two authors. The corresponding author (K.Y.C.) had the final decision on articles included if a consensus could not be reached between authors responsible for screening. This systematic review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and checklist.34 Steps in the selection process, from identification, screening, eligibility to the inclusion of articles, are shown in Figure 1.

Figure 1 Flowchart of the article selection process.

From the literature search, 381 articles were identified, of which 87 were obtained from PubMed, 182 were from Scopus, 3 from Cochrane Library and 109 from Web of Science. A total of 155 duplicate articles were identified and removed. Of the 226 articles screened, 202 articles were excluded based on the selection criteria, whereby 51 articles did not contain primary data (3 book chapters, 2 commentary and 46 review articles), 147 articles and 2 conference abstracts presented topics irrelevant to the current review, a conference abstract had been published as a full-length research article and another conference abstract did not contain sufficient experiment details (Supplementary Material). Finally, 24 articles fulfilling all criteria mentioned were included in the review.

The included studies were published between 2006 and 2020. Seven studies were in vitro experiments using mouse bone marrow macrophages (BMMs), RAW264.7, RAW D and MG63 osteoblast cell lines3541 while 19 studies were in vivo studies using Sprague Dawley/Sprague Dawley albino rats, Wistar/Wistar albino rats, Balb/c mice, lipopolysaccharide (LPS)-resistant C3H/HEJ mice, C57BL/6J mice and ICR mice.35,38,4258 No human studies on this topic were reported.

Six in vitro studies focused on the effects of CA on osteoclast differentiation from haematopoietic cells using macrophage colony-stimulating factor (M-CSF), receptor activator of NF-B (RANK) ligand (RANKL) or TNF-,3539,41 while one in vitro study focused on the effect of CA on osteoblasts using MG63 osteoblast cell line.40 Four in vitro studies used CA doses between 0.15 M.35,37,38,40 Ang et al.36 used doses between 00.3 M and Sandra et al.41 and Sandra and Ketherin39 used a dose of 10 g/mL (55.5 M). The treatment period was 57 days for the differentiation of osteoclasts.

For animal studies, Duan et al.,55 Zawawi et al.,58 William et al.,51 Wu et al.,38 Zych et al.49 and Folwarczna et al.48,52 used CA or its derivatives at doses between 0.550 mg/kg via oral or intraperitoneal (i.p.) administration. Ucan et al.,57 Erdem et al.,53 Cicek et al.,54 Yigit et al.,45 Yildiz et al.50 and Tolba et al.56 used doses between 1020 mol/kg/day (2.845.69 mg/kg/day) via i.p. administration. Kizilda et al.4244 and Kazanciolu et al.46,47 used the dose of 10 mmol/kg/day (2.843 g/kg/day) for an i.p. administration, Kazanciolu et al.47 employed 50100 mmol/kg/day (14.2228.43 g/kg/day) for a localised administration, while Ha et al.35 used a collagen sponge soaked with CAPE with the final dose of 250 g/mouse. For oral administration, first-pass effect might affect the enteric absorption of CA or its derivatives.59 For i.p. administration, the injection is commonly performed at the lower left or right quadrant of the abdomen. The peritoneum can absorb the compounds fast and reach systemic circulation with greater bioavailability with fewer handling errors.60

The bone-related disease models used included ovariectomy (OVX)- or glucocorticoids (dexamethasone)-induced osteoporosis, polyethylene particle-induced bone defect and osteolysis, electromagnetic force (EMF)-stimulated bone loss, osteotomy- or anti-collagen antibody-induced arthritis (CAIA) and rapid maxillary expansion (RME) and LPS-induced periodontitis. The endpoints studied included bone microstructure, histomorphometry, bone remodelling and oxidative status. The effects of CA and its derivatives on bone remodelling have been summarized in Table 1.

Melguizo-Rodrguez et al. reported that 24-hour CA (1 M) incubation increased the number of MG63 osteoblast cells compared with control.40 Gene expression studies revealed that CA increased the expression of osteoblast-related genes such as bone morphogenetic protein-2 and -7 (BMP-2 and BMP-7), transforming growth factor-beta 1 (TGF-1), transforming growth factor-beta receptor 1, 2 and 3 (TGF-R1, TGF-R2 and TGF-R3) and osteoblastogenesis genes including Runt-related transcription (RUNX-2), alkaline phosphatase (ALP), collagen type 1 (COL-I), osterix (OSX) and osteocalcin (OSC).40 Additionally, pretreatment of CA (10 g/mL or 55.5 M) on RAW D cells for 2 h also significantly inhibited the RANKL and TNF-induced osteoclastogenesis with the suppression of p38 MAPK phosphorylation and tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells (OCLs) formation.39 Similarly, pretreatment of CA (0.1, 1 and 10 g/mL or 0.555, 5.55 and 55.5 M) on RAW D cells and BMMs for 3 days significantly inhibited the RANKL and TNF-induced osteoclastogenesis and NF-B activity in RAW-D cells and RANKL, TNF and M-CSF-induced osteoclastogenesis in BMMs.41

On the other hand, CAPE treatment (00.3 M; 57 days) suppressed the formation of TRAP-positive OCLs on RANKL-treated RAW264.7 cells and BMMs.36 Apoptosis occurred in CAPE-treated RAW264.7 cells with the disruption of the microtubule network in OCLs.36 Similarly, Kwon et al. reported that CAPE treatment (0.15 M) for 5 days suppressed OCLs formation from RANKL-stimulated RAW264.7 cells.37 Another study by Ha et al. treating M-CSF and RANKL-stimulated BMMs with CAPE (05 M for 57 days) also showed decreased OCLs formation in a concentration-dependent manner.35 The amount of TRAP-positive OCLs was decreased upon 0.1 and 0.5 M CAPE treatment by 30% and 95% respectively.35 No OCL formation was observed upon 1 M CAPE treatment.35 The anti-osteoclastogenic activities of CAPE are mainly contributed by its anti-inflammatory and antioxidant properties. Mechanistically, CAPE reduces superoxide anion generation by downregulating the nicotinamide adenine dinucleotide phosphate oxidase 1 (Nox1) expression through the interruption of nuclear factor-kappa B (NF-B) and c-Jun N-terminal kinase (JNK) signalling pathways.37 CAPE suppresses RANKL-mediated activation of the NF-B pathway by downregulating NF-B p65 subunit expression and its nuclear translocation,37 suppressing nuclear factor of activated T cells (NFAT) activities36 and degradation of NF-B inhibitor (IB),36,37 as well as inducing the degradation of IB kinase (IKK).37 CAPE also suppresses the expression and activation of JNK and its downstream transcription factors, such as c-Fos and c-Jun, which subsequently interrupt the protein activator-1 (AP-1) complex formation.37 Additionally, CAPE suppressed RANKL-induced activation of the Nox1 by inhibiting the Nox p47PHOX subunit translocation to the cell membrane and downregulation of Ras-related C3 botulinum toxin substrate 1 (Rac1) expression.37

On the other hand, Wu et al. reported that CADPE (0.15 M for 7 days) also concentration-dependently reduced OCL formation in the M-CSF and RANKL-stimulated BMMs and RAW264.7 cells.38 Mechanistic and characterisation examination revealed that CADPE suppressed RANKL-induced tumour necrosis factor receptor-associated factor 6 (TRAF6) activation and protein kinase B (PKB or also known as Akt) and activation of major MAPKs including ERK, JNK and p38.38 Subsequently, CADPE suppressed downstream expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), nuclear translocation of c-Fos protein and expression of osteoclastic markers, such as TRAP and cathepsin K, possibly through the non-receptor tyrosine kinase c-Src signalling.38 Interestingly, CADPE did not significantly affect the NF-kB signalling pathway and M-CSF-induced proliferation and differentiation of BMMs.

Supplementation of CA in animal models of bone loss yielded heterogeneous findings.48,49,52 This observation might be attributable to oral administration. Folwarczna et al. reported that CA (5 and 50 mg/kg, by stomach tube for 4 weeks) improved the bone mechanical properties by increasing the width of the trabecular metaphysis of the femur and decreasing the transverse growth in endosteal of the femur in OVX rats.48 Folwarczna et al. then demonstrated that CA (10 mg/kg/day; oral administration for 4 weeks) could reduce the width of tibial periosteal and endosteal osteoid compared with untreated OVX rats.52 However, CA did not promote or reduce the resorption of compact bone in the tibia of OVX-induced osteoporotic rats as evidenced by negligible changes of bone mass, bone mineral mass, bone mass/body mass ratio and bone mineral mass/body mass ratio.52 On the other hand, Zych et al. reported that CA at a similar dose (10 mg/kg/day; by stomach tube for 4 weeks) worsened the bone mechanical properties of healthy female Wistar Cmd:(WI)WU rats by decreasing the load of fracture at the femoral neck, decreasing the width of periosteal osteoid in the tibia and decreasing the width of the epiphysis and metaphysis trabecular in the femur compared with the negative control group.49

CAPE is the most extensively studied caffeic acid derivative in animal studies. The beneficial effects on new bone formation and healing upon systemic administration of CAPE had been reported.46,47,53,57 Erdem et al. reported that a low dose of CAPE (10 mol/kg; i.p. injection for 22 days) increased new bone formation and bone strength by increasing maximum torsional fracture momentum and degree of rigidity compared with negative control in rats that underwent unilateral femoral lengthening (osteotomy).53 Similarly, a 30-day i.p. injection of CAPE (10 mol/kg/day) also increased bone healing level in Sprague Dawley rats with cranial critical size bone defect.57 A higher dose of CAPE (10 mmol/kg/day, i.p. for 20 days) also further promoted the RME procedure-induced new bone formation in midpalatal suture of male Sprague Dawley rats.47 Similarly, a longer treatment period of CAPE (10 mmol/kg/day; i.p. injection for 28 days) also significantly promoted bone healing by increasing the total new bone areas in surgical-induced calvarial defects of male Wistar rats compared with the negative control.46 However, localised administration of CAPE (28 days) on surgical-induced calvarial defects by pre-mixing 50 and 100 mmol/kg CAPE solutions with gelatin sponges did not significantly improve the new bone formation.46

Localised and systemic administration of CAPE was reported to be beneficial in reducing osteolysis and bone loss.35,4245,50,5456,58 Ha et al. reported that collagen sponge implant impregnated with 250 g CAPE and RANKL could reduce osteoclastogenesis with significantly lesser TRAP-stained area in mouse calvariae compared with implants with RANKL only.35 Subcutaneous injection of CAPE (1 mg/kg/day for 10 days) reduced the polyethylene particle-induced calvarial osteolysis, surface bone resorption and TRAP-positive cells formation with an increase of bone volume (BV) on LPS-resistant C3H/HEJ female mice.58 However, no significant changes were observed in carboxy-terminal cross-linked type 1 collagen (CTX-1) and osteoclast-associated receptor levels among untreated and CAPE-treated rats with calvarial osteolysis.58

Similarly, Duan et al. reported that lower dose and frequency of CAPE injection (0.5 mg/kg twice a week; i.p. injection for 4 weeks) also increased the BV and trabecular number (Tb.N) due to the decrease of bone osteoclast formation (evidenced by decreased osteoclast number/bone perimeter) in OVX mice.55 Tolba et al. also reported that i.p. injection of CAPE (10 and 20 mol/kg) for 3 weeks increased femur weight and length in rats with dexamethasone-induced bone loss.56 The preservation of skeletal health in their study was associated with an improved antioxidant defence, such as higher levels of glutathione (GSH) and superoxide dismutase (SOD), and the reduction of malondialdehyde (MDA, lipid peroxidation product).56 This event led to an increase of osteoblastogenesis indicated by upregulation of RUNX-2 and ALP (osteoblast marker) levels56 On the other hand, decreased RANKL/osteoprotegerin (OPG) ratio was observed with CAPE treatment, indicating the suppression of osteoclastogenesis, which was further confirmed by lower acid phosphatase level and TRAP activity.56 In another study by Yildiz et al., CAPE (10 mol/kg/day; i.p. injection for 22 days) also increased the spine and femur BMD in rats with EMF-induced bone loss.50 Similarly, Cicek et al. reported a longer treatment of CAPE (10 mol/kg/day; i.p. injection for 28 days) also significantly improved the mechanical strength of cortical bone by increasing the breaking force, bending strength and total fracture energy in rats with EMF-induced bone loss compared with negative control.54

Additionally, a study by Wu et al. treated mice with an OVX-induced bone loss with a moderately high dose of CADPE (10 mg/kg; i.p. injection) every 2 days for 3 months.38 Results showed that CADPE could increase the BV fraction (BV/TV) and Tb.N, as well as decreased trabecular spacing (Tb.Sp) compared with the negative control.38 The improvement in the bone structure was contributed by reduced osteoclast number and eroded surface on the bone.38 Assessment of bone remodelling markers also revealed that serum TRAP5b and CTX-1 levels were reduced in CADPE-treated group compared with the negative control.38

On the other hand, CAPE was effective in reducing periodontitis-related bone loss and osteolysis.4245 CAPE (10 mol/kg/day, i.p. for 14 days) significantly reduced the subgingival ligature placement-induced periodontitis-mediated articular bone loss, histopathological features and severity of periodontal inflammation with lesser polymorphonuclear cells (PMNLs) infiltration in the junctional epithelium and connective tissues among Wistar albino rats.45 CAPE also suppressed the periodontitis-upregulated interleukin (IL)-1, IL-6, IL-10, TNF, MDA levels and the percentage of gingival apoptosis with the parallel restoration of periodontitis-downregulated GSH and glutathione peroxidase (GPx).45 Administration of high-dose CAPE (10 mmol/kg/day; i.p. for 15 days) in streptozotocin (STZ)-induced diabetic male Sprague Dawley rats reduced RANKL-positive osteoclast number, IL-1 levels, oxidative stress index (OSI), alveolar bone loss and histological analysis score in LPS-induced periodontitis. The treated rats also suffered lesser inflammatory reactions, ulcers and hyperemia.42 Similar changes of osteoclast number, IL-1 and OSI were observed in male Sprague Dawley rats with chronic stress and LPS-induced periodontitis treated with CAPE (10 mmol/kg/day, i.p. for 14 days).44 In addition, CAPE also increased the mesial and distal periodontal bone supports (MPBS and DPBS) in these rats.44 The effects of CAPE were sustained with a longer treatment period of CAPE (10 mmol/kg/day, i.p. for 28 days) on male Sprague Dawley rats with LPS-induced periodontitis.43

In contrast to the above findings, Williams et al. reported that subcutaneous injection of CAPE (1 mg/kg; at day 3, 7 and 10) did not reduce paw inflammation or bone loss in CAIA mice.51 Cartilage and bone degradation, as well as TRAP-positive cells on the bone surface and soft tissues, were still apparent in the supplemented CAIA group compared with the normal control.51

This systematic review found that although CA and its derivatives is a potential anti-osteoporosis agent by suppressing the formation of osteoclasts and their bone resorption activity, it worsened bone mechanical properties in some cases. The anti-osteoclastogenesis action of CA and its derivatives was mediated by the antioxidant activities, which blocked RANKL-induced TRAF6/Akt and MAPK signalling, as well as M-CSF/c-Src signalling. In animals, CA and its derivatives (mainly CAPE) prevented bone resorption in rodent calvariae when implanted in situ, facilitated the healing of bone defects, preserved bone structure and improved mechanical strength in osteoporosis models induced by OVX, dexamethasone, osteotomy, LPS-mediated periodontitis and EMF. However, CA did not alter bone resorption in OVX-induced osteoporotic rats and worsened the mechanical properties in normal rats. Additionally, CAPE did not suppress bone loss in rats with CAIA-induced bone loss.

Osteoblasts are bone-forming cells derived from bone marrow mesenchymal stem cells and are responsible for the synthesis, secretion and mineralisation of bone matrix.61 The expression of osteoblast markers was increased following CA or CAPE supplementation, an indication that CA and CAPE stimulated osteoblast proliferation, differentiation and maturation.40,56 Osteoblasts and osteocytes regulate the formation of osteoclasts through RANKL/OPG axis. Osteoblasts and osteocytes synthesise RANKL, which binds to RANK to activate the canonical pathway for osteoclastogenesis. They also secrete OPG, which is a decoy receptor for RANKL to suppress osteoclastogenesis. The production of RANKL is stimulated under conditions such as oestrogen deficiency62 and oxidative stress.63 Osteoclastogenesis can also be stimulated via a non-canonical pathway, for instance, through the binding of TNF with TNF receptor I or II.64 Glucocorticoids are potential modulators of RANKL/OPG axis, whereby dexamethasone is shown to downregulate OPG levels in osteoblasts.65 Tolba et al. showed that the RANKL/OPG level reduced in rats induced with dexamethasone with CAPE treatment.56 Other cellular studies showed that CA and its derivatives suppressed RANKL- and TNF-induced formation of OCLs from haematopoietic cells,3539 indicating that CA and its derivatives suppressed both canonical and non-canonical osteoclastogenesis.

The complex formed by the binding of RANKL to RANK causes the recruitment of the adaptor molecules tumour necrosis factor receptor-associated factors (TRAFs), including TRAF6.66 This event leads to the activation of several downstream signalling pathways, including c-Src/Akt/phosphatidylinositol 3-kinase and MAPKs (ERK/p38/JNK). CADPE was shown to suppress RANKL-induced activation of TRAF6 activation and the subsequent signalling pathways in multiple osteoclast progenitors, such as BMMs,38 RAW264.738 and RAW D cells.39 Sandra and Ketherin suggested that the downregulation of p38 is the key step of CA-mediated osteoclastogenesis.39 Upon activation, p38 initiates osteoclastogenesis by inducing NF-B and NFATc1 expression.67,68 Inhibition of p38 MAPK reduces RANKL (canonical) and TNF-induced (non-canonical) osteoclast formation.69

The NF-B pathway is another signalling pathway downstream of TRAFs critical for osteoclast differentiation and bone reabsorption activity. Upon activation, IKK (consisting of IKK, IKK and IKK) phosphorylates and degrades IB, which enables translocation of NF-B p65/p50 heterodimers into the nucleus to allow transcription of osteoclast-related genes.70 Kwon et al. demonstrated that the anti-osteoclastogenesis effects of CAPE were mediated via the degradation of total IKK, thereby preventing the phosphorylation and degradation of IB and subsequently suppresses the nuclear translation of p65.37 On the other hand, Wu et al. reported that CADPE did not affect phosphorylation or degradation of IB, as well as nuclear translocation, and DNA-binding activity of p65.38 This observation suggests that compared with CAPE, CADPE does not influence the NF-B signalling pathway.

ROS are one of the important secondary signals in the early stages of osteoclast differentiation.71,72 These ROS are mainly produced as superoxide anions by Nox1.73 Blocking of Nox1 ameliorates ROS production and the downstream MAPKs (JNK, p38 and ERK) and NF-B activation74 and subsequently suppresses the osteoclast formation.71 The reduction of Nox 1 and Rac1 expression by CAPE is accompanied by RANKL-downstream signalling, denoting that anti-osteoclastogenesis effects of CAPE are dependent on suppression of Nox1-mediated superoxide anion production. Besides, dexamethasone has been reported to increase the expression of oxidative stress-related genes in human osteoblasts.75 Tolba et al. showed that CAPE increased GSH and SOD but reduced MDA in the bone of the rats exposed to dexamethasone, indicating an improvement of redox status in the skeletal environment.56 Additionally, CAPE also reduced the OSI and bone loss with an improvement of bone support in rats with LPS-induced periodontitis.

NFATc1 is the master regulator of osteoclast-related gene expression, and it is activated by c-Fos and NF-B.76 Ha et al. observed that CAPE inhibited the recruitment of NF-B to NFATc1 promoter, and the combined effect of NF-B inhibition on c-Fos and NFATc1 may have caused CAPE to suppress osteoclastogenesis effectively.35 Holland et al. demonstrated a new fluorinated derivative of CAPE possesses potent anti-osteoclastogenic properties on RAW 264.7 cells by downregulating NFATc1 via suppression of c-Fos and NF-B signalling pathways.77 Besides, this new fluorinated CAPE also exhibits improved stability with a 2-fold higher potency than CAPE.77 On the other hand, although CADPE did not alter NF-B signalling, it still could suppress NFATc1 and other osteoclast-related markers, indicating other mechanisms of suppression could be involved, for instance, c-Src and MAPKs signalling pathways.38

Matrix metalloproteinases (MMPs), including gelatinases (MMP-2 and MMP-9) are examples of zinc-dependent extracellular matrix-degrading enzymes, which actively participate in bone resorption.78 MMPs are expressed as inactive proenzymes or zymogens that can be activated by several mediators including AP-1, NF-B, TNF and TGF.78 Currently, there is no study conducted to investigate the inhibitory effects of CA and CAPE on osteoclastic MMPs activity and its subsequent linkage in bone resorption; interestingly, CA and CAPE were reported to inhibit MMP-9 activity in human hepatocellular carcinoma HEP3B cells.79,80 This observation renders an interesting research gap in osteoclastic MMP inhibition upon CA and its derivatives treatment.

Suppression of osteoclastogenesis by CA or its derivatives have significant therapeutic potential against bone disorders induced by excessive bone resorption. Bone loss after osteotomy is a rapid process that affects both fractured and unfractured bone and may be incompletely reversible.81 CAPE was reported to improve bone formation and mechanical strength of bone in osteotomy.53 Exposure to EMF radiation caused by high-voltage transmission lines and transformers could affect bone health through decreased BMD, serum calcium and ALP level leading to the increase of bone resorption.82 CAPE increased the spine and femur BMD levels50 and increased mechanical strength of bones54 in rats exposed to EMF radiation. Total hip arthroplasty without cement often caused osteolysis induced by polyethylene particles.83 CAPE was shown by Zawawi et al. to prevent calvarial bone resorption in a murine polyethylene particle-induced osteolysis model.58 Therefore, biomaterials impregnated with CA or its derivatives could be adopted to prevent osteolysis in the arthroplasty procedure. CA has been incorporated in chitosan/(3-chloropropyl) trimethoxysilane scaffold for hard-tissue engineering applications and this adopted material exhibits antibacterial and anticancer effects.84 Ucan et al. observed that CAPE increased cranial bone healing in rats with critical size bone defect, suggesting that it could be administered systematically or locally to treat bone fracture/defect healing.57

Similarly, CAPE also effectively reduced the articular bone loss, inflammatory cytokines production and oxidative stress in rats with LPS-mediated periodontitis. Additionally, Wu et al.38 and Duan et al.55 demonstrated that CADPE prevented the ovariectomy-induced bone loss by suppressing osteoclast activity in a mouse model, while Folwarczna et al. showed increased width of trabecular metaphysis in the femur of OVX rats.48 Similarly, Tolba et al. showed improved bone formation and skeletal health in rats with dexamethasone-induced bone loss upon receiving CAPE.56 Additionally, CA and its derivatives may be involved in oestrogen production and signalling. Zych et al. reported that an oral administration of CA (10 mg/kg/day for 4 weeks) significantly restored the serum oestradiol levels in OVX rats.85 Interestingly, CA at 10 and 100 M did not cause any alteration in calcium content in the femoral-diaphyseal and metaphyseal ex vivo culture, suggesting its bone-protecting effect may not involve calcium metabolism and regulation.86 Additionally, CAPE was reported as a selective human oestrogen receptor agonist with the EC50 value of 3.72 M in oestrogen-responsive element transcription.87 A recent in silico study by Zhao et al. suggested potential osteoimmunological effects of CAPE, which may explain its biological activities on both immune and skeletal systems.88 However, the findings from this modelling study requires further validation through in vitro and in vivo models. As oestrogen deficiency due to menopause and glucocorticoids present the most significant cause of primary and secondary osteoporosis globally, CA and its derivatives have the potential to be used as an adjuvant therapy to existing osteoporosis management strategies. The mechanisms of action of CA and its derivatives in osteoclastogenesis have been summarized in Figure 2.

Figure 2 Mechanism of action of caffeic acid and its derivatives.

Abbreviations: , decrease or downregulate; ?, unknown mechanism; Akt, protein kinase B; AP-1, activator protein 1; CA, caffeic acid; CADPE; caffeic acid 3,4-dihydroxy-phenethyl ester; CAPE, caffeic acid phenethyl ester; c-Src, cellular sarcoma tyrosine kinase; ERK1/2, extracellular signal-regulated kinases 1/2; GM-CSF, granulocyte-macrophage colony-stimulating factor; Grb2, growth factor receptor-bound protein 2; IFN-, interferon-gamma; IL, interleukin; IL1R, interleukin-1 receptor; IB, NF-B inhibitor protein; IKK, IB kinase; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; M-CSF-R, M-CSF receptor; MAPKs, mitogen-activated protein kinases; NFAT, nuclear factor of activated T cells; NF-B, nuclear factor kappa B; NIK, MAPK kinase kinase 14; Nox1, nicotinamide adenine dinucleotide phosphate oxidase 1; OPG, osteoprotegerin; PI3k, phosphoinositide 3-kinase; Rac1, Ras-related C3 botulinum toxin substrate 1; RANK, receptor activator of NF-B; RANKL, receptor activator of NF-B ligand; ROS, reactive oxygen species; TAK, MAPK kinase kinase 7; TLR4, Toll-like receptor 4; TNF, tumour necrosis factor-alpha; TNFR1/2, TNF receptor 1/2; TRAF2, tumour necrosis factor receptor-associated factor 2; TRAF6; tumour necrosis factor receptor-associated factor 6.

Regardless of the positive effects of CA on bone status, some studies have reported negative effects associated with supplementation of CA and its derivatives. CA supplementation did not affect the bone resorption52 and reduced transverse growth of endosteal in femur48 of rats with OVX-induced osteoporosis. In normal rats, CA supplementation even negatively affected their bone mechanical properties.49 Moreover, CAPE supplementation has been reported to stimulate the synthesis of PGE2,89 which mediates osteoclastogenesis through RANKL stimulation and activation of the NF-B pathway.90 This event will eventually increase TRAP-positive OCLs. Similarly, Williams et al. showed that CAPE did not suppress osteoclastogenesis in rats with CAIA.51

In term of safety, the International Agency for Cancer Research classifies CA as Class 2B (possibly carcinogenic to humans),91 and it was reported to induce renal tubular cell hyperplasia, forestomach hyperplasia, renal cell adenoma and forestomach cancer in rodents.9294 CA has been reported to be non-mutagenic and non-clastogenic.91 Therefore, its carcinogenicity may involve epigenetic modification. Human toxicity and carcinogenicity of CA and its derivatives remain unknown. CA also showed anti-implantation activity in pregnant mice at a median effective dose of 4.26 mg/kg/day.95 Similarly, 5 mg/kg/day and 150 mg/kg of CA in mice demonstrated anti-implantation activity in early pregnancy.96 On the other hand, 0.15 mg/kg/day, 5 mg/kg/day and 150 mg/kg/day of CA for 21 days in mice showed no maternal toxicity, foetal teratogenesis or post-natal effects on pup development and mortality.96 The same experiment stated that the no-observed-adverse-effect level of CA for pregnant female mice was 0.15 mg/kg/day.96 Therefore, high-dose CA should be cautioned in humans, especially pregnant women.

Several common limitations can be identified from the studies reviewed. Most studies did not adopt a positive control to compare against the anti-osteoclastogenesis or anti-osteoporosis effect of CA. Therefore, the therapeutic effects of CA and currently available anti-resorptive therapy cannot be compared. Although osteoblastogenesis and bone formation are also important in bone remodelling, evidence of CA on these processes is limited in the literature. The actions of CA in humans cannot be confirmed due to the lack of human clinical trials. These aspects can be improved in future studies.

The current review also has several limitations. We only considered articles indexed by PubMed, Scopus, Cochrane Library and Web of Science; therefore, non-indexed articles could be overlooked. We only selected articles studying CA or its derivatives as a single compound to understand its mechanism of action properly without other interference, but not a mixture of compounds or natural products rich in CA. CA are present in foods, and interaction with other compounds in the food matrix might alter its absorption, bioavailability and action on the target tissue. Moreover, the heterogeneous findings of CA in bone loss reduction upon oral administration further emphasise these possibilities.

The current preclinical evidence agrees that CA and its derivatives exert promising skeletal protective effects by inhibiting osteoclastogenesis and bone resorption, but literature on bone formation is limited. Notwithstanding that, the skeletal effects of CA and its derivatives in models of normal bone health should be investigated because the limited studies available show undesirable effects. Human clinical trials to validate the skeletal effects of CA are lacking. Therefore, a well-planned clinical trial should be conducted to confirm the potential of CA as an antiresorptive agent. This information is critical for CA and its derivatives to be incorporated as part of the strategies to prevent bone loss.

The researchers are funded by Universiti Kebangsaan Malaysia through Research University Grant (GUP-2020-021). S.O.E. and K.L.P. are post-doctoral researchers funded by Universiti Kebangsaan Malaysia through FPR-1 and RGA-1 grants.

The authors report no conflicts of interest in this work.

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31. Laranjinha J, Vierira O, Almeida L, et al. Inhibition of metmyoglobin/H2O2-dependent low density lipoprotein lipid peroxidation by naturally occurring phenolic acids. Biochem Pharmacol. 1996;51(4):395402. doi:10.1016/0006-2952(95)02171-X

32. Meyer AS, Donovan JL, Pearson DA, et al. Fruit hydroxycinnamic acids inhibit human low-density lipoprotein oxidation in vitro. J Agric Food Chem. 1998;46(5):17831787.

33. Fukumoto LR, Mazza G. Assessing antioxidant and prooxidant activities of phenolic compounds. J Agric Food Chem. 2000;48(8):35973604. doi:10.1021/jf000220w

34. Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: the prisma statement. PLoS Med. 2009;6(7):e1000097. doi:10.1371/journal.pmed.1000097

35. Ha J, Choi H-S, Lee Y, et al. Caffeic acid phenethyl ester inhibits osteoclastogenesis by suppressing NF kappaB and downregulating NFATc1 and c-Fos. Int Immunopharmacol. 2009;9(6):774780. doi:10.1016/j.intimp.2009.03.001

36. Ang ES, Pavlos NJ, Chai LY, et al. Caffeic acid phenethyl ester, an active component of honeybee propolis attenuates osteoclastogenesis and bone resorption via the suppression of RANKL-induced NF-kappaB and NFAT activity. J Cell Physiol. 2009;221(3):642649. doi:10.1002/jcp.21898

37. Kwon YB, Wang FF, Jang HD. Anti-osteoclastic effect of caffeic acid phenethyl ester in murine macrophages depends upon the suppression of superoxide anion production through the prevention of an active-nox1 complex formation. J Nutr Biochem. 2018;58:158168. doi:10.1016/j.jnutbio.2018.03.023

38. Wu X, Li Z, Yang Z, et al. Caffeic acid 3,4-dihydroxy-phenethyl ester suppresses receptor activator of NF-B ligandinduced osteoclastogenesis and prevents ovariectomy-induced bone loss through inhibition of mitogen-activated protein kinase/activator protein 1 and Ca2+nuclear factor of activated T-cells cytoplasmic 1 signaling pathways. J Bone Miner Res. 2012;27(6):12981308. doi:10.1002/jbmr.1576

39. Sandra F, Ketherin K. Caffeic acid inhibits RANKL and TNF--induced phosphorylation of p38 mitogen-activated protein kinase in RAW-D cells. Indones Biomed J. 2018;10(2):140143. doi:10.18585/inabj.v10i2.437

40. Melguizo-Rodrguez L, Manzano-Moreno FJ, Illescas-Montes R, et al. Bone protective effect of extra-virgin olive oil phenolic compounds by modulating osteoblast gene expression. Nutrients. 2019;11(8):1722. doi:10.3390/nu11081722

41. Sandra F, Kukita T, Tang QY, et al. Cafeic acid inhibits NFB activation of osteoclastogenesis signaling pathway. Indones Biomed J. 2011;3(3):216222. doi:10.18585/inabj.v3i3.153

42. Kzlda A, Arabac T, Albayrak M, et al. A biochemical and immunohistochemical study of the effects of caffeic acid phenethyl ester on alveolar bone loss and oxidative stress in diabetic rats with experimental periodontitis. Biotech Histochem. 2020;95(6):456463. doi:10.1080/10520295.2020.1718756

43. Kzlda A, Arabac T, Albayrak M, et al. Therapeutic effects of caffeic acid phenethyl ester on alveolar bone loss in rats with endotoxin-induced periodontitis. J Dent Sci. 2019;14(4):339345. doi:10.1016/j.jds.2019.03.011

44. Kzlda A, Arabac T, Albayrak M, et al. Evaluation of caffeic acid phenethyl ester administration in chronically stressed rats with experimental periodontitis. Cumhur Dent J. 2019;22(1):114120.

45. Yiit U, Krzolu FY, Uuz AC, et al. Is caffeic acid phenethyl ester more protective than doxycycline in experimental periodontitis? Arch Oral Biol. 2017;81:6168. doi:10.1016/j.archoralbio.2017.04.017

46. Kazancioglu HO, Bereket MC, Ezirganli S, et al. Effects of caffeic acid phenethyl ester on wound healing in calvarial defects. Acta Odontol Scand. 2015;73(1):2127. doi:10.3109/00016357.2014.942876

47. Kazancioglu HO, Aksakalli S, Ezirganli S, et al. Effect of caffeic acid phenethyl ester on bone formation in the expanded inter-premaxillary suture. Drug Des Devel Ther. 2015;9:64836488. doi:10.2147/DDDT.S97797

48. Folwarczna J, Pytlik M, Zych M, et al. Effects of caffeic and chlorogenic acids on the rat skeletal system. Eur Rev Med Pharmacol Sci. 2015;19(4):682693.

49. Zych M, Folwarczna J, Pytlik M, et al. Administration of caffeic acid worsened bone mechanical properties in female rats. Planta Med. 2010;76(5):407411. doi:10.1055/s-0029-1240603

50. Yildiz M, Cicek E, Cerci SS, et al. Influence of electromagnetic fields and protective effect of cape on bone mineral density in rats. Arch Med Res. 2006;37(7):818821. doi:10.1016/j.arcmed.2006.03.006

51. Williams B, Tsangari E, Stansborough R, et al. Mixed effects of caffeic acid phenethyl ester (CAPE) on joint inflammation, bone loss and gastrointestinal inflammation in a murine model of collagen antibody-induced arthritis. Inflammopharmacology. 2017;25(1):5568. doi:10.1007/s10787-016-0306-z

52. Folwarczna J, Zych M, Burczyk J, et al. Effects of natural phenolic acids on the skeletal system of ovariectomized rats. Planta Med. 2009;75(15):15671572. doi:10.1055/s-0029-1185904

53. Erdem M, Gulabi D, Sen C, et al. Effects of caffeic acid phenethyl ester and melatonin on distraction osteogenesis: an experimental study. SpringerPlus. 2014;3:8. doi:10.1186/2193-1801-3-8

54. Cicek E, Gokalp O, Varol R, et al. Influence of electromagnetic fields on bone fracture in rats: role of cape. Biomed Environ Sci. 2009;22(2):157160. doi:10.1016/S0895-3988(09)60039-8

55. Duan W, Wang Q, Li F, et al. Anti-catabolic effect of caffeic acid phenethyl ester, an active component of honeybee propolis on bone loss in ovariectomized mice: a micro-computed tomography study and histological analysis. Chin Med J (Engl). 2014;127(22):39323936.

56. Tolba MF, El-Serafi AT, Omar HA. Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo: impact on oxidative stress and RANKL/OPG signals. Toxicol Appl Pharmacol. 2017;324:2635. doi:10.1016/j.taap.2017.03.021

57. Uan MC, Koparal M, Aaayak S, et al. Influence of caffeic acid phenethyl ester on bone healing in a rat model. J Int Med Res. 2013;41(5):16481654. doi:10.1177/0300060513490613

58. Zawawi MS, Perilli E, Stansborough RL, et al. Caffeic acid phenethyl ester abrogates bone resorption in a murine calvarial model of polyethylene particle-induced osteolysis. Calcif Tissue Int. 2015;96(6):565574. doi:10.1007/s00223-015-9982-8

59. Renwick A. First-pass metabolism within the lumen of the gastrointestinal tract. Presystem Drug Eliminat. 2013;1:1.

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

By daniellenierenberg

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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BrainStorm’s Covid-19 ARDS treatment improves lung function in study – Clinical Trials Arena

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BrainStorm Cell Therapeutics has announced that its NurOwn (MSC-NTF cell) derived exosomes provided significant improvement in lung function and histology in an acute respiratory distress syndrome (ARDS) mouse model, in a preclinical study.

Mesenchymal stem cell (MSC)-derived exosomes can penetrate deep into tissues and deliver immunomodulatory molecules effectively.

A type of respiratory failure, ARDS is linked to Covid-19 and is mediated by dysregulated cytokine production.

Intratracheal administration of NurOwn derived exosomes provided a statistically significant reduction in lung disease severity score, the study data showed.

Furthermore, improvements in lipopolysaccharide (LPS)-induced ARDS markers like lung function, fibrin presence, neutrophil accumulation, cytokine expression and oxygenation levels in the blood, were observed.

These improvements were significantly superior to those noticed following nave MSC-derived exosome administration.

BrainStorm Research and Development vice-president Dr Revital Aricha said: These exciting preclinical data suggest that NurOwn derived exosomes have the potential to treat Covid-19-induced ARDS or other severe respiratory complications and that they are more effective than exosomes isolated from nave MSCs at combatting the various symptoms of the syndrome.

This publication in a highly regarded journal provides important validation for the scientific advances and significance of BrainStorms preclinical research programs, including on our exosome-based technology platform.

The NurOwn technology platform (autologous MSC-NTF cells) represents a promising investigational therapeutic approach to targeting disease pathways important in neurodegenerative disorders.

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MSC-NTF cells are made from autologous, bone marrow-derived MSCs expanded and separated ex vivo.

Brainstorm CEO Chaim Lebovits said: While our primary focus is on advancing NurOwn towards regulatory approval in ALS, we continue to evaluate the potential of our exosome-based platform to address unmet medical needs.

In December 2019, the company received a recommendation from the independent Data Safety Monitoring Board (DSMB) to continue the Phase II clinical trial of NurOwn in progressive multiple sclerosis patients.

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

By daniellenierenberg

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

By daniellenierenberg

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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A Study of Cord Blood Transplantation in Children and Young Adults with Blood Cancers and Non-Cancerous Blood Disorders – On Cancer – Memorial Sloan…

By daniellenierenberg

Full TitleCord Blood Transplantation in Children and Young Adults with Hematologic Malignancies and Non-Malignant DisordersPurpose

The transplantation of stem cells from umbilical cord blood is a treatment for some blood cancers and non-cancerous blood or metabolic disorders. Patients routinely receive high doses of chemotherapy and sometimes radiation before receiving the stem cells to help make room in the bone marrow for new blood stem cells to grow, prevent the body from rejecting the transplanted cells, and help kill any abnormal blood cells in the body. However, the combination of these treatments can have serious side effects.

Researchers are doing this study to find out whether a combination of the chemotherapy drugs clofarabine, fludarabine, and busulfan (without radiation) is a safe and effective treatment for children and young adults receiving cord blood transplants for blood cancers or non-cancerous blood or metabolic disorders. These three drugs are given intravenously (by vein).

To be eligible for this study, patients must meet several criteria, including but not limited to the following:

For more information about this study and to inquire about eligibility, please contact 1-833-MSK-KIDS.

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Adipose Derived Stem Cell Therapy Market Analysis and Forecast, 2020-2026 Coherent Market Insights | BioRestorative Therapies, Inc., Celltex…

By daniellenierenberg

The Adipose Derived Stem Cell Therapy Market Research Report is a resource, which provides current as well as upcoming technical and financial details of the industry to 2027. This report gives you so important and essentials data of Market size, share, trends, Growth, applications, forecast and cost analysis. Delivery development in North America, China, Europe, and South East Asia, Japan as well as in the Globe. The report proves to be indispensable when it comes to market definition, classifications, applications and engagements. The market report also computes the market size and revenue generated from the sales. The industry analysis report presents the key statistics on the market status of global and regional manufacturers and also acts as a valuable source of leadership and direction. What is more, theAdipose Derived Stem Cell Therapy market report analyses and provides historic data along with the current performance of the market

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.

This Press Release will help you to Know the Volume, growth with Impacting Trends. Get SAMPLE PDF (Including Full TOC, Table & Figures) at:

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Global Adipose Derived Stem Cell Therapy Market competition by Top Key Players: 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.

Adipose Derived Stem Cell Therapy Market section by Region:

The Middle East and Africa North AmericaSouth AmericaEuropeAsia-Pacific

Segmentation: The report has been separated into different categories, such as product type, application, end user, and region. Every segment is evaluated based on the CAGR, share and growth potential. In the regional analysis, the report highlights the prospective region, which should generate opportunities in the global Adipose Derived Stem Cell Therapy market in the years to come. This segmented analysis will surely prove to be a useful tool for readers, stakeholders and market participants to get a full picture of the Adipose Derived Stem Cell Therapy global market and its growth potential in the years to come.

TheAdipose Derived Stem Cell TherapyMarket report offers a plethora of insights which include:

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Important Information that can be extracted from the Report:

Assessment of the COVID-19 impact on the growth of the Adipose Derived Stem Cell Therapy MarketSuccessful market entry strategies formulated by emerging market playersPricing and marketing strategies adopted by established market playersCountry-wise assessment of the Adipose Derived Stem Cell Therapy Market in key regionsYear-on-Year growth of each market segment over the forecast period 2026

TheAdipose Derived Stem Cell TherapyMarket report considers the following years to predict market growth:

The GlobalAdipose Derived Stem Cell TherapyMarket is displayed in 13 Chapters:

Chapter 1: Market Overview, Drivers, Restraints and OpportunitiesChapter 2: Market Competition by ManufacturersChapter 3: Production by RegionsChapter 4: Consumption by RegionsChapter 5: Production, By Types, Revenue and Market share by TypesChapter 6: Consumption, By Applications, Market share (%) and Growth Rate by ApplicationsChapter 7: Complete profiling and analysis of ManufacturersChapter 8: Manufacturing cost analysis, Raw materials analysis, Region-wise manufacturing expensesChapter 9: Industrial Chain, Sourcing Strategy and Downstream BuyersChapter 10: Marketing Strategy Analysis, Distributors/TradersChapter 11: Market Effect Factors AnalysisChapter 12: Market ForecastChapter 13:Adipose Derived Stem Cell Therapy Research Findings and Conclusion, Appendix, methodology and data source

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Alterations of NK Cell Phenotype in the Disease Course of Multiple Myeloma – DocWire News

By daniellenierenberg

This article was originally published here

Cancers (Basel). 2021 Jan 10;13(2):E226. doi: 10.3390/cancers13020226.

ABSTRACT

Accumulating evidence demonstrates important roles for natural killer (NK) cells in controlling multiple myeloma (MM). A prospective flow cytometry-based analysis of NK cells in the blood and bone marrow (BM) of MM patient subgroups was performed (smoldering (SMM), newly diagnosed (ND), relapsed/refractory, (RR) and post-stem cell transplantation (pSCT)). Assessments included the biomarker expression and function of NK cells, correlations between the expression of receptors on NK cells with their ligands on myeloma cells, and comparisons between MM patient subgroups and healthy controls. The most striking differences from healthy controls were found in RR and pSCT patients, in which NK cells were less mature and expressed reduced levels of the activating receptors DNAM-1, NKG2D, and CD16. These differences were more pronounced in the BM than in blood, including upregulation of the therapeutic targets TIM3, TIGIT, ICOS, and GITR. Their expression suggests NK cells became exhausted upon chronic encounters with the tumor. A high expression of SLAMF7 on blood NK cells correlated with shorter progression-free survival. This correlation was particularly evident in ND patients, including on mature CD56dim NK cells in the BM. Thus, our NK cell analysis identified possible therapeutic targets in MM and a biomarker with prognostic potential for disease progression.

PMID:33435153 | DOI:10.3390/cancers13020226

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

By daniellenierenberg

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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Stem Cell Assay Market | Know the aspects that will serve as game-changers for the market – BioSpace

By daniellenierenberg

Stem cell assay refers to the procedure of measuring the potency of antineoplastic drugs, on the basis of their capability of retarding the growth of human tumor cells. The assay consists of qualitative or quantitative analysis or testing of affected tissues and tumors, wherein their toxicity, impurity, and other aspects are studied.

With the growing number of successful stem cell therapy treatment cases, the global market for stem cell assays will gain substantial momentum. A number of research and development projects are lending a hand to the growth of the market. For instance, the University of Washingtons Institute for Stem Cell and Regenerative Medicine (ISCRM) has attempted to manipulate stem cells to heal eye, kidney, and heart injuries. A number of diseases such as Alzheimers, spinal cord injury, Parkinsons, diabetes, stroke, retinal disease, cancer, rheumatoid arthritis, and neurological diseases can be successfully treated via stem cell therapy. Therefore, stem cell assays will exhibit growing demand.

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Another key development in the stem cell assay market is the development of innovative stem cell therapies. In April 2017, for instance, the first participant in an innovative clinical trial at the University of Wisconsin School of Medicine and Public Health was successfully treated with stem cell therapy. CardiAMP, the investigational therapy, has been designed to direct a large dose of the patients own bone-marrow cells to the point of cardiac injury, stimulating the natural healing response of the body.

Newer areas of application in medicine are being explored constantly. Consequently, stem cell assays are likely to play a key role in the formulation of treatments of a number of diseases.

Global Stem Cell Assay Market: Overview

The increasing investment in research and development of novel therapeutics owing to the rising incidence of chronic diseases has led to immense growth in the global stem cell assay market. In the next couple of years, the market is expected to spawn into a multi-billion dollar industry as healthcare sector and governments around the world increase their research spending.

The report analyzes the prevalent opportunities for the markets growth and those that companies should capitalize in the near future to strengthen their position in the market. It presents insights into the growth drivers and lists down the major restraints. Additionally, the report gauges the effect of Porters five forces on the overall stem cell assay market.

Global Stem Cell Assay Market: Key Market Segments

For the purpose of the study, the report segments the global stem cell assay market based on various parameters. For instance, in terms of assay type, the market can be segmented into isolation and purification, viability, cell identification, differentiation, proliferation, apoptosis, and function. By kit, the market can be bifurcated into human embryonic stem cell kits and adult stem cell kits. Based on instruments, flow cytometer, cell imaging systems, automated cell counter, and micro electrode arrays could be the key market segments.

In terms of application, the market can be segmented into drug discovery and development, clinical research, and regenerative medicine and therapy. The growth witnessed across the aforementioned application segments will be influenced by the increasing incidence of chronic ailments which will translate into the rising demand for regenerative medicines. Finally, based on end users, research institutes and industry research constitute the key market segments.

The report includes a detailed assessment of the various factors influencing the markets expansion across its key segments. The ones holding the most lucrative prospects are analyzed, and the factors restraining its trajectory across key segments are also discussed at length.

Global Stem Cell Assay Market: Regional Analysis

Regionally, the market is expected to witness heightened demand in the developed countries across Europe and North America. The increasing incidence of chronic ailments and the subsequently expanding patient population are the chief drivers of the stem cell assay market in North America. Besides this, the market is also expected to witness lucrative opportunities in Asia Pacific and Rest of the World.

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Global Stem Cell Assay Market: Vendor Landscape

A major inclusion in the report is the detailed assessment of the markets vendor landscape. For the purpose of the study the report therefore profiles some of the leading players having influence on the overall market dynamics. It also conducts SWOT analysis to study the strengths and weaknesses of the companies profiled and identify threats and opportunities that these enterprises are forecast to witness over the course of the reports forecast period.

Some of the most prominent enterprises operating in the global stem cell assay market are Bio-Rad Laboratories, Inc (U.S.), Thermo Fisher Scientific Inc. (U.S.), GE Healthcare (U.K.), Hemogenix Inc. (U.S.), Promega Corporation (U.S.), Bio-Techne Corporation (U.S.), Merck KGaA (Germany), STEMCELL Technologies Inc. (CA), Cell Biolabs, Inc. (U.S.), and Cellular Dynamics International, Inc. (U.S.).

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