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."

More here:
'I was excited to help somebody': Montana Western's Dylan Pope reflects on donating bone marrow - MontanaSports

Bone Marrow Stem Cells Comments Off on ‘I was excited to help somebody’: Montana Western’s Dylan Pope reflects on donating bone marrow – MontanaSports | January 22nd, 2021

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...

Bone Marrow Stem Cells Comments Off on Global Bone Marrow-Derived Stem Cells (BMSCS) Market 2021 Overview, Trends, Growth Factors and Leading Players With Detailed Analysis of Industry… | January 22nd, 2021

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

Bone Marrow Stem Cells Comments Off on Brave Evie Hodgson from Sleights finally has bone marrow transplant after one last ‘twist in the tale’ – Yorkshire Live | January 22nd, 2021

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.

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

Bone Marrow Stem Cells Comments Off on Mesoblast Limited: Is Stemcell Therapy Ready For Prime Time? – Sick Economics | January 22nd, 2021

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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41. Atwell S, Ridgway JB, Wells JA, Carter P. Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library. J Mol Biol. 1997;270:2635. doi:10.1006/jmbi.1997.1116

42. Pan H, Mostoslavsky G, Eruslanov E, Kotton DN, Kramnik I. Dual-promoter lentiviral system allows inducible expression of noxious proteins in macrophages. J Immunol Methods. 2007;329(12):3144. doi:10.1016/j.jim.2007.09.009

43. Biagi E, Bambacioni F, Gaipa G, et al. Efficient lentiviral transduction of primary human acute myelogenous and lymphoblastic leukemia cells. Haematologica. 2001;86(1):1316.

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45. Liberg D, nnervik P, Riva M, Larsson L, Forsberg G, Wachenfeldt K. Antibody Blockade of IL1RAP Signaling Reduces Metastasis in a Breast Cancer Model. Annual Meeting of the American Association for Cancer: McCormick Place North/South Chicago, Illinois, USA; 2018.

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

Bone Marrow Stem Cells Comments Off on [Full text] Identification and Targeting of ThomsenFriedenreich and IL1RAP | OTT – Dove Medical Press | January 22nd, 2021

Introduction

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Figure 1 Distribution of 60 isolated pathogenic bacteria pathogen.

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

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

Table 2 Univariate Analysis of Risk Factors for BSI

Table 3 Multivariate Analysis of Risk Factors for BSI

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

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

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

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

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

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

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

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

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

The authors report no conflicts of interest in this work.

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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.

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

Bone Marrow Stem Cells Comments Off on [Full text] Clinical Analysis of Bloodstream Infections During Agranulocytosis Aft | IDR – Dove Medical Press | January 22nd, 2021

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

Bone Marrow Stem Cells Comments Off on BrainStorm Announces the Publication of Preclinical Data Highlighting the Potential of a NurOwn Derived Exosome-Based Treatment for COVID-19 ARDS -… | January 22nd, 2021

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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

Bone Marrow Stem Cells Comments Off on Cancer requires more tutoring, with Meyer continuing to Teaching Cancer a lesson – News – vintontoday.com | January 22nd, 2021

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

Bone Marrow Stem Cells Comments Off on [Full text] Effects of Caffeic Acid and Its Derivatives on Bone: A Systematic Revi | DDDT – Dove Medical Press | January 22nd, 2021

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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18. Mavrokokki T, Cheng A, Stein B, Goss A. Nature and frequency of bisphosphonate-associated osteonecrosis of the jaws in Australia. J Oral Maxillofacial Surg. 2007;65:415423. doi:10.1016/j.joms.2006.10.061.

19. Gutta R, Louis PJ. Bisphosphonates and osteonecrosis of the jaws: science and rationale. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol. 2007;104:186193. doi:10.1016/j.tripleo.2006.12.004.

20. Ruggiero SL, Dodson TB, Assael LA, Landesberg R, Marx RE, Mehrotra B. American Association of Oral and Maxillofacial Surgeons position paper on bisphosphonate-related osteonecrosis of the jaws2009 update. J Oral Maxillofacial Surg. 2009;67:212. doi:10.1016/j.joms.2009.01.009.

21. Patel S, Choyee S, Uyanne J, et al. Non-exposed bisphosphonate-related osteonecrosis of the jaw: a critical assessment of current definition, staging, and treatment guidelines. Oral Dis. 2012;18:625632. doi:10.1111/j.1601-0825.2012.01911.x.

22. Yarom N, Fedele S, Lazarovici TS, Elad S. Is exposure of the jawbone mandatory for establishing the diagnosis of bisphosphonate-related osteonecrosis of the jaw? J Oral Maxillofacial Surg. 2010;68:705. doi:10.1016/j.joms.2009.07.086.

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24. Junquera L, Gallego L. Nonexposed bisphosphonate-related osteonecrosis of the jaws: another clinical variant? J Oral Maxillofacial Surg. 2008;66:15161517. doi:10.1016/j.joms.2008.02.012.

25. Fedele S, Porter SR, DAiuto F, et al. Nonexposed variant of bisphosphonate-associated osteonecrosis of the jaw: a case series. Am J Med. 2010;123:10601064. doi:10.1016/j.amjmed.2010.04.033.

26. Schiodt M, Reibel J, Oturai P, Kofod T. Comparison of nonexposed and exposed bisphosphonate-induced osteonecrosis of the jaws: a retrospective analysis from the Copenhagen cohort and a proposal for an updated classification system. Oral Surg Oral Med Oral Pathol Oral Radiol. 2014;117:204213. doi:10.1016/j.oooo.2013.10.010.

27. Gnant M, Mlineritsch B, Stoeger H, et al. Adjuvant endocrine therapy plus zoledronic acid in premenopausal women with early-stage breast cancer: 62-month follow-up from the ABCSG-12 randomised trial. Lancet Oncol. 2011;12:631641. doi:10.1016/S1470-2045(11)70122-X.

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33. Wood J, Bonjean K, Ruetz S, et al. Novel antiangiogenic effects of the bisphosphonate compound zoledronic acid. J Pharmacol Exp Ther. 2002;302:10551061. doi:10.1124/jpet.102.035295.

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42. Krger TB, Herlofson BB, Landin MA, Reseland JE. Alendronate alters osteoblast activities. Acta Odontol Scand. 2016;74:550557. doi:10.1080/00016357.2016.1217041.

43. Bouquot J, McMahon R The histopathology of chronic ischemic bone disease (ON) parameters and disease classification. Tucson, Arizona: Proceedings of the Annual Meeting of the American Association of Oral & Maxillofacial Pathology; 2010.

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48. Favia G, Pilolli GP, Maiorano E. Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: an analysis of 31 cases with confocal laser scanning microscopy. Bone. 2009;45:406413. doi:10.1016/j.bone.2009.05.008.

49. Paparella ML, Brandizzi D, Santini-Araujo E, Cabrini RL. Histopathological features of osteonecrosis of the jaw associated with bisphosphonates. Histopathology. 2012;60:514516. doi:10.1111/j.1365-2559.2011.04061.x.

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51. Glueck CJ, McMahon RE, Bouquot J, et al. Thrombophilia, hypofibrinolysis, and alveolar osteonecrosis of the jaws. Oral Surg Oral Med Oral Pathol Oral Radiol Endodontol. 1996;81:557566. doi:10.1016/S1079-2104(96)80047-3.

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

Bone Marrow Stem Cells Comments Off on [Full text] Osteonecrosis of the Jaw Beyond Bisphosphonates: Are There Any Unknown | CCIDE – Dove Medical Press | January 22nd, 2021

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.

GlobalData's TMT Themes 2021 Report tells you everything you need to know about disruptive tech themes and which companies are best placed to help you digitally transform your business.

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

Bone Marrow Stem Cells Comments Off on BrainStorm’s Covid-19 ARDS treatment improves lung function in study – Clinical Trials Arena | January 22nd, 2021

Regenerative medicine contributed to patient care in 2020 more than ever before, bolstered by synergies in research, practice and education. Mayo Clinics Center for Regenerative Medicine is at the forefront of a biotherapy revolution in which health care advances from treating disease to restoring health.

The centrality of the body to regenerate itself is paving the way for new horizons in regenerative care. The triad of protecting against disease, preventing disease progression and promoting healing is at the core of the regenerative vision, says Andre Terzic, M.D., Ph.D., director of Mayo Clinics Center for Regenerative Medicine. To this end, the regenerative toolkit has grown more robust over the past year with new technologies now available to boost the bodys ability to repair and restore health of an organ and importantly of the patient as a whole.

The convergence of research, practice and education, empowered by strong innovation and advanced biomanufacturing, is creating an increased level of readiness for applying validated regenerative science to new areas of health care, Dr. Terzic says.

Practice advancement

A deeper understanding of the biology of health and disease is driving the ongoing regenerative medicine evolution.

The remarkable progress in science that is advancing our fundamental comprehension of both health and disease has guided the informed and responsible development of patient-ready curative strategies, says Dr. Terzic.

New discoveries at Mayo Clinic that may shape future practice include:

Validating safety and efficacy of

stem cell therapy for heart failure. The largest regenerative medicine clinical trial to date for heart failure, spanning 39 medical centers and 315 patients from 10 countries, validated the long-term safety of stem cell therapy. The late-stage research found stem cell therapy shows particular benefit for patients with advanced left ventricular enlargement. This Mayo Clinic-led study offers guidance on which patients are most likely

to respond to stem cell therapy for heart failure.

Uncovering stem cell activation of healing. Mayo Clinic researchers uncovered stem cell-activated molecular mechanisms of healing after a heart attack. Stem cells restored the makeup of failing cardiac muscle back to its condition before the heart attack, providing an intimate blueprint of how they may work to heal diseased tissue. This research offers utility to delineate and interpret complex regenerative outcomes.

Discovering a molecular light switch. Mayo Clinic research discovered a molecular switch that turns on a substance that repairs neurological damage. This early research could bolster a therapy approved by the Food and Drug Administration, and that could lead to new strategies for treating diseases of the central nervous system such as multiple sclerosis.

The federal regulatory environment is making it possible to more seamlessly integrate new discoveries into the practice. The 21st Century Cures Act, for example, seeks to create an accelerated path to market for safe, validated procedures that could provide new therapies for patients with serious conditions.

To read the rest of this article on the Center for Regenerative Medicine blog, visit http://www.regenerativemedicineblog.mayoclinic.org.

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Regenerative medicine is advancing health care in diverse ways - Hometown Focus

Cardiac Stem Cells Comments Off on Regenerative medicine is advancing health care in diverse ways – Hometown Focus | January 22nd, 2021

There's no better time than a national lockdown to test out the coolest new skincare/makeup/hair trends and snazzy innovations in beauty tech from the comfort of your own home. That said, there's nothing more frustrating than spending upwards of 100 on a snazzy new hair treatment or at-home facial system, only to find out it doesn't *actually* work.

Enter GLAMOUR Tries: the weekly Instagram series which sees GLAMOUR editors do all of the time-consuming (and expensive) work for you.

We've been busy trying out all of the (sometimes) wacky but always wonderful beauty crazes to take the internet by storm - from the FOREO's UFO 2 Smart Mask Treatment to Sarah Chapman's 138 Meso-Melt Infusion at-home facial and Toni&Guy's Hello Day! Secret Volumising Crimper. These are the products that every veteran beauty sleuth is talking about - but that you want to do a bit of research into before buying.

Thanks to GLAMOUR Tries, you won't need to waste your hard-earned pennies on testing these innovations yourself. We're getting in their first, giving you the lowdown and making sure you invest in products that genuinely work. Like what you see? You can shop all of the products seen on GLAMOUR tries, with the click of a button, down below. Don't say we don't treat you.

On GLAMOUR Tries this week, Social Media Assistant Luca Wetherby-Matthews tried the Korean beauty technique of slugging. In short, it involves putting Vaseline on your face. Intrigued? Us too.

Vaseline, 2.99, Boots

The science behind it is that petroleum jelly acts as a moisture barrier, locking in your skin's moisture and preventing any hydration from escaping overnight. You can choose where you want to put the Vaseline... you could do it all over you face, or, if you've got oilier skin you could avoid your T-Zone and target the areas where your skin is dry. You could even target your under eyes or fine lines... the choice is yours!

You want to apply the Vaseline after you've carried out your normal skincare routine - yes, even after your face serum and moisturiser - as this is your final step. You're then going to sleep in it overnight and wake up (hopefully) with a transformed complexion.

So how did it go? "It wasn't as weird as I was expecting it to be," Luca said. "It does feel like you're putting something on that you shouldn't be putting on, but not too bad, so we're going to sleep in it and see what happens in the morning."

"There is a slight difference in my skin," Luca said when she woke up. The bits that were the most dry, like my forehead, I can feel are definitely smoother. So I think as far as skin treatments go, it's *super* cheap to buy a pot of Vaseline and despite its reputation of not being as chic as other products, I think it does the job."

Vaseline, 2.99, Boots

Would you give slugging a go? Have you already tried it? Let us know your thoughts over on Instagram @glamouruk.

Link:
I Tried The Slugging Skincare Trend & Here's What Happened - GLAMOUR UK

Skin Stem Cells Comments Off on I Tried The Slugging Skincare Trend & Here’s What Happened – GLAMOUR UK | January 22nd, 2021

Vancouver, British Columbia, Jan. 19, 2021 (GLOBE NEWSWIRE) -- The global industrial nitrogen market is forecast to be valued at USD 29.36 Billion by 2027growing at a CAGR of 5.7%, according to a current analysis by Emergen Research. Nitrogen gas is used in a wide range of applications and end-uses, some of which include laser cutting, food packaging, coffee production, chemicals, electronics, breweries, oil and gas industries, and laboratories. Some among the primary factors driving growth of industrial liquid nitrogen market include major improvements in industrial gas industry, surge in demand from various end-use industries such as metal cutting and fabrication, manufacturing, and food & beverages industry, and growing demand from pharmaceuticals and medical industry.

However, increasingly stringent safety regulations, and structural and regulatory restrictions are some factors expected to hamper market growth to some extent. High production cost and labor force risk driven by accidental leakage of gas are other factors to consider.

In addition, demand for nitrogen for applications in cryopreservation, cryotherapies, and in cryosurgeries for removing skin tags, moles, and treating skin cancer is expected to remain consistent over the forecast period. Wide applications in preserving blood in blood banks, food freezing, rubber and plastic deflashing and grinding, cooling, preserving of biological samples, metal treating, pulverization, and other temperature-associated applications is also expected to support demand and drive market growth.

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Companies Profiled in Industrial Nitrogen Market Report Include:Praxair, Air Products and Chemicals, Air Liquide, Linde, Taiyo Nippon Sanso, Cryotec, Bhuruka Gases Limited, Sudanese Liquid Air Company, Cross Country Infrastructure Services Inc., and Canair Nitrogen Inc.

Key Highlights of Report and Developments

Check Our Prices@ https://www.emergenresearch.com/select-license/477For the purpose of this report, Emergen Research has segmented the global industrial nitrogen market based on form, production technology, distribution and transportation, end-user and regions as follows:

Click to access the Report Study, Read key highlights of the Report and Look at Projected Trends: https://www.emergenresearch.com/industry-report/industrial-nitrogen-market

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Spherical graphite market size was valued at USD 2,435.8 Million in 2019 and is forecasted to reach USD 9,598.8 Million by 2027 at a CAGR of 18.6%. The spherical graphite market is observing a double-digit growth attributed to its increasing usage in lithium-ion battery production.

Sodium dichromate market size was valued at USD 759.2 Million in 2019 and is forecasted to reach USD 1,242.4 Million by 2027 at a CAGR of 6.3%. The sodium dichromate market is observing high demand attributed to its increasing application in pigment, metal finishing, chromium compounds preparation, leather tanning, and wood preservative.

Acoustic insulation market size was valued at USD 12.94 Billion in 2019 and is forecasted to reach USD 19.64 Billion by 2027 at a CAGR of 5.3%. The acoustic insulation market is observing high demand attributed to its increasing application in building & construction, automotive, aerospace, and manufacturing.

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Industrial Nitrogen Market to Reach Value of USD 29.36 Billion by 2027 | Global Analysis, Statistics, Revenue, Demand and Trend Analysis Research...

Skin Stem Cells Comments Off on Industrial Nitrogen Market to Reach Value of USD 29.36 Billion by 2027 | Global Analysis, Statistics, Revenue, Demand and Trend Analysis Research… | January 22nd, 2021

California's ballot measures often reveal much about the broader U.S. policy environment. This is particularly true of the approval by the state's voters in November of Proposition 14, The California Stem Cell Research, Treatments, and Cures Initiative of 2020. Proposition 14 extends the 2004 ballot Proposition 71, which established the California Institute for Regenerative Medicine (CIRM) and authorized $3 billion in state-issued bonds for CIRM to fund stem cell and regenerative research and medicine (restricted to California). Proposition 14, which authorizes $5.5 billion over the next 10 years to continue CIRM's work, succeeded in part by informing voters of CIRM's successes and that its conflict-of-interest provisions are extremely strong. This state-level action is critical because, contrary to opponents' opinions, the overall policy environment for human stem cell research in the United States is in some ways worse now than when Proposition 71 passed.

Since 2004, CIRM has funded groundbreaking work on immune disorders, cancer, spinal cord injury, diabetes, and more. The result has been more than 90 stem cellrelated clinical trials (directly or indirectly supported by CIRM), almost 3000 scientific papers, and contributions to two cancer therapies approved by the U.S. Food and Drug Administration. The lives of many patients have improved because of CIRM. Notably, many CIRM-funded clinical trials rely on human embryonic or fetal stem cells, whereas the federal government currently does not fund any clinical trials using these types of cells.

Proposition 71 was motivated largely in response to restrictions on human embryonic stem cell research in the United States in 2004. However, although research was limited to a small number of human embryonic stem cell lines, there was no formal ban on federal funding of research on such stem cells. In addition, in 2004 there were no restrictions on federal funding of human fetal stem cell and tissue research; however, there is now near-complete blockage of federal funding for such research. And federal funding for human embryonic stem cell research is again at risk. On 4 September 2020, 22 Republican senators and 72 Republican House members wrote to President Trump requesting an end to all federal funding of human embryonic stem cell research. Could President Trump impose a ban that would be difficult to revoke? Or, could Republican senators manufacture a ban by legislative maneuvering on a budget reconciliation vote, which requires 60% support? Such maneuvering created the effectively permanent 1995 Dickey-Wicker amendment, which prohibits federal funding of any research in which human embryos are created or destroyed. Dickey-Wicker has limited research on in vitro fertilization methods and stalled progress on understanding early human development. It has not solved the problem of the many, perhaps 1 million frozen embryos in the United States that will not be used for in vitro fertilization and will be destroyed without benefit if not used for research. Vital long-term research is greatly harmed by the U.S. policy environment, with the likely outcome that many young scientists will avoid research using human embryonic stem cells and human fetal tissue.

Restrictions on valuable, ethical research appear particularly fool-hardy during a deadly pandemic. Research on viruses such as HIV and SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) can benefit greatly from work using mice that utilize human fetal stem cells and tissues to generate a human-like immune system. These mice allow evaluation of a human immune system in the contexts of infection mechanisms, generation of immunity, and drug response. These studies can be supported with Proposition 14 funds in California, but not with federal funds. It is crucial for the incoming Biden administration to evaluate the need for federal funding in these important areas with high-quality scientific input and evidence.

California's vote on Proposition 14 should also help the rest of the country appreciate the need to increase investments in biomedical research at the U.S. National Institutes of Health and other federal agencies. Current biomedical research expenditures amount to only a tiny fraction of the costs of disease, so an objective evaluation of appropriately increased research funding relative to disease costs is warranted. Once again, California is showing the way.

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Stem cells on the ballot - Science Magazine

Spinal Cord Stem Cells Comments Off on Stem cells on the ballot – Science Magazine | January 19th, 2021

Special delivery: Stem cells can be modified to produce a therapeutic protein in the brain.

Laguna Design / Science Photo Library

Two unpublished studies detail improved methods for delivering gene therapies to the brain: One involves a type of stem cell that can produce gene-altering proteins on-site; the other taps an engineered virus to target neurons efficiently and noninvasively.

Researchers presented the work virtually on Monday and Tuesday at the 2021 Society for Neuroscience Global Connectome.

One of the biggest hurdles for targeted gene therapy is getting enough treatment to the right spot. In the first study, researchers overcame this obstacle by developing stem cells that produce a therapeutic protein inside the brain.

The team is using the approach to develop a treatment for Angelman syndrome, which is caused by mutation in or deletion of the maternal copy of the gene UBE3A. Because the paternal copy of the gene is typically silent, loss of the maternal copy results in an absence of UBE3A protein. People with Angelman syndrome usually have intellectual disability and motor impairments, and many are autistic.

The researchers had previously used modified stem cells to produce a protein that can activate the paternal copy of UBE3A. Transplanting the cells into the brains of Angelman syndrome model mice boosts levels of UBE3A protein, they found. However, the treatment required multiple direct injections into the animals brains.

In the new work, they instead tried injecting the cells into a pocket of cerebrospinal fluid at the base of the skull an approach that is less invasive and can be performed multiple times. They compared the results with direct injection into the animals hippocampus. In both cases, the mice had UBE3A expression in the brain for up to three weeks.

Mice that received direct injection of the stem cells had fewer Angelman syndrome traits than controls, as measured by their motor skills.

This suggests that though the new route is effective, it may not provide a high enough dosage, says Peter Deng, a postdoctoral researcher inKyle Finkslab at the University of California, Davis, who presented the work. And because the transplanted cells produce protein for only a limited period of time, the effects are temporary a limitation the team is addressing.

Deng and his colleagues also found that monkeys treated with the stem cells had the therapeutic protein throughout their brain and spinal cord three weeks after injection, which suggests the approach has potential for treating people.

The second approach presented at the conference improves the delivery of a more permanent form of gene therapy that uses adeno-associated viruses (AAVs).

Researchers typically inject these viruses directly into the brain, and the viruses usually only affect cells immediately surrounding the injection site.

Youre required to use a ton of the virus to penetrate the whole brain, says Jerzy Szablowski, assistant professor of neuroengineering at Rice University in Houston, Texas, who presented the work.

One potential workaround is to inject the AAV into the blood and use focused ultrasound to temporarily open up the blood-brain barrier, allowing the AAV to cross into the brain. Sometimes with this approach, however, the virus also inserts itself into other organs.

In their new work, the team developed AAVs that more easily cross the blood-brain barrier and more selectively target neurons than previous versions do. As a result, the new AAVs can be given in lower doses, reducing the amount of tissue affected outside the brain, Szablowski says.

To identify the most efficient AAV, Szablowski and his colleagues designed 2,100 new viruses, injected them all into the bloodstream of mice and applied focused ultrasound to the animals skulls. The mice had been engineered so that AAVs that successfully inserted themselves into a neuron got tagged with a marker. The team performed genomic sequencing on the mouse brains a few weeks later and read out the levels of viruses.

Compared with the previously most effective AAV, the top five newly identified AAVs targeted twice as many cells in the brain (including more neurons), and nearly half as many cells outside the brain, the researchers found.

The approach could be used to more efficiently deliver treatments for conditions such as Angelman syndrome or Parkinsons disease, the team says.

Read more reports from the 2021 Society for Neuroscience Global Connectome.

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New gene therapy methods deliver promise - Spectrum

Spinal Cord Stem Cells Comments Off on New gene therapy methods deliver promise – Spectrum | January 19th, 2021

18 January 2021

A new way of controlling the immune system's 'natural killer' cells has been identified, which could help prevent rejection of transplanted cells.

Natural killer cells act as a front-line defence against foreign cells in the body. Overcoming this defence has represented a major challenge to transplant, cancer immunotherapy and regenerative medicine. The new research provides a possibility to disguise transplanted cells so that they are 'invisible' to this immune defence.

'As a cardiac surgeon, I would love to put myself out of business by being able to implant healthy cardiac cells to repair heart disease', said the study's lead author Professor Tobias Deuse, from the Department of Surgery at University of California, San Francisco. 'And there are tremendous hopes to one day have the ability to implant insulin-producing cells in patients with diabetes or to inject cancer patients with immune cells engineered to seek and destroy tumours. The major obstacle is how to do this in a way that avoids immediate rejection by the immune system'.

Professor Deuse and his colleagues genetically engineered cells to express the protein CD47 and found that these were not attacked by natural killer cells. They discovered that this was because the CD47 protein activates another protein found in the natural killer cells called SIRP.

SIRP was known to be important in other immune cell responses, but scientists had not previously been able to identify it in natural killer cells. 'All the literature said that natural killer cells don't have this checkpoint, but when we looked at cells from human patients in the lab we found SIRP there, clear as day', said corresponding author Professor Sonja Schrepfer.

The team investigated whether this finding could be used to help transplanted stem cells to avoid immune rejection. To do this, they genetically engineered human cells to express rhesus monkey CD47 and transplanted these cells into monkeys. They found that the engineered cells activated the SIRP on natural killer cells, which prevented them from being attacked.

In the future the same procedure could be performed in reverse, expressing human CD47 in pig cardiac cells, for instance, to prevent them from activating natural killer cells when transplanted into human patients.

The research team aims to create a 'hypoimmune' cell line for use in regenerative medicine that will be able to avoid immune rejection.

'Currently engineered cell therapies for cancer and fledgling forms of regenerative medicine all rely on being able to extract cells from the patient, modify them in the lab, and then put them back in the patient. This avoids rejection of foreign cells, but is extremely laborious and expensive', said Professor Schrepfer. 'Our goal in establishing a hypoimmune cell platform is to create off-the shelf products that can be used to treat disease in all patients everywhere'.

The research was recently published in the Journal of Experimental Medicine.

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PFFFIKON, Switzerland, Jan. 19, 2021 /PRNewswire/ -- DiNAQOR, a gene therapy platform company,today announcedthat it has acquired EHT Technologies GmbH, a Germany-based engineered heart tissue (EHT) technology platform company. Financial terms of the transaction were not disclosed.

EHT Technologies was founded in 2015 based upon research on human induced pluripotent stem cells (hiPSC) at the University Medical Center Hamburg-Eppendorf. Cardiomyocytes derived from hiPSC are an innovative research technology for cardiac drug development programs. Engineered heart tissues are three-dimensional, hydrogel-based muscle constructs that can be generated from isolated heart cells of chicken, rat, mouse, human embryonic stem cells and hiPSC. Proof-of-concept studies have shown that EHT can be transduced efficiently with adeno-associated virus (AAV) vectors, including AAV9, validating the use of this platform for gene therapy applications.

"EHT Technologies' proprietary hiPSC platform for disease modeling is a perfect complement to DiNAQOR's research and development efforts and leaps forward our ability to develop creative approaches for treating heart diseases in the future. EHT's intellectual property and know-how is industry-leading and we are excited to be able to harness its platform at DiNAQOR," commented Johannes Holzmeister, M.D., Chairman and CEO at DiNAQOR.

"After more than 25 years of development, I'm very excited that our engineered heart tissue technology is making the transition from an academic research model to a drug development tool. The combined application of human cardiomyocytes and a versatile, 3D in vitro assay will facilitate development and reduce reliance on animal studies. The hiPSC-derived EHT assay has great potential for the development of innovative cardiovascular therapeutics and DiNAQOR is the perfect fit for this enterprise," commented Professor Thomas Eschenhagen, M.D., co-founder of EHT Technologies. Professor Eschenhagen serves on DiNAQOR's Scientific Advisory Board.

"The EHT technology will accelerate the advancement of our discovery pipeline and bridge the translational gap between the animal model and human disease. We are proud that DiNAQOR is on the forefront of implementing this innovative technology to expedite new therapies into the clinic," said Valeria Ricotti, M.D., Chief Medical Officer at DiNAQOR.

About DiNAQORFounded in 2019,DiNAQOR is a global gene therapy platform company focused on advancing novel solutions for patients suffering from heart disease.The company is headquartered in Pfffikon, Switzerland, with additional presence in London, England and Hamburg, Germany. For more information visitwww.dinaqor.com.

ContactKWM CommunicationsKellie Walsh[emailprotected] or Stephanie Marks[emailprotected]

SOURCE DiNAQOR

DiNAQOR: A global gene therapy platform company

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DiNAQOR Acquires EHT Technologies GmbH to Advance Engineered Heart Tissue R&D Capabilities - PRNewswire

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Genmab Announces that Janssen has been Granted U.S. FDA Approval for DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) for Patients with Newly Diagnosed Light-chain (AL) Amyloidosis

Company Announcement

Copenhagen, Denmark; January 15, 2021 Genmab A/S (Nasdaq: GMAB) announced today that the U.S. Food and Drug Administration (U.S. FDA) has approved the use of DARZALEX FASPRO (daratumumab and hyaluronidase-fihj), a subcutaneous formulation of daratumumab, in combination with bortezomib, cyclophosphamide, and dexamethasone (VCd) for the treatment of adult patients with newly diagnosed light-chain (AL) amyloidosis. A supplemental Biologics License Application (sBLA) for this indication was submitted by Janssen Biotech, Inc. (Janssen), in September 2020. The U.S. FDA reviewed the submission of data for approval in this indication under their Real-Time Oncology Review (RTOR)1 pilot program and Project Orbis2. Continued approval for this indication may be contingent upon verification and description of clinical benefit in a confirmatory trial(s). DARZALEX FASPRO is not indicated and is not recommended for the treatment of patients with light-chain (AL) amyloidosis who have NYHA Class IIIB or Class IV cardiac disease or Mayo Stage IIIB outside of controlled clinical trials. In August 2012, Genmab granted Janssen an exclusive worldwide license to develop, manufacture and commercialize daratumumab.

AL amyloidosis is a devastating and potentially fatal blood disorder that, until now, did not have any U.S. FDA-approved therapies. This makes todays approval of DARZALEX FASPRO a critical step forward for patients in the U.S. in dire need of treatment options, said Jan van de Winkel, Ph.D., Chief Executive Officer of Genmab.

The approval was based on data from the Phase 3 ANDROMEDA (AMY3001) study of daratumumab and hyaluronidase-fihj in combination with VCd as treatment for patients with newly diagnosed AL amyloidosis.

The most common adverse reactions (20%) were upper respiratory tract infection, diarrhea, peripheral edema, constipation, fatigue, peripheral sensory neuropathy, nausea, insomnia, dyspnea and cough. Serious adverse reactions occurred in 43% of patients who received DARZALEX FASPRO in combination with VCd. Serious adverse reactions that occurred in at least 5% of patients in the D-VCd arm were pneumonia (9%), cardiac failure (8%) and sepsis (5%). Fatal adverse reactions occurred in 11% of patients. Fatal adverse reactions that occurred in more than one patient included cardiac arrest (4%), sudden death (3%), cardiac failure (3%) and sepsis (1%).3

Among patients who received DARZALEX FASPRO in combination with VCd, 72% of patients had baseline cardiac involvement with Mayo Cardiac Stage I (3%), Stage II (46%) and Stage III (51%). Serious cardiac disorders occurred in 16% of patients (8% of patients with Mayo Cardiac Stage I and II and 28% of patients with Stage III). Serious cardiac disorders in more than 2% of patients included cardiac failure (8%), cardiac arrest (4%) and arrhythmia (4%). Fatal cardiac disorders occurred in 10% of patients (5% of patients with Mayo Cardiac Stage I and II and 19% of patients with Stage III) who received DARZALEX FASPRO in combination with VCd. Fatal cardiac disorders that occurred in more than one patient in the D-VCd arm included cardiac arrest (4%), sudden death (3%) and cardiac failure (3%).3

Full prescribing information will be available at http://www.DARZALEX.com.

Genmab will receive a milestone payment of USD 30 million in connection with the first commercial sale of DARZALEX FASPRO in this indication, which is expected to occur quickly after approval. The milestone will be reflected in Genmabs 2021 guidance, which will be published on February 23, 2021.

About the ANDROMEDA (AMY3001) studyThe Phase 3 study (NCT03201965) included 388 patients newly diagnosed with AL amyloidosis. Patients were randomized to receive treatment with either daratumumab and hyaluronidase-fihj in combination with bortezomib (a proteasome inhibitor), cyclophosphamide (a chemotherapy), and dexamethasone (a corticosteroid) or treatment with VCd alone. The primary endpoint of the study was the percentage of patients who achieve hematologic complete response.

About Light-chain (AL) AmyloidosisAmyloidosis is a disease that occurs when amyloid proteins, which are abnormal proteins, accumulate in tissues and organs. When the amyloid proteins cluster together, they form deposits that damage the tissues and organs. AL amyloidosis most frequently affects the heart, kidneys, liver, nervous system and digestive tract. Until now there were no approved therapies for AL amyloidosis in the U.S., though it is currently being treated with chemotherapy, dexamethasone, stem cell transplants and supportive therapies.4 It is estimated that there are approximately 3,000 to 4,000 new cases of AL amyloidosis diagnosed annually in the U.S.5

About DARZALEX (daratumumab)DARZALEX (daratumumab) has become a backbone therapy in the treatment of multiple myeloma. DARZALEX intravenous infusion is indicated for the treatment of adult patients in the United States: in combination with carfilzomib and dexamethasone for the treatment of patients with relapsed/refractory multiple myeloma who have received one to three previous lines of therapy; in combination with bortezomib, thalidomide and dexamethasone as treatment for patients newly diagnosed with multiple myeloma who are eligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of patients with multiple myeloma who have received at least one prior therapy; in combination with pomalidomide and dexamethasone for the treatment of patients with multiple myeloma who have received at least two prior therapies, including lenalidomide and a proteasome inhibitor (PI); and as a monotherapy for the treatment of patients with multiple myeloma who have received at least three prior lines of therapy, including a PI and an immunomodulatory agent, or who are double-refractory to a PI and an immunomodulatory agent.6 DARZALEX is the first monoclonal antibody (mAb) to receive U.S. Food and Drug Administration (U.S. FDA) approval to treat multiple myeloma.

DARZALEX is indicated for the treatment of adult patients in Europe via intravenous infusion or subcutaneous administration: in combination with bortezomib, thalidomide and dexamethasone as treatment for patients newly diagnosed with multiple myeloma who are eligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of adult patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; for use in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone, for the treatment of adult patients with multiple myeloma who have received at least one prior therapy; and as monotherapy for the treatment of adult patients with relapsed and refractory multiple myeloma, whose prior therapy included a PI and an immunomodulatory agent and who have demonstrated disease progression on the last therapy7. Daratumumab is the first subcutaneous CD38 antibody approved in Europe for the treatment of multiple myeloma. The option to split the first infusion of DARZALEX over two consecutive days has been approved in both Europe and the U.S.

In Japan, DARZALEX intravenous infusion is approved for the treatment of adult patients: in combination with lenalidomide and dexamethasone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with bortezomib, melphalan and prednisone for the treatment of patients with newly diagnosed multiple myeloma who are ineligible for autologous stem cell transplant; in combination with lenalidomide and dexamethasone, or bortezomib and dexamethasone for the treatment of relapsed or refractory multiple myeloma. DARZALEX is the first human CD38 monoclonal antibody to reach the market in the United States, Europe and Japan. For more information, visit http://www.DARZALEX.com.

DARZALEX FASPRO (daratumumab and hyaluronidase-fihj), a subcutaneous formulation of daratumumab, is approved in the United States for the treatment of adult patients with newly diagnosed light-chain (AL) amyloidosis in combination with bortezomib, cyclophosphamide, and dexamethasone. It is also approved in the U.S. for the treatment of adult patients with multiple myeloma: in combination with bortezomib, thalidomide, and dexamethasone in newly diagnosed patients who are eligible for ASCT; in combination with bortezomib, melphalan and prednisone in newly diagnosed patients who are ineligible for ASCT; in combination with lenalidomide and dexamethasone in newly diagnosed patients who are ineligible for ASCT and in patients with relapsed or refractory multiple myeloma who have received at least one prior therapy; in combination with bortezomib and dexamethasone in patients who have received at least one prior therapy; and as monotherapy, in patients who have received at least three prior lines of therapy including a PI and an immunomodulatory agent or who are double-refractory to a PI and an immunomodulatory agent.8 DARZALEX FASPRO is co-formulated with recombinant human hyaluronidase PH20 (rHuPH20), Halozyme's ENHANZE drug delivery technology. .DARZALEX FASPRO is the first subcutaneous CD38 antibody approved in the U.S. for the treatment of multiple myeloma and the first and only approved treatment for patients with AL amyloidosis in the U.S.

Daratumumab is a human IgG1k monoclonal antibody (mAb) that binds with high affinity to the CD38 molecule, which is highly expressed on the surface of multiple myeloma cells. Daratumumab triggers a persons own immune system to attack the cancer cells, resulting in rapid tumor cell death through multiple immune-mediated mechanisms of action and through immunomodulatory effects, in addition to direct tumor cell death, via apoptosis (programmed cell death).6,9,10,11,12

Daratumumab is being developed by Janssen Biotech, Inc. under an exclusive worldwide license to develop, manufacture and commercialize daratumumab from Genmab. A comprehensive clinical development program for daratumumab is ongoing, including multiple Phase III studies in smoldering, relapsed and refractory and frontline multiple myeloma settings. Additional studies are ongoing or planned to assess the potential of daratumumab in other malignant and pre-malignant diseases in which CD38 is expressed, such as amyloidosis and T-cell acute lymphocytic leukemia (ALL). Daratumumab has received two Breakthrough Therapy Designations from the U.S. FDA for certain indications of multiple myeloma, including as a monotherapy for heavily pretreated multiple myeloma and in combination with certain other therapies for second-line treatment of multiple myeloma.

About Genmab Genmab is an international biotechnology company with a core purpose to improve the lives of patients with cancer. Founded in 1999, Genmab is the creator of multiple approved antibody therapeutics that are marketed by its partners. The company aims to create, develop and commercialize differentiated therapies by leveraging next-generation antibody technologies, expertise in antibody biology, translational research and data sciences and strategic partnerships. To create novel therapies, Genmab utilizes its next-generation antibody technologies, which are the result of its collaborative company culture and a deep passion for innovation. Genmabs proprietary pipeline consists of modified antibody candidates, including bispecific T-cell engagers and next-generation immune checkpoint modulators, effector function enhanced antibodies and antibody-drug conjugates. The company is headquartered in Copenhagen, Denmark with locations in Utrecht, the Netherlands, Princeton, New Jersey, U.S. and Tokyo, Japan. For more information, please visit Genmab.com.

Contact: Marisol Peron, Corporate Vice President, Communications & Investor Relations T: +1 609 524 0065; E: mmp@genmab.com

For Investor Relations: Andrew Carlsen, Senior Director, Investor RelationsT: +45 3377 9558; E: acn@genmab.com

This Company Announcement contains forward looking statements. The words believe, expect, anticipate, intend and plan and similar expressions identify forward looking statements. Actual results or performance may differ materially from any future results or performance expressed or implied by such statements. The important factors that could cause our actual results or performance to differ materially include, among others, risks associated with pre-clinical and clinical development of products, uncertainties related to the outcome and conduct of clinical trials including unforeseen safety issues, uncertainties related to product manufacturing, the lack of market acceptance of our products, our inability to manage growth, the competitive environment in relation to our business area and markets, our inability to attract and retain suitably qualified personnel, the unenforceability or lack of protection of our patents and proprietary rights, our relationships with affiliated entities, changes and developments in technology which may render our products or technologies obsolete, and other factors. For a further discussion of these risks, please refer to the risk management sections in Genmabs most recent financial reports, which are available on http://www.genmab.com and the risk factors included in Genmabs most recent Annual Report on Form 20-F and other filings with the U.S. Securities and Exchange Commission (SEC), which are available at http://www.sec.gov. Genmab does not undertake any obligation to update or revise forward looking statements in this Company Announcement nor to confirm such statements to reflect subsequent events or circumstances after the date made or in relation to actual results, unless required by law.

Genmab A/S and/or its subsidiaries own the following trademarks: Genmab; the Y-shaped Genmab logo; Genmab in combination with the Y-shaped Genmab logo; HuMax; DuoBody; DuoBody in combination with the DuoBody logo; HexaBody; HexaBody in combination with the HexaBody logo; DuoHexaBody; HexElect; and UniBody. DARZALEX and DARZALEX FASPRO are trademarks of Janssen Pharmaceutica NV.

1 Real-Time Oncology Review Pilot Program. https://www.fda.gov/about-fda/oncology-center-excellence/real-time-oncology-review-pilot-program Accessed September 20202 Project Orbis. U.S. Food and Drug Administration. https://www.fda.gov/about-fda/oncology-center-excellence/project-orbis. Accessed September 2020.3DARZALEX FASPRO Prescribing Information. Horsham, PA: Janssen Biotech, Inc.4 Mayo Clinic website: http://www.mayoclinic.com/health/amyloidosis/DS004315 Research and Markets, Amyloidosis Treatment Market Size, Share & Trends Analysis Report by Treatment (Stem Cell Transplant, Chemotherapy, Supportive Care, Surgery, Targeted Therapy), By Country, And Segment Forecasts, 2018 - 20256 DARZALEX Prescribing information, August 2020 https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761036s029lbl.pdf Last accessed August 20207 DARZALEX Summary of Product Characteristics, available at https://www.ema.europa.eu/en/medicines/human/EPAR/darzalex Last accessed June 20208 DARZALEX FASPRO Prescribing information, May 2020. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/761145s000lbl.pdf Last accessed May 20209 De Weers, M et al. Daratumumab, a Novel Therapeutic Human CD38 Monoclonal Antibody, Induces Killing of Multiple Myeloma and Other Hematological Tumors. The Journal of Immunology. 2011; 186: 1840-1848.10 Overdijk, MB, et al. Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma. MAbs. 2015; 7: 311-21.11 Krejcik, MD et al. Daratumumab Depletes CD38+ Immune-regulatory Cells, Promotes T-cell Expansion, and Skews T-cell Repertoire in Multiple Myeloma. Blood. 2016; 128: 384-94.12 Jansen, JH et al. Daratumumab, a human CD38 antibody induces apoptosis of myeloma tumor cells via Fc receptor-mediated crosslinking. Blood. 2012; 120(21): abstract 2974

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Genmab Announces that Janssen has been Granted U.S. FDA Approval for DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) for Patients with Newly Dia...

Cardiac Stem Cells Comments Off on Genmab Announces that Janssen has been Granted U.S. FDA Approval for DARZALEX FASPRO (daratumumab and hyaluronidase-fihj) for Patients with Newly Dia… | January 19th, 2021

As the air continues to dry and temperatures drop, the yearly battle against dry hands and skin has officially begun. New research from Northwestern University has discovered new evidence deep within the skin about the mechanisms controlling skin renewal and repair.

Skins barrier function gives it the unique ability to fight winter woes and keep water for our bodies. The outer layer of the skin, the skin, is constantly turning over to replace damaged or dead cells, creating new cells to reinforce the barrier function and heal damage. The gene regulatory mechanisms that control epidermis turnover remain incompletely understood.

Every month were covered with a new layer of the epidermis, said Northwesterns Xiaomin Bao, who headed the study. The next question is what does that procedure involve?

The newspaper will be published on Jan. 19 in the journal Nature Communications.

GeneticjunkThe scientific community has developed a wide breadth of knowledge about proteins, the workhorses of various cellular activities. Many mysteries remain about the nature of introns, non-coding segments of DNA that make up 24% of the human genome.

Regardless of the general belief that introns are nothing butgenetic junk, they actually play critical roles in modulating RNA transcription throughout a tissues lifespan. RNA transcription is the first step of gene expression, in which the data from DNA is copied into RNA, which is then subsequently used as a template for synthesizing proteins to drive the specific function of a cell.

Based on where transcription terminates within an intron or at the conclusion of a gene an epidermal stem cell will either remain a stem cell or become a specified cell barrier function. Bao said while its well-known that transcription ends at the end of a gene, her labs research found conflicting data.

Future research could have implications for carcinoma research.

Technology critical to the discovery of the phenomenonSkin cells are gaining popularity with researchers in part because of their regenerative properties and readiness to grow in cultures. This allows researchers to apply an assortment of state-of-the-art technologies. By growing skin cells and regenerating skin tissue in a petri dish, the Bao Lab can experiment with this fast-growing tissue to determine molecular mechanisms and regulatory elements within DNA.

Technology development is an integral driver that enabled us to uncover this new phenomenon, Bao said.

The group used a novel genomic technique that maps where transcription stops. The integration of proteomic approaches identified RNA-binding proteins which read specific regulatory sequences in the introns.

The team further leveraged CRISPR technologies to delete genomic sequences in the intron, which provided direct evidence demonstrating that the crucial roles of introns in modulating gene expression.

Before this study, mechanisms between introns to govern the switch between a skin stem cell and a terminally differential state (for example, a cell that participates in forming a skin barrier), were unknown. Most studies ignored introns, despite them accounting for 10 to 20 times more sequences than the protein-coding regions (exons) in the human genome.

The study demonstrates that different genes may involve different sets of RNA-binding proteins to recognize the regulatory sequences in their introns. These RNA-binding proteins help to mature RNAdecide whether to cut transcription early or dismiss termination sites within an intron during differentiation due to changes in protein availability.

We are only starting to appreciate the roles of the intron in human health and diseases, Bao said.

Results of this study could have broader impacts because, according to Bao, the procedures regulating skin cells are almost definitely not restricted to skin cells. Future research on other systems, including other epithelial tissues, will likely uncover similar patterns.

We are extremely hopeful that what weve found is the first step to knowing what we have ignored in the past, Bao said. My students also wish to know more about the RNA binding proteins which provide specificity in governing which site to use to terminate transcription.

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Research finds new proof about the systems controlling skin repair and regeneration - Microbioz India

Skin Stem Cells Comments Off on Research finds new proof about the systems controlling skin repair and regeneration – Microbioz India | January 19th, 2021