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Emerging Quadruplets, Novel Targets, and Immunotherapy Advances Personalized Medicine in Multiple Myeloma – OncLive

By daniellenierenberg

The future is quite bright for multiple myeloma. We are really homing in on the best regimen for frontline therapy in transplant-eligible and -ineligible [patient populations], Martin said. We are also closer with our recommendations to figuring out how to treat early-relapsed multiple myeloma. We have a variety of novel drugs that are approved for use to treat [patients with] late relapse. That [setting] has been our unmet medical need, [historically].

Martin, a clinical professor of medicine in the Adult Leukemia and Bone Marrow Transplantation Program; associate director of the Myeloma Program; and co-leader of the Cancer Immunology and Immunotherapy Program at the Helen Diller Family Comprehensive Cancer Center of the University of California, San Francisco; added that there are several very exciting therapies under investigation in clinical trials, including BiTEs. [These therapies] are showing unprecedented responses in very refractory patients, [including] the triple-class exposed patients, which is amazing.

He spoke with OncLive during an Institutional Perspectives in Cancer webinar on multiple myeloma. He chaired the virtual meeting which covered updates in frontline, early-, and late-relapsed multiple myeloma, immunotherapy in multiple myeloma, and frontline and relapsed/refractory amyloidosis.

Martin discussed the latest news in frontline, early relapsed, and heavily pretreated multiple myeloma, including the growing promise of quadruplets, emerging targets beyond BCMA, and the potential emergence of quadruplets, venetoclax (Venclexta), and antiviral therapy in amyloidosis.

Martin: For frontline therapy in multiple myeloma, we break [our algorithm] up [according to] patients who are fit and [unfit. Patients who are fit] can likely go to stem cell transplant. A quadruplet is going to be where we are headed, and it is going to be [a quadruplet using] the 3 different classes of drugs: a monoclonal antibody, an immunomodulatory drug [IMiD], and a proteasome inhibitor [PI], together with a steroid. [The combination of] those 4 classes of drugs [were evaluated] in the GRIFFIN [NCT02874742] and Cassiopeia trials [NCT02541383]. The GRIFFIN trial looked at daratumumab [Darzalex], lenalidomide [Revlimid], bortezomib [Velcade], and dexamethasone, whereas the Cassiopeia trial looked at daratumumab, thalidomide [Thalomid], and dexamethasone. Both [trials] showed spectacular early responses for induction therapy to [the respective] quadruplets.

Another study looked at daratumumab [plus] carfilzomib [Kyprolis], lenalidomide, and dexamethasone [KRd]. That trial too showed unprecedented early responses as frontline therapy. More studies are looking at other CD38[-directed monoclonal antibodies], like isatuximab-irfc [Sarclisa], together with lenalidomide, as well as KRd.

These quadruplets are showing fast and deep responses after 4 cycles [of treatment]. For patients who are transplant eligible, [treatment with a quadruplet] prepares them for transplant quite well. They can go into transplant with a nice, deep response and, hopefully, [derive] a deeper response after remission.

The question exists of whether the quadruplets and other therapies may take away the need for autologous stem cell transplant. Right now, transplant is still part of frontline therapy and is especially useful in patients who have high-risk disease.

In the transplant-ineligible population, the MAIA trial [NCT02252172] looked at daratumumab plus lenalidomide and dexamethasone vs lenalidomide and dexamethasone. The triplet has shown a median progression-free survival [PFS] approaching 60 months; that is just amazing for frontline therapy. We will see if quadruplets are needed in the transplant-ineligible setting.

We have several trials testing quadruplet therapy in the transplant-eligible population. Both daratumumab and isatuximab are being combined with IMiDs, PIs, and dexamethasone in a randomized fashion [vs triplet therapy]. We will see what the winner is. It will be interesting as we move forward, but right now, if we start that triplet therapy, we expect a PFS of 60 months, which is just amazing.

When we think about early relapse, what becomes important is what patients were on when they became relapsed or refractory. If they were on an IMiD, most of the time it was lenalidomide as maintenance therapy. We would then consider that patient lenalidomide refractory. In that scenario, we would use a CD38[-directed monoclonal antibody] plus pomalidomide [Pomalyst] and dexamethasone or a CD38[-directed monoclonal antibody] plus a PI and dexamethasone.

The data with daratumumab plus pomalidomide and dexamethasone, as well as isatuximab plus pomalidomide and dexamethasone, are quite good. Truthfully, my favorite [approach] is that if the patient is on an IMiD, I give an antibody together with a PI. The IKEMA [NCT03275285] and CANDOR [NCT03158688] studies have shown deep and durable responses with a CD38[-directed monoclonal antibody] plus carfilzomib and dexamethasone in the early-relapsed setting.

The CANDOR study showed a PFS of about 28 months. We still need longer follow-up from the IKEMA study to see what the PFS is going to be, but it is certainly going to be at least 28 months. Specifically, [in the IKEMA] study we showed that 30% of patients had achieved minimal residual disease [MRD] negativity with the triplet combination in the early-relapsed setting. Its unprecedented to see these deep responses with evidence of MRD negativity.

If patients have not received a CD38[-directed monoclonal antibody] as part of frontline therapy, that is what the first component should be to add for first relapse. The other regimens, which weve used before and are good, include pomalidomide, bortezomib, and dexamethasone, or pomalidomide, carfilzomib, and dexamethasone. There are multiple other choices, but those are my favorites.

In early-to-mid relapse, we usually use a ping-pong approach where we go back and forth between the categories of agents. Eventually, after 2 or 3 lines of therapy, patients have been exposed to what I call the big 5, which are lenalidomide, bortezomib, carfilzomib, pomalidomide, and a CD38-directed antibody. This is a setting which had been our unmet medical need.

We now have 3 agents that are FDA approved for that group of patients. We have selinexor [Xpovio] plus dexamethasone, which was approved based on the STORM trial [NCT02336815]. That doublet can be used in the [originally indicated] twice-weekly [dose], or given once weekly, which is much better tolerated. Often, we combine [selinexor] with another agent, such as bortezomib, carfilzomib, pomalidomide, or, even, daratumumab, so it is a kind of pick-your-partner [agent] in that regard. There are toxicities associated with selinexor, and we must follow patients closely. We cant just give them the therapy and see them in 4 weeks. We must follow their sodium closely because some patients need salt replacement, hydration, and anti-emetics.

The second [agent approved for triple-class refractory multiple myeloma] is belantamab mafodotin-blmf [Blenrep], which is an antibody-drug conjugate that targets BCMA. The poison is MMAF, which is associated with thrombocytopenia and ocular toxicity. We found that when belantamab mafodotin is used as a single agent without a steroid, the response rate was just over 30%. Patients who respond have durable responses upward of 10 or 12 months. We just have to watch patients for ocular toxicity because [belantamab mafodotin] can cause keratitis on the surface of the eye. Patients must see an ophthalmologist before each dose of belantamab mafodotin, which is dosed every 3 weeks. In my experience, [keratitis] usually occurs after the second or third dose. Most patients respond after the first or second dose, so we can see if the patient responds, and then continue or modify the regimen. We can lengthen the dose out to every 4 weeks or every 6 weeks or drop the dose from 2.5 mg/kg to 1.9 mg/kg.

Lastly, we have a new drug called melphalan flufenamide [melflufen; Pepaxto], which is a lipophilic, alkylator-based therapy. The lipophilic component gets the drug fast into cells, but it can be cleaved off the alkylator by aminopeptidases. In fact, normal cells dont have many aminopeptidases, so [melflufen] gets in and out of normal cells relatively quickly; however, the drug gets in myeloma cells, the lipophilic component is cleaved off, and the alkylator gets trapped inside the cell. [Melflufen] is [administered as] one flat dose of 40 mg every 4 weeks with weekly dexamethasone. It is tolerable; the big adverse effect [AE] is blood count suppression. Weve seen response rates in the 25% to 30% range.

The newest [therapy] on the block in what is available for patients who have had 4 prior lines of therapy is the CAR T-cell therapy ide-cel. It is BCMA directed, the original vector was known as bb2121. It is now FDA approved.

The rollout [of ide-cel] has been a little slow in terms of slot allocation, and it has been difficult for centers across the country to get patients on slots. We are hoping that the slot availability will increase over the next few months.

That said, for patients who are triple-class refractory and have had 4 prior lines of therapy, [ide-cel] is a perfect therapy. The CAR T cells have to be done at a licensed CAR T-cell center, of which there are only about 70 in the United States. That comes with some overhead because patients must move to the center and remain there for the first 30 days of therapy because of the significant toxicities associated with CAR T-cell therapy. [These AEs] are mostly cytokine release syndrome [CRS], which happens 80% to 90% of the time, and some neurotoxicity, which is reported in around 15% to 20% of patients. Patients must be followed closely and require initial hospitalization between 7 to 14 days. Then, patients must stay local [for follow-up].

There is a lot of overhead, but it is a one-and-done treatment. We collect their T cells, give them lymphodepletion, give them back the T cells, and patients are off therapy. The median PFS for ide-cel is about 12 months, so hopefully patients get 12 months of free time where they dont need therapy and have truly good quality of life, which is quite nice.

The nice thing about immunotherapy is that multiple targets are being investigated. BCMA was our first target, but we have others, such as GPRC5D and FcRH5. We have multiple different CAR T-cell therapies currently in research studies to try to build upon ide-cel.

We also have BiTEs, in which one arm binds to BCMA or whatever the target is on the myeloma cell, and the other arm looks for the immune cell in the local environment. Most of the other arms bind to CD3 on T cells to activate the T cells. [BiTEs] are a little bit different in terms of how they bind to the myeloma cell and how much they activate the T cell by binding to CD3.

That said, in the early research, most of these therapeutics as single agents have shown response rates on the order of 60% to 80%. Thats, again, unprecedented for single agents. These therapeutics are quite impressive in terms of response rates, but they are also associated with CRS and mild neurotoxicity. They require initial dosing in the hospital and patients are usually hospitalized for 7 to 10 days for step-up dosing. After that, [treatment] can be done in the outpatient setting with intermittent dosing. BiTEs vary from dosing weekly and then less frequently to every 3 weeks. Coming back to the center every 3 weeks is reasonable, even for patients who live outside the research center.

In San Francisco, we have patients coming in every 3 weeks to get their therapy and then they head back home, which is nice. However, it is ongoing therapy and patients must continue their therapy rather than receive a one-and-done treatment. This is because BiTEs are off-the-shelf products. There is not a collection and manufacturing step. These drugs are going to be given in the community eventually once they are approved. These drugs will be used in many more patients compared with CAR T-cell therapy just because of the logistics of CAR T-cell therapies, so BiTEs are exciting.

These advances [observed in multiple myeloma] have also spilled over to amyloidosis. We now have great frontline therapy for amyloidosis, as well as many irons in the fire [evaluating] ways we can treat relapsed amyloidosis. Weve had a troubled past [with] antiviral therapy in amyloidosis. However, there is renewed interest in this and, certainly, there are patients with amyloidosis who would benefit from antiviral therapy.

There is a lot of work going on in amyloidosis currently. The ANDROMEDA study [NCT03201965] has shown in randomized fashion that daratumumab plus bortezomib, cyclophosphamide, and dexamethasone [VCd] results in better organ response rates and PFS vs VCd alone, which had really been our standard therapy in amyloidosis. Going forward, patients with amyloidosis should receive this quadruplet as frontline therapy.

Patients with amyloidosis also have a high incidence of 11;14 translocations [t11;14]. Some case reports [have read out] of patients being treated with venetoclax. Ongoing research avenues are going to further investigate venetoclax with or without the combination of other drugs. Venetoclax will have a strong response rate in patients with amyloidosis and will be used for initial relapse. Eventually, [venetoclax] might be used in patients with t11;14, but those studies are being done. Approval for that is a long way down the road.

Also down the road for amyloidosis are BiTEs. BCMA is on the surface of plasma cells in amyloidosis, also, [as in multiple myeloma]. There is also a renewed interest in antiviral therapy in amyloidosis. The amyloid proteins deposit in the cell and cause significant organ toxicity, especially in the [heart] and kidneys. Antiviral therapy may enhance and quicken organ responses to improve survival for patients, including those with severe cardiac amyloidosis.

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Emerging Quadruplets, Novel Targets, and Immunotherapy Advances Personalized Medicine in Multiple Myeloma - OncLive

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cardiovascular disease | Symptoms, Causes, Treatment …

By daniellenierenberg

Cardiovascular disease, any of the diseases, whether congenital or acquired, of the heart and blood vessels. Among the most important are atherosclerosis, rheumatic heart disease, and vascular inflammation. Cardiovascular diseases are a major cause of health problems and death.

This micrograph shows a cross section of a coronary artery narrowed by an atherosclerotic plaque (purplish matter inside the artery). The extensive buildup of plaque impedes the flow of blood through the artery and to the heart's tissues.

Britannica Quiz

44 Questions from Britannicas Most Popular Health and Medicine Quizzes

How much do you know about human anatomy? How about medical conditions? The brain? Youll need to know a lot to answer 44 of the hardest questions from Britannicas most popular quizzes about health and medicine.

Life depends on the functioning of the heart; thus, the heart is involved in all death, but this does not account for its prominence in causing death. To some degree, as medical science advances, more people are saved from other illnesses only to die from one of the unsolved and uncontrolled disorders of the cardiovascular system. Some forms of cardiovascular diseases are becoming less frequent causes of death, and continued research and preventive measures may provide even greater benefits. However, changes in lifestyle and diet, including the adoption of more sedentary lifestyles and the consumption of fried foods and foods high in sugar, have resulted in increases in the incidence of otherwise preventable cardiovascular-related illness and death.

Heart disease as such was not recognized in non-technological cultures, but the beating heart and its relationship to death have always been appreciated. Sudden death, now usually attributed to heart disease, was recognized as early as the 5th century bce by the Greek physician Hippocrates and was noted to be more common in the obese. The role of disease in affecting the heart itself did not become apparent until the 17th century, when examination of the body after death became acceptable.

Gradually, the involvement of the heart valves, the blood vessels, and the heart muscle was observed and categorized in an orderly fashion. The circulation of the blood through the heart was described in 1628 by the British physician William Harvey. The recognition of the manifestations of heart failure came later, as did the ability to diagnose heart ailments by physical examination through the techniques of percussion (thumping), auscultation (listening) with the stethoscope, and other means. It was not until early in the 20th century that the determination of arterial blood pressure and the use of X-rays for diagnosis became widespread.

In 1912 James Bryan Herrick, a Chicago physician, first described what he called coronary thrombosis (he was describing symptoms actually caused by myocardial infarction). Angina pectoris had been recorded centuries earlier. Cardiovascular surgery in the modern sense began in the 1930s, and open-heart surgery began in the 1950s.

The exact incidence of heart disease in the world population is difficult to ascertain, because complete and adequate public health figures for either prevalence or related deaths are not available. Nonetheless, in the 21st century, in many parts of the world, cardiovascular disease was recognized as a leading cause of death. In the more technologically developed countries of the worldsuch as the United Kingdom and most continental European countriesarteriosclerotic heart disease (heart disease resulting from thickening and hardening of the artery walls) was one of the most common forms of cardiovascular disease. In the early 21st century in the United States, an estimated one-half of the adult population was affected by some form of cardiovascular disease; while heart disease and stroke accounted for a significant proportion of this disease burden, high blood pressure was the most common condition. In other areas of the world, such as the countries of Central Africa, other forms of heart disease, often nutritional in nature, were a common cause of death. In Asia and the islands of the Pacific, hypertensive cardiovascular disease, disease involving high blood pressure, constituted a major health hazard.

The hearts complicated evolution during embryological development presents the opportunity for many different types of congenital defects to occur. Congenital heart disease is one of the important types of diseases affecting the cardiovascular system, with an incidence of about 8 per 1,000 live births. In most patients the causes appear to fit in the middle of a continuum from primarily genetic to primarily environmental.

Of the few cases that have a genetic nature, the defect may be the result of a single mutant gene, while in other cases it may be associated with a chromosomal abnormality, the most common of which is Down syndrome, in which about 50 percent of afflicted children have a congenital cardiac abnormality. In the even smaller number of cases of an obvious environmental cause, a variety of specific factors are evident. The occurrence of rubella (German measles) in a woman during the first three months of pregnancy is caused by a virus and is associated in the child with patent ductus arteriosus (nonclosure of the opening between the aorta and the pulmonary artery). Other viruses may be responsible for specific heart lesions, and a number of drugs, including antiepileptic agents, are associated with an increased incidence of congenital heart disease.

In most cases, congenital heart disease is probably caused by a variety of factors, and any genetic factor is usually unmasked only if it occurs together with the appropriate environmental hazard. The risk of a sibling of a child with congenital heart disease being similarly affected is between 2 and 4 percent. The precise recurrence can vary for individual congenital cardiovascular lesions.

Prenatal diagnosis of congenital cardiovascular abnormalities is still at an early stage. The most promising technique is ultrasonography, used for many years to examine the fetus in utero. The increasing sophistication of equipment has made it possible to examine the heart and the great vessels from 16 to 18 weeks of gestation onward and to determine whether defects are present. Amniocentesis (removal and examination of a small quantity of fluid from around the developing fetus) provides a method by which the fetal chromosomes can be examined for chromosomal abnormalities associated with congenital heart disease. In many children and adults the presence of congenital heart disease is detected for the first time when a cardiac murmur is heard. A congenital cardiovascular lesion is rarely signaled by a disturbance of the heart rate or the heart rhythm.

Congenital cardiac disturbances are varied and may involve almost all components of the heart and great arteries. Some may cause death at the time of birth, others may not have an effect until early adulthood, and some may be associated with an essentially normal life span. Nonetheless, about 40 percent of all untreated infants born with congenital heart disease die before the end of their first year.

Congenital heart defects can be classified into cyanotic and noncyanotic varieties. In the cyanotic varieties, a shunt bypasses the lungs and delivers venous (deoxygenated) blood from the right side of the heart into the arterial circulation. The infants nail beds and lips have a blue colour due to the excess deoxygenated blood in the system. Some infants with severe noncyanotic varieties of congenital heart disease may fail to thrive and may have breathing difficulties.

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cardiovascular disease | Symptoms, Causes, Treatment ...

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FDA Approves Expanded Indication for Merck’s KEYTRUDA (pembrolizumab) in Locally Advanced Cutaneous Squamous Cell Carcinoma (cSCC) – Business Wire

By daniellenierenberg

KENILWORTH, N.J.--(BUSINESS WIRE)--Merck (NYSE: MRK), known as MSD outside the United States and Canada, today announced that the U.S. Food and Drug Administration (FDA) has approved an expanded label for KEYTRUDA, Mercks anti-PD-1 therapy, as monotherapy for the treatment of patients with locally advanced cutaneous squamous cell carcinoma (cSCC) that is not curable by surgery or radiation. This approval is based on data from the second interim analysis of the Phase 2 KEYNOTE-629 trial, in which KEYTRUDA demonstrated an objective response rate (ORR) of 50% (95% CI, 36-64) (n=54), including a complete response rate of 17% and a partial response rate of 33% in the cohort of patients with locally advanced disease. Among the 27 responding patients, 81% had a duration of response (DOR) of six months or longer, and 37% had a DOR of 12 months or longer. In June 2020, KEYTRUDA was granted its first indication in cSCC, as monotherapy for the treatment of patients with recurrent or metastatic disease that is not curable by surgery or radiation.

This approval is great news for these patients and further demonstrates Mercks commitment to the skin cancer community. KEYTRUDA has shown meaningful efficacy in patients with locally advanced or recurrent or metastatic cutaneous squamous cell carcinoma that cannot be cured by surgery or radiation, said Dr. Vicki Goodman, vice president, clinical research, Merck Research Laboratories. This expanded indication reinforces the role of KEYTRUDA in this cancer type, which is the second most common form of non-melanoma skin cancer.

Immune-mediated adverse reactions, which may be severe or fatal, can occur in any organ system or tissue and can affect more than one body system simultaneously. Immune-mediated adverse reactions can occur at any time during or after treatment with KEYTRUDA, including pneumonitis, colitis, hepatitis, endocrinopathies, nephritis, dermatologic reactions, solid organ transplant rejection, and complications of allogeneic hematopoietic stem cell transplantation. Important immune-mediated adverse reactions listed here may not include all possible severe and fatal immune-mediated adverse reactions. Early identification and management of immune-mediated adverse reactions are essential to ensure safe use of KEYTRUDA. Based on the severity of the adverse reaction, KEYTRUDA should be withheld or permanently discontinued and corticosteroids administered if appropriate. KEYTRUDA can also cause severe or life-threatening infusion-related reactions. Based on its mechanism of action, KEYTRUDA can cause fetal harm when administered to a pregnant woman. For more information, see Selected Important Safety Information below.

Data Supporting the Approval

The approval was based on data from KEYNOTE-629 (ClinicalTrials.gov, NCT03284424), a multicenter, multi-cohort, non-randomized, open-label trial that enrolled patients with recurrent or metastatic cSCC or locally advanced cSCC. The trial excluded patients with autoimmune disease or a medical condition that required immunosuppression.

Patients received KEYTRUDA 200 mg intravenously every three weeks until documented disease progression, unacceptable toxicity or a maximum of 24 months. Patients with initial radiographic disease progression could receive additional doses of KEYTRUDA during confirmation of progression unless disease progression was symptomatic, rapidly progressive, required urgent intervention, or occurred with a decline in performance status.

Assessment of tumor status was performed every six weeks during the first year and every nine weeks during the second year. The major efficacy outcome measures were ORR and DOR as assessed by blinded independent central review (BICR) according to RECIST v1.1, modified to follow a maximum of 10 target lesions and a maximum of five target lesions per organ.

Among the 54 patients with locally advanced cSCC treated, the study population characteristics were: median age of 76 years (range, 35 to 95), 80% age 65 or older; 72% male; 83% white, 13% race unknown; 41% Eastern Cooperative Oncology Group (ECOG) performance status (PS) of 0 and 59% ECOG PS of 1. Twenty-two percent received one or more prior lines of therapy; 63% received prior radiation therapy.

The ORR was 50% (95% CI, 36-64), including a complete response rate of 17% and a partial response rate of 33%, for patients treated with KEYTRUDA. After a median follow-up of 13.4 months, the median DOR had not yet been reached (range, 1.0+ to 17.2+ months). Among the 27 responding patients, 81% had a DOR of six months or longer, and 37% had a DOR of 12 months or longer.

Among the 159 patients with advanced cSCC (recurrent or metastatic or locally advanced disease) enrolled in KEYNOTE-629, the median duration of exposure to KEYTRUDA was 6.9 months (range, 1 day to 28.9 months). Adverse reactions occurring in patients with recurrent or metastatic cSCC or locally advanced cSCC were similar to those occurring in 2,799 patients with melanoma or non-small cell lung cancer treated with KEYTRUDA as a single agent. Laboratory abnormalities (Grades 3-4) that occurred at a higher incidence included lymphopenia (10%) and decreased sodium (10%).

About KEYTRUDA (pembrolizumab) Injection, 100 mg

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

Merck has the industrys largest immuno-oncology clinical research program. There are currently more than 1,500 trials studying KEYTRUDA across a wide variety of cancers and treatment settings. The KEYTRUDA clinical program seeks to understand the role of KEYTRUDA across cancers and the factors that may predict a patient's likelihood of benefitting from treatment with KEYTRUDA, including exploring several different biomarkers.

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

Melanoma

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

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

Non-Small Cell Lung Cancer

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

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

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

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

Head and Neck Squamous Cell Cancer

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

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

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

Classical Hodgkin Lymphoma

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

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

Primary Mediastinal Large B-Cell Lymphoma

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

Urothelial Carcinoma

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

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

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

Microsatellite Instability-High or Mismatch Repair Deficient Cancer

KEYTRUDA is indicated for the treatment of adult and pediatric patients with unresectable or metastatic microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR)solid tumors that have progressed following prior treatment and who have no satisfactory alternative treatment options

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

Microsatellite Instability-High or Mismatch Repair Deficient Colorectal Cancer

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

Gastric Cancer

KEYTRUDA, in combination with trastuzumab, fluoropyrimidine- and platinum-containing chemotherapy, is indicated for the first-line treatment of patients with locally advanced unresectable or metastatic HER2-positive gastric or gastroesophageal junction (GEJ) adenocarcinoma. This indication is approved under accelerated approval based on tumor response rate and durability of response. Continued approval for this indication may be contingent upon verification and description of clinical benefit in the confirmatory trials.

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

Esophageal Cancer

KEYTRUDA is indicated for the treatment of patients with locally advanced or metastatic esophageal or GEJ (tumors with epicenter 1 to 5 centimeters above the GEJ) carcinoma that is not amenable to surgical resection or definitive chemoradiation either:

Cervical Cancer

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

Hepatocellular Carcinoma

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

Merkel Cell Carcinoma

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

Renal Cell Carcinoma

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

Tumor Mutational Burden-High Cancer

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

Cutaneous Squamous Cell Carcinoma

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

Triple-Negative Breast Cancer

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

Selected Important Safety Information for KEYTRUDA

Severe and Fatal Immune-Mediated Adverse Reactions

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

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

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

Immune-Mediated Pneumonitis

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

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

Immune-Mediated Colitis

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

Hepatotoxicity and Immune-Mediated Hepatitis

KEYTRUDA as a Single Agent

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

KEYTRUDA with Axitinib

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

Immune-Mediated Endocrinopathies

Adrenal Insufficiency

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

Hypophysitis

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

Thyroid Disorders

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

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

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

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

Immune-Mediated Nephritis With Renal Dysfunction

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

Immune-Mediated Dermatologic Adverse Reactions

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

Other Immune-Mediated Adverse Reactions

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

Infusion-Related Reactions

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

Complications of Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

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

Increased Mortality in Patients With Multiple Myeloma

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

Embryofetal Toxicity

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

Adverse Reactions

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

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FDA Approves Expanded Indication for Merck's KEYTRUDA (pembrolizumab) in Locally Advanced Cutaneous Squamous Cell Carcinoma (cSCC) - Business Wire

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2 Reasons to Buy Intellia — and 1 Big Reason Why I Won’t – The Motley Fool

By daniellenierenberg

Clinical trial data supporting the safety of the CRISPR-Cas9 genomic editing tool was presented on Monday by Intellia Therapeutics (NASDAQ:NTLA) for its lead product, NTLA-2001. The data was highly encouraging. However, despite NTLA-2001's positive early results as a potential treatment for the rare disease transthyretin (TTR) amyloidosis, there's still a long way to go before Intellia could bring it to market.

In transthyretin amyloidosis, cells in the liver produce misfolded TTR proteins, which accumulate throughout the body, causing debilitating complications that can involve the digestive system, nervous system, and heart. Once symptoms appear, they grow progressively worse, and the disease leads to death within a median of 4 to 17 years among patients with nervous system involvement, and 2 to 6 years among patients with cardiac involvement.

NTLA-2001 edits the genes in those liver cells, removing the segment that produces those lethal misfolded proteins.

Worldwide, an estimated 250,000-550,000 people suffer from some form of amyloidosis.

IMAGE SOURCE: GETTY IMAGES.

An interim readout from Intellia's ongoing phase 1 trial found that a single high-dose infusion of NTLA-2001 led to an 87% mean reduction in the amount of misfolded TTR in patients' bloodstreams, with a maximum reduction of 96% by day 28 in one patient. Encouragingly, no serious adverse events were observed in the six study participants. While this is a small pilot study, in previous studies of NTLA-2001 in mice, the maximum reductions in TTR persisted for 12 months after a single treatment.

All of this data provides an early indication that CRISPR gene therapies are safe and efficacious as treatments for at least some genetic diseases.

There are other treatments on the market for TTR amyloidosis, but one thing that would set CRISPR apart is the relative simplicity of administering it. And that factor could lead insurers to favor CRISPR treatments for certain rare and debilitating diseases such as TTR amyloidosis and hemophilia.

For example, Alnylam's (NASDAQ:ALNY) RNA-silencing therapy Onpattro requires an infusion every three weeks at a clinician's office. Ionis Pharmaceuticals' (NASDAQ:IONS) Tegsedi requires regular injections, though they can be self-administered. Both are priced in the neighborhood of $345,000 per year, and Onpattro comes with the additional costs associated with going to a medical office and having an infusion set up. Then there is Pfizer's (NYSE:PFE) once-daily oral medication Vyndamax, which costs $225,000 annually.

As a one-time infusion, gene therapy may become a compelling option for both patients and insurers, particularly given the high prices of currently available treatments. Though TTR amyloidosis treatments are a niche market, in 2020, Onpattro generated sales of $306 million, Tegsedi just under $70 million, and Vyndamax $429 million. Assuming that Intellia charges more for NTLA-2001 -- a one-time treatment with bluebird bio's (NASDAQ:BLUE) gene therapy for beta-thalassemia, Zynteglo, costs about $1.8 million -- TTR amyloidosis treatment could easily become a multibillion-dollar addressable market for the biotech.

Notably, CRISPR therapy for TTR amyloidosis may also put less stress on the healthcare system than the lentivirus and adenovirus gene therapies that are further along in clinical trials. Consider, for instance, Zynteglo, which requires a significant amount of effort and processing prior to treatment. First, physicians must extract stem cells from the patient, which must then be transported to and treated by bluebird bio. In the meantime, the patient undergoes "myeloablative conditioning" -- essentially knocking down the patient's bone marrow in preparation for a transplant of the edited stem cells, which will contain a repaired version of the gene that (when mutated) causes beta-thalassemia. This complicated process requires treatment at a qualified transplant center.

By comparison, for TTR amyloidosis, NTLA-2001 requires pre-medication with steroids and antihistamines. That's it. No prolonged patient preparation at the hospital. No bone marrow suppression. No shipping the patient's stem cells to a lab. The relative simplicity of administering CRISPR therapies is just one reason for the degree of excitement they are generating.

It may also give them a lower total cost of treatment than current gene therapies, which could make these therapies more palatable to insurers. If NTLA-2001 pans out, we may see a new biotech boom, with Intellia leading the charge.

Before investors get their hopes up too much, remember that these results were from a six-person, phase 1 trial, and that Intellia now holds a market cap of roughly $11 billion. In fact, its valuation rose by about $2.8 billion in a single trading session after the interim trial data was made public. That gain was more than the current $2.1 billion market cap of bluebird bio, which already has an approved gene therapy on the market as well as a CAR-T therapy, and has two more candidates in phase 3 trials.

For further context, bluebird bio announced phase 1 results for Zynteglo in December 2014. While Zynteglo was approved for use in the EU in late 2019, bluebird bio faced some backlash on pricing, and the company isn't selling it in Germany because the two sides could not agree on pricing.

Moreover, the NTLA-2001 study excluded patients who had previously received RNA-silencing therapy, and none of these patients had previously taken Vyndamax either. How previous treatments will affect the way patients respond to NTLA-2001 is not yet known. And with hundreds of millions of dollars in revenue annually on the line, it is doubtful that Alynam, Ionis, or Pfizer will surrender this market without a fight.

In sum, Intellia will still need to conduct several years of trials, leap many regulatory hurdles, and outmaneuver an array of rivals stand before it can declare the CRISPR-Cas9 platform a winner. Not only that, but -- recognizing that future studies won't be cheap -- Intellia has already proposed another public offering of $400 million worth of common stock this week, diluting its current shareholders.

So while long-term Intellia shareholders have reason to celebrate, let bluebird bio serve as a cautionary tale. That biotech was once flying high on positive trial data, hitting a market cap of around $15.5 billion in March 2018. Since then, its shares have nose-dived by more than 80%. This despite the fact that it now has two approved therapies and two more candidates in phase 3 trials.

As such, I would be concerned about investing new money in Intellia now. I suspect it will soon reach its peak for the foreseeable future. Biotech investing can be gut-wrenchingly fickle, and investors may want to consider taking a basket approach to high-risk clinical-stage biotechs, rather than investing too heavily in a single player.

This article represents the opinion of the writer, who may disagree with the official recommendation position of a Motley Fool premium advisory service. Were motley! Questioning an investing thesis -- even one of our own -- helps us all think critically about investing and make decisions that help us become smarter, happier, and richer.

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Global Autologous Stem Cell Based Therapies Market 2021 Outlook and Study of Top Players Regeneus, Mesoblast, Pluristem Therapeutics Inc, US STEM…

By daniellenierenberg

MarketQuest.biz has recently added a report titled Global Autologous Stem Cell Based Therapies Market 2021 by Company, Regions, Type and Application, Forecast to 2026 describes the crucial aspects of the market by conducting an in-depth analysis of the current trend, emerging threats, and future market assessment. The report presents a thorough study of the current trends leading to this vertical trend in various regions. The report highlights important details related to global Autologous Stem Cell Based Therapies market share, market size, applications, and statistics. It sheds light on market dynamics and demonstrates a superior forecast for the development of the market.

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The report familiarizes prominent market leaders with the latest market trends, challenges, opportunities, industry information, and market share. The report content includes technology, industry drivers, geographic trends, market statistics, market forecasts, producers, and raw material/equipment suppliers. Segmentation and sub-segmentation is a consolidation of global Autologous Stem Cell Based Therapies industry segment, type segment, channel segment, and many more. Further, the report is expanded to provide thorough insights into each segment.

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This report is aimed toward guiding people towards an apprehensive, better, and clearer knowledge of the market. The global Autologous Stem Cell Based Therapies market growth analysis is provided for the international markets including development trends, competitive landscape analysis, investment plan, business strategy, opportunity, and key regions development status. The market is meant to point out substantial growth during the forecast period of 2021-2026.

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Global Autologous Stem Cell Based Therapies Market 2021 Outlook and Study of Top Players Regeneus, Mesoblast, Pluristem Therapeutics Inc, US STEM...

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Disease-modifying therapies for osteoarthritis | DDDT – Dove Medical Press

By daniellenierenberg

Why is the Development of Disease-Modifying Osteoarthritis Drugs (DMOADs) Required?Disease Burden

Osteoarthritis (OA) is the most prevalent arthritis globally and represents a major challenge for twenty-first century health care systems.1,2 The Global Burden of Disease 2020 report showed an increase of 9.3% and 8.2% in the age-standardized OA point prevalence and annual incidence rate from 1990 to 2017.3 The prevalence rises with increasing age; in the USA (United States of America), OA was found in 13.9% of adults aged 25 years and 33.6% for those aged 65 years respectively in 2005.4 The lifetime risk of having symptomatic knee OA is about 40% in men and 47% in women, and the risk increases to 60.5% among obese persons.5 By the year 2040, an estimated 25.9% of the total adult population will have doctor-diagnosed arthritis in the USA.6

Globally, 80% of patients with OA suffer from limitations in movement, and 25% from difficulty in performing their major daily activities of life; representing a significant impact of OA on functional impairment and disability.7 In terms of economic burden, mean per-person earnings losses caused by OA were, on average, 7548 US$ per year from 2008 to 2011.8 The mean all-cause health care utilization of working-age patients with OA is $14,521 US$ per year.9 The socio-economic costs of OA were reported to range between 0.25% and 0.50% of a countrys GDP.10 In an individual patient data meta-analysis, the pooled estimate for premature mortality revealed a 23% increased risk (95% CI 1.07, 1.42) in patients with knee OA and a 20% increased risk (95% CI 1.04, 1.37) in hip OA.11

Current OA treatment options are focused on symptomatic improvement in pain and joint function and include paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, and intra-articular medications such as steroids and hyaluronic acids.14 Surgical treatments are typically indicated only for patients with end-stage OA, as a last resort. Recently, paracetamol and opioids are only conditionally or not recommended by several scientific organisations,12,13 highlighting the importance of finding new effective treatments for OA. In addition, outcomes for patients with OA are usually suboptimal and patients remain vulnerable to the clinical consequences of the disease on pain and physical function.14

OA was previously regarded as a degenerative disorder resulting from cartilage damage;15 however, the development and utilization of modern imaging methods revealed that it results from the failure of the joint organ with a heterogeneous involvement of the whole joint structures, including cartilage damage, subchondral bone remodeling, synovial inflammation and osteophyte development.16 Therefore, OA can be defined as a complex heterogeneous syndrome with multiple joint tissue involvement of varying severity. In part as a consequence, it is a huge challenge to develop a single one size fits all therapy that may be suitable and effective for all patients with OA.17

The central hallmark in the pathologic process of OA disease is the progressive deterioration in the biological, structural and mechanical properties and function of the joint tissues, and an effective medical treatment should possess the ability to delay these processes or ideally even halt them completely. Such pharmaceutical agents that will alter the natural history of disease progression by arresting joint structural change and ameliorating symptoms, either by reducing pain or improving physical function are termed as DMOADs.18

Currently, regulatory bodies such as US Food and Drug Administration (FDA)19 and the European Medicines Agency (EMA)20 have not approved any drug as an effective DMOAD, as the approval guide requires a potential DMOAD to demonstrate a slowing in the loss of knee or hip joint space width (JSW) on x-ray with associated symptomatic improvement.17 Therefore, current OA trials for DMOAD development pipeline need to meet both clinically meaningful symptom improvement with concomitant structural benefits according to US FDAs published draft industry guidance on structural endpoints for OA published in 2018.18

Because OA is characterised by its extraordinary inter-patient variability in clinical and structural manifestations, identification of patient/disease subtypes appropriate for targeted therapy is probably one of the promising ways forward in drug development research.21,22 In addition, structural changes in OA result from complex interactions among different pathobiological pathways, which implicate a variety of catabolic factors and cytokines in the different joint tissues (molecular cross-talk).23 Therefore, a new model of classifying OA based on pathophysiological disease subtypes is needed.

These subtypes can be clinical phenotypes or molecular/mechanistic endotypes.24 A clinical phenotype can be defined as a group of observable traits (ie aetiologic factors, risk factors) that can identify and characterize a subtype in a defined population.25,26 In other words, these subgroups of patients have similar clinically observable characteristics for better identifying individuals who are at higher risk of progression (prognostic) or who are more likely to respond to a specific intervention (prescriptive).27,28

An endotype is a disease subtype defined by distinct pathophysiologic mechanisms, including cellular, molecular and biomechanical signalling pathways.29 Therefore, the endotype is distinct from a phenotype, and indicates the presence of a well-defined molecular mechanism. A given clinical phenotype of OA may comprise overlapping molecular endotypes (ie, different mechanisms giving rise to the same manifestation at varying degrees during different phases of the disease).24

From the point of view of targeted drug discovery, where identifying and directing the right pathobiological mechanism and structural manifestations of disease is key for success, drug development in OA should be based on the endotypes as the basis of the main drivers of OA disease.30 In this review, we will, therefore, focus on currently ongoing phase 2 and 3 clinical trials of active drug development (Figure 1) related to three main molecular/mechanistic endotypes: 1) Cartilage-driven endotype, 2) Bone-driven endotype, 3) Inflammation-driven endotype. While each drug has been assigned to and is discussed under one endotype based on its predominant activity, a particular therapeutic may have broader endotype-effects and where present, these are duly noted.

Figure 1 Active drugs related to the three main molecular or mechanistic OA endotypes (phase 2 and 3).

One author (WMO) conducted electronic and manual searches on the https://clinicaltrials.gov/ for identifying ongoing phase 2/3 clinical trials in active drug development pipelines, as well as electronic database searches in the PubMed and Embase via Ovid for published reports of phase-2/3 clinical trials results from the inception of these databases to 31st March 2021 using the following MESH or keywords: osteoarthritis OR osteoarthrosis AND DMOAD/ OR structure modification OR disease-modifying osteoarthritis drugs/.

Cartilage damage is considered as a central part of OA disease process, which involves a variety of catabolic and reparative mechanisms at the molecular level. The pharmaceutical drugs in phase 2 and 3 stages of development for cartilage-driven endotype are summarized in Table 1.

Matrix-degrading enzymes in the joint such as collagenases and aggrecanases are responsible for proteolysis of extracellular matrix components such as type II collagen and aggrecan, which is the most abundant proteoglycan in cartilage.31 Proteinases such as matrix metalloproteinase 13 (MMP13) and ADAMTS5 (a Disintegrin And Metalloproteinase with ThromboSpondin-motif-5) are involved in cartilage destruction and progression of cartilage damage in OA pre-clinical models.32,33 The potential benefits of MMP inhibitors in preserving the OA joint have been investigated. However, in patients with knee OA, broad-spectrum MMP inhibitors such as PG-116800 showed reversible musculoskeletal toxicities in a dose-dependent manner without clinical benefits, leading to the termination of further development of this drug.34

S201086/GLPG1972 is a potent and highly selective active site inhibitor of ADAMTS5. It possesses an excellent selectivity profile in animal models and high stability in dog and human liver microsomes and hepatocytes.35 Phase-1 clinical studies revealed favorable pharmacokinetics as well as a strong and consistent target engagement in both healthy subjects and OA patients (n=171).36 In a phase-2 study (Roccella study) which investigated the efficacy and safety profile of three different once-daily oral doses of GLPG1972/S201086 (n=932), the change in cartilage thickness [in mm (SD)] of central medial tibiofemoral compartment of the target knee via quantitative MRI was 0.116 (0.27) for the placebo group and 0.068 (0.20), 0.097 (0.27) and 0.085 (0.22), for the low, medium and high dose, respectively. There was no statistically significant difference versus placebo in both MRI and clinical outcome measures.37 Another ADAMTS5-targeting agent, M6495 an anti-ADAMTS5 Nanobody (Ablynx), showed an acceptable safety profile and dose-dependent effects in a phase-1 study.38

Sprifermin is a recombinant human fibroblast growth factor 18 (FGF18) which binds to fibroblast growth factor receptor-3 (FGFR-3) in cartilage.39 It stimulates the proliferation of articular chondrocytes and induces hyaline extracellular matrix synthesis in rat OA models.40 At the cellular level, intermittent administration may transiently promote an anabolic effect, while continuous administration may stimulate other signalling pathways, leading to a weaker effect.41

Lohmander et al reported in 2014 that intra-articular (IA) sprifermin administration did not improve medial tibiofemoral cartilage-thickness over 12 months quantified by MRI (n=168) possibly as follow-ups were too short for detection of the full disease-modifying effect of treatment.39 However, a significant dose-dependent response was detected in total and lateral tibiofemoral cartilage-thickness and radiographic JSW over 12 months. The authors speculated that the dynamic loading implicated in predominantly medial tibiofemoral involvement seems to impede attempts to prevent cartilage loss or regenerate cartilage tissue. Sprifermin had no major local or systemic adverse events compared with placebo. Conference abstracts published in 2015 and 2016 reported the structure-modifying effects on cartilage thickness and bone marrow lesions (BMLs) on MRI on 12-month follow-up, using post-hoc analyses of the same study.42,43

In another clinical trial in which Sprifermin was administered up to 300 g for advanced knee OA, it was reported in 2016 that no significant benefits were detected for cartilage outcomes on histology, synovitis, effusion, BMLs on MRI and JSW on X-ray. However, the study was underpowered as MRI was only available in 30 out of 52 patients and the follow-up period was only 24 weeks, which may be too short for capturing the structure-modifying effects.44

In a 5-year, phase 2 dose-finding, multicenter randomized clinical trial [FGF18 Osteoarthritis Randomized Trial with administration of Repeated Doses (FORWARD) study], the effects of Sprifermin on changes in total femorotibial joint cartilage thickness (n=549) on MRI was evaluated at 2-year follow-up (NCT01919164). Hochberg et al reported in 2019 that three once-weekly IA injection of 100 g sprifermin provided a significant improvement in total femorotibial joint cartilage thickness [0.05 mm (95% CI, 0.03 to 0.07 mm)] for participants administered every 6 months and [0.04 mm (95% CI, 0.02 to 0.06 mm)] for participants administered every 12 months, compared with the placebo saline injection provided every 6 months (0.02 mm).45 No significant improvement in total WOMAC scores was detected, compared with placebo. The most frequently reported treatment-emergent adverse event was arthralgia and showed no difference from the placebo group (43%). An exploratory analysis of the same study at 3 year-follow-up (n=442) reveals significant differences (0.05 mm [95% CI, 0.030.07 mm]) in total femorotibial joint cartilage thickness over MRI between Sprifermin (100 g of Sprifermin every 6 months) and placebo (saline every 6 months).45 However, the clinical significance of a 0.05-mm increase of cartilage thickness in this study remains unclear in terms of reducing risk for knee replacement, delaying time towards knee replacement, or both.46 No significant change in total WOMAC scores in this study may be attributed to using intra-articular saline injections as a control since the IA saline injection may act as an active placebo,47 masking symptomatic benefits. In addition, a large number of patients with low baseline pain and/or high baseline cartilage thickness may result in a potential floor effect on symptoms as 32% of this study had <40/100 points on WOMAC pain score at baseline and 50% had medial minimum joint space width (mJSW) >4.0 mm on baseline X-rays. Therefore, analysis of a more selective subgroup, featuring baseline characteristics associated with rapid structural and symptomatic OA progression should be investigated. In a 2019 ACR conference abstract, it was reported that in a subgroup at risk (n=161) of structural and symptomatic progression with a baseline medial or lateral mJSW between 1.5 and 3.5 mm and WOMAC pain score of 4090 out of 100, WOMAC pain was significantly improved on 3 year follow-up [8.8 (22.4, 4.9)] in the group administered with the 100 g Sprifermin (n=34) compared with the placebo (n=33)48 suggesting that, in this subgroup, the drug effect reaches the absolute minimal clinically important improvement for the WOMAC pain subscore which ranges 69.49

In a recent 2020 paper using a post-hoc analysis of the same data from the FORWARD study, thinning/thickening scores and ordered values of femorotibial cartilage thickness change on MRI over 24 months were analyzed by applying location-independent (ie not region-specific) analysis methodology in the knee joint.50 With administration of 100g Sprifermin every 6 months cartilage thickening is more than double [856m (717 to 996) vs 356m (313 to 398)] and cartilage thinning almost reduced to [432m (521 to 343) vs 335m (381 to 288)] that in healthy reference subjects from the Osteoarthritis Initiative dataset (n=82). The authors concluded that the finding supported the evidence of substantial structure-protective action of Sprifermin. However, as this is a post-hoc analysis, further study will be required to confirm its structure-modifying effect.

At a molecular level, the regulation of Wnt signalling determines osteoblast and chondrocyte lineage specification and their homeostasis.51 Increased Wnt signaling predisposes MSCs to an osteogenic lineage fate and induces generation of metalloproteinases which can cause cartilage degradation in OA.52 Increased expression and activation of the Wnt pathway in articular cartilage chondrocytes in OA similarly promotes cartilage degradation, while elevated Wnt signalling in subchondral bone enhances bone formation and sclerosis.5355 Therefore, pharmacological modulation of Wnt signaling might have potential benefits in repairing osteochondral dysregulation detected in OA disease process. Moreover, increased Wnt signaling in the synovium may potently lead to the OA progression via increased production of MMPs as well as activation of osteoclast differentiation and enhanced subchondral bone turnover.56,57

Lorecivivint (SM04690) is a small-molecule CLK/DYRK1A inhibitor that blocks Wnt signalling at the transcriptional level.58 It showed induction of chondrogenesis and reduction in cartilage degradation in preclinical studies.5860 In a 52-week, multicenter, phase-2 trial (n=455) (NCT02536833), the primary end point, a significant improvement in the WOMAC pain score compared with placebo at week 13, was not met, compared with IA placebo saline injection, However, at 52-week follow-up, intra-articular administration of 0.07 mg demonstrated a significant benefit in pain and functional scores [between-group difference versus placebo, 8.73, 95% CI (17.44, 0.03) and 10.26, 95% CI (19.82, 0.69)], as well as improvement in mJSW on X-rays [between-group difference versus placebo, +0.39 mm, 95% CI (0.06, 0.72)] in patients with unilateral knee OA. Serious adverse events were reported in 17 (3.7%) patients.61 The most common SAEs included infections and cardiac disorders and were deemed unrelated to the study drug by the investigators.62

Another phase-2 trial evaluated in 700 patients for 24 weeks was completed (NCT03122860) where the 0.07 mg lorecivivint treatment group demonstrated more favorable reductions in both WOMAC indices as compared with placebo.63 Recently, the investigators reported the safety data after the combined analysis of the two trials, which included 848 Lorecivivint-treated and 360 control subjects in total. The incidence of adverse effects or serious adverse effects was similar in treatment (41.3% and 2.4%) and control groups (38.3% and 1.1%), respectively. The most commonly reported AE in both groups was arthralgia (7.6% vs 7.2%).64 Two small phase-2 (NCT03727022, NCT03706521) and three phase-3 (NCT03928184, NCT04385303, NCT04520607) trials are still active.

Transforming growth factor- (TGF-) induces extracellular matrix protein synthesis and modulates cartilage development. A variety of TGF- signalling pathways are crucial for early cartilage growth, maintaining cartilage homeostasis in later life and may also possess anti-inflammatory and immunosuppressive properties.65 Impaired TGF- function in cartilage might be related to an increased susceptibility to OA.66 However, the biological effect of TGF- is under complex control, and may switch from being protective in normal joints to detrimental in OA as a result of changes in the predominant cell-surface receptors and intra-cellular signalling pathways in various joint tissues (cartilage, bone, synovium).67 In addition, osteocyte TGF- signaling could regulate the osteogenic and osteoclastic activity of mesenchymal stem cells and may be associated with the remodeling of subchondral bone in advanced OA.68

TissueGene-C (TG-C) uses a cell-mediated cytokine gene therapy approach and includes non-irradiated allogeneic human chondrocytes and irradiated allogeneic human GP2-293 cells in a ratio of 3:1, retrovirally transduced to promote TGF-beta1 transcription (hChonJb#7 cells).6971 A recent study reported as a possible mechanism of action that TG-C induced an M2 macrophage-dominant pro-anabolic micro-environment in a rat model, thereby providing a beneficial effect on cartilage regeneration.72 At one-year follow-up after a single IA administration, there were significant improvements in pain, sports activities and quality of life but structure-modifying effects on the cartilage were insignificant (n=156).73 In a phase-2 trial (NCT01221441) including 57 patients in the treatment group and 29 patients in the placebo group, the TG-C administration caused less progression (47.9% vs 34.6%; adjusted RR 0.7, 95% CI 0.51.1) of cartilage damage than placebo over 12-months.69 In a phase-3 trial (NCT02072070) which included 163 patients, symptomatic benefit was detected.74

The two pivotal phase-3 trials (NCT03203330, NCT03291470) had been on hold in April 2019 while the regulators were investigating chemistry, manufacturing, and control issues related with the potential mislabeling of ingredients.75 This clinical hold was lifted in April 2020, and trial enrollments have been reinitiated later in 2020.76 Recently, analysis of the safety data from an observational long-term safety follow-up trial showed that there is no evidence to suggest that injection of TG-C was associated with increased risk of cancer nor generated any long-term safety concerns over an average 10 years.71

Senescence is characterized mainly by altered responses to cellular stress and proliferation arrest of cells.77 Senescent cells (SnCs) are a newly implicated factor in the OA pathogenic process78 by promoting pathological age-related deterioration via the production of proinflammatory cytokines, chemokines, extracellular proteases, and growth factors (termed the senescence-associated secretory phenotype (SASP))79 and altering the function of neighbouring cells (termed secondary or paracrine senescence).80 Therefore, senotherapeutics which are directed at SnCs are an emerging therapy for treating diseases related to ageing. Senotherapeutics can be classified into of 3 types: 1) senolytics which kill and destroy SnCs selectively; 2) senomorphics which modulate or even reverse the phenotype of SnCs to those of young cells by blocking SASP; 3) senoinflammation, the immune system-mediated clearance of SnCs.81 Several senolytic pharmaceutical drugs such as Fisetin and UBX0101 are emerging.

Fisetin is a polyphenol extracted from fruits and vegetables and shows potential senolytic and anti-inflammatory activities.82 Fisetin inhibited IL-1-induced MMP13 and ADAMTS5 expression in human OA chondrocytes in vitro, and reduced cartilage damage along with subchondral bone thickening and synovitis in a mouse OA model induced by destabilization of the medial meniscus (DMM).83 Two phase-2 clinical trials (NCT 04210986, NCT04815902) are under investigation in patients with knee OA and estimated to be completed in 2022 and 2025, respectively.

UBX0101 is a small molecule inhibitor of the MDM2/p53 protein interaction, which possesses a potent senolytic candidate. In a preclinical study, UBX0101 improved chondrogenesis in human OA tissue in vitro, and in an anterior cruciate ligament transection (ACLT) OA model in mice UBX0101 attenuated SnCs by stimulating apoptosis, and reduced cartilage damage and joint pain.84 The amount SnCs in human OA synovial tissues positively correlated with knee pain, disease severity and synovitis severity.85 A phase-1 study (n=48) revealed that a single intra-articular injection of UBX0101 at different doses up to 4 mg had a favorable safety profile and dose-dependent, clinically meaningful improvements in pain on Numeric Rating Scale (010) [3.95 (95% CI, 4.74, 3.16)] and WOMAC function [1.05 (95% CI, 1.36,-0.74)] compared with placebo injection. Recently, UNITY Biotechnology announced 12-week data from UBX0101 Phase-2 Clinical Study (NCT04129944) which did not detect a significant change in pain and function in 183 patients with painful knee OA.86 A follow-up observational study of the previous trial (NCT04349956) was terminated in November 2020 due to failure to meet the trial outcomes.

Subchondral change in OA involves an uncoupled remodelling process, which is characterized by both increased osteoblast activation and bone formation but simultaneously macrophage infiltration and osteoclast formation.87 Activation of osteoclasts can result in pain genesis through developing acidic conditions at the osteochondral junction, thereby activating acid-sensing receptors of sensory neurons.88,89 Subchondral bone also undergoes remarkable alterations in both composition and structural organization, leading to adverse effects on the overlying articular cartilage.90 Therefore, targeting the pathways that modify subchondral bone turnover is an attractive option for DMOAD research.89 The pharmaceutical drugs in phase 2 and 3 stages of development for bone-driven endotype are summarized in Table 2.

Table 2 The Registered Phase 2/3 Clinical Trials on Compounds with Potential Disease-Modifying Effects on Subchondral Bone

Cathepsin K is a cysteine protease which induces bone resorption and cartilage damage through the breakdown of key bone matrix proteins.91,92 Cathepsin K knock out mice had attenuated cartilage damage in OA induced by DMM, and inhibition of Cathepsin K in rabbits by daily oral dosing with L-006235 reduced cartilage damage and subchondral bone remodelling in an ACLT model of OA.93,94

MIV-711 is a selective cathepsin K inhibitor, and in a 6-month phase 2 clinical trial (NCT02705625) (n=244), significantly reduced femoral bone disease progression and reduced cartilage loss, although there was no improvement in pain outcome.95 Infrequent musculoskeletal symptoms, infections and rashes were reported. A further 6-month open-level extension study showed the maintenance of structural benefit with symptomatic improvement (n=50).96 However, as most of the participants in the extension sub-study were selected because their symptoms did not worsen, a treatment benefit may be due to positive selection bias.95

Recombinant human PTH, teriparatide, is a 134 amino-acid fragment acquired from human PTH). Its anabolic action on bone production is used for osteoporosis management. In OA, it exhibits the ability to maintain articular cartilage health,97 stimulate the synthesis of extracellular matrix and induce chondrocyte proliferation in pre-clinical injury-induced OA models.98 PTH can increase subchondral bone mineral density, which could exert a negative effect on OA progression. In this sense, PTH could be an excellent drug in OA patients with osteoporosis and low subchondral sclerosis.99 Additionally, intermittent parathyroid hormone treatment attenuates OA pain in a DMM model, in association with inhibiting subchondral sensory innervation, subchondral bone deterioration, and articular cartilage degeneration.100 A phase-2 study is currently ongoing to evaluate the efficacy of PTH in knee OA participants (NCT03072147).

TPX-100 is a novel 23-amino-acid peptide derived from MEPE, a member of the Small Integrin-Binding Ligand, N-linked Glycoprotein (SIBLING) protein family, involved in subchondral bone remodeling.101 TPX-100 provided symptomatic improvements in patellofemoral OA knees administered with 4 weekly 200 mg injections compared with placebo injection in the contralateral knees (n=93), but only 14% of knees showed changes in cartilage thickness/volume measured on MRI over 12 months with no evidence of structural modification. No drug-related SAEs occurred in this study.102 Another 2020 OARSI conference abstract reported a statistically significant decrease in pathologic bone shape change in the femur at both 6 and 12 months using 3D femoral bone shape change.103

Antiresorptive drugs have shown reduction in bone remodeling and improvement in trabecular microarchitecture and bone mineralization. In clinical trials investigating the structure-modifying effects of bisphosphonates (alendronate, risedronate, zoledronic acid), the results are inconsistent across the studies and their outcomes presented a great heterogeneity.17,104 In a recent systematic review including preclinical studies (n=26) over the past two decades (20002020), these drugs showed better chondroprotective effects at high doses with a dose-dependent manner as well as depending on the timing of treatment initiation in relation to OA stage (time-dependency).105 Therefore, these agents may still be of potential benefits in certain OA endotypes with high rates of subchondral bone turnover. This phenotype-dependency has been demonstrated in pre-clinical research, where bisphosphonates are differentially effective in reducing pain and not only bone but also cartilage pathology in OA models with high versus low bone turnover.106109 Recently, clodronate (n=74)110 and neridronate (n=64)111 have been successfully used for the treatment of knee and hand OA, with an interesting efficacy on BMLs, although the sample sizes are small. An individual patient data meta-analysis for examining their efficacy in specific knee OA subtypes is still ongoing.112

In a multicentre, randomised controlled trial involving knee OA patients with significant knee pain and MRI-detected BMLs (n = 223), 2 annual infusions with 5 mg of zoledronic acid (the most potent of all bisphosphonates) did not significantly reduce cartilage volume loss, knee pain or BML size although the study was designed for detecting effects on the bone-driven subgroup with BMLs which may likely have potential benefits from this therapy.113 It was noted that more knee replacement procedures were performed in the zoledronic acid group compared with the placebo group (9% vs 2%) in contrast with other population-based studies.114,115

Another study involving Osteoarthritis Initiative (OAI) female participants (n=346) showed that bisphosphonate therapy may be protective of radiographic knee OA progression in nonoverweight patients with earlystage OA.116 Currently, a Phase 3 study (NCT04303026) to examine its effects in hip OA is ongoing. A phase 2 study examining the effects of another anti-resorptive, denosumab, in hand OA is expected to finish in 2021 (NCT02771860).

Vitamin D has a direct impact on cartilage by inducing proteoglycan synthesis in mature chondrocytes,117 and enhances chondrocyte viability and reduces their inflammatory cytokine synthesis through activating AMPK/mTOR and autophagy.118 Active vitamin D administration reduced cartilage degradation and inflammation in models of OA in mice and rats induced by meniscal injury/meniscectomy and ACLT.118120 Out of two recently published systematic reviews, one review showed the association of vitamin D deficiency with knee OA in patients but inconsistent evidence for its role in the prevention of incidence and progression of radiographic OA,121 while the other argued that inconsistent results may be attributed to factors such as severity of knee OA, baseline level of serum vitamin D, duration of treatment, and vitamin D dosages.122 There is a need for multicentric and well-conducted randomized studies using larger samples to determine its efficacy. A small Phase 4 clinical trial is currently active (NCT04739592).

Synovial inflammation (synovitis) is an important contributing factor to the OA pathogenesis through increased local production of pro-inflammatory cytokines, chemokines, and mediators of joint tissue damage123,124 which may be amenable to a range of anti-inflammatory drugs commonly used in inflammatory rheumatic diseases. The pharmaceutical drugs in phase 2 and 3 stages of development for inflammation-driven endotype are summarized in Table 3.

Diacerein is a purified anthraquinone derivative. It involves an inhibitory action on IL-1 and its signalling pathway, possesses an anticatabolic effect on OA tissues and reduces generation of metalloproteases.125 In animal models of OA (sheep meniscectomy, canine ACLT, rabbit ACLT and partial meniscectomy) diacerein has generally shown limited long-term effect on cartilage composition or pathology, but some evidence of reducing synovitis.126129 In a 2014 Cochrane review, the authors concluded that diacerein demonstrated only a minimal symptomatic improvement in patients with unclear benefits in JSW on X-rays, compared with placebo. Diarrhoea was the main adverse event with an absolute difference of 26%.130

The EMAs Pharmacovigilance Risk Assessment Committee suspended diacerein across Europe in 2013 due to its harms overweighing benefits,131 and then re-evaluated the drug in 2014, suggesting that it remain available with restrictions to limit risks of severe diarrhoea and hepatotoxicity.132 In 2016, the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO) reported that diacerein had efficacy similar to that of NSAIDs with slower onset of action, suggesting that it might have some benefits for patients with contraindication to NSAID.133

Recently, results of a phase-3 clinical trial (NCT02688400) were reported where the authors explored the comparative efficacy and safety of diacerein vs celecoxib in patients with moderate and severe knee OA using a non-inferiority trial design [(6-months of diacerein 50 mg once daily for 1 month and twice daily thereafter (n = 187), or celecoxib 200 mg once daily (n = 193)]. Diacerein was non-inferior to celecoxib in reducing pain, stiffness, or functional limitations. The diacerein group had a higher number of emergent AEs (26.3%) compared with the celecoxib group (17.4%), mainly due to higher diarrhoea events (10.2% vs 3.7%). One patient in the diacerein group had three SAEs (abdominal pain, elevated transaminase and gamma-glutamyl transferase, collectively suggestive of hepatitis) which resolved spontaneously following drug withdrawal.134

In in vitro and in vivo preclinical studies, interleukin-1 (IL-1), tumor necrosis factor- (TNF-), IL-6, IL-15, IL-17, and IL-18 exhibit pro-inflammatory actions, leading to the initiation and progression of cartilage damage and joint inflammation. So far, IL-1 and TNF- have been the most extensively studied cytokines in pre-clinical research.135,136 Despite this favorable evidence in animal OA models, most clinical trials investigating the disease-modifying effects demonstrated by inhibitors of IL-1 and TNF- in OA patients failed to meet the primary and secondary endpoints such as in cases of Gevokizumab (XOMA-052),137 AMG108,138 Lutikizumab (ABT-981),139,140 anakinra,141 adalimumab142144 and etanercept.145 In a meta-analysis evaluating the efficacy of disease-modifying anti-rheumatic drugs in OA, neither IL1-inhibitors nor TNF-inhibitors possess symptomatic benefits irrespective of the joint site affected or the inflammatory phenotype (erosive or non-erosive OA).146

These failed trial results may suggest the implication of a more complicated interaction among various cytokines in the OA pathogenic process. One of the reasons for failure may be that the clinical trials were designed to detect an effect on symptoms rather than on joint structure, which is conversely the main outcome evaluated in preclinical studies, or that they are underpowered or have not followed participants for long enough to find meaningful structural effects such as proposed in the recent CANTOS trial.147 In a recent exploratory analysis of the CANTOS trial involving patients with elevated high-sensitivity C-reactive protein (hs-CRP) levels 2 mg/L and a history of myocardial infarction (n=10061), IL-1 inhibition using canakinumab may render a substantial reduction of THR/TKR rates as well as OA-related symptoms on an averaged 3.7 years follow-up.147 Although the study had some positives such as a large sample size and long-term follow-up, it was not primarily designed to investigate the DMOAD efficacy of canakinumab and many relevant OA outcomes were missing, necessitating further confirmatory studies.

IL-6 can increase the risk of radiographic OA and associated with knee cartilage damage,148 suggesting the potential role of low-level inflammation in the pathogenesis of OA. IL-6R blockage with tocilizumab contributes to cartilage preservation and increases bone volume in a mouse model of ischemic osteonecrosis,149 and reduced cartilage lesions, osteophyte formation and synovitis in DMM-induced OA in mice.150 However, male IL-6 knock out mice have increased cartilage damage and age-related OA.151 In local joint tissues, IL-6 classic signaling produces structure-protective effects, while trans-signaling leads to catabolic effects.152 This finding might suggest that selective inhibition of IL-6 trans-signaling could be a superior treatment strategy as this may inhibit deleterious IL-6 effects in OA, while maintaining protective IL-6 signaling via the classic pathway.153 Recently, in a phase-3 trial evaluating the efficacy of tocilizumab in hand OA for 12 weeks (n=104), it revealed no more effectiveness than placebo for pain relief (7.9 vs 9.9 on VAS score in the tocilizumab and placebo groups).154

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. It also possesses chondroprotective effects, via reduced production of matrix metalloproteases155 as well as inhibition of chondrocyte apoptosis.156 Therefore, IL-10 could have potential benefits in OA management, both for pain improvement and suppression of the cartilage-damaging processes. Currently, there is a phase-2 clinical trial evaluating the safety and efficacy of a single injection of XT-150 (a plasmid DNA with a variant of human IL-10 transgene) in patients with knee OA (NCT04124042), and it is estimated to be complete in 2022.

In this section, we briefly put forward the reasons for failures in OA clinical trials and possible steps to overcome these barriers (Figure 2).

Figure 2 Reasons for DMOAD trial failures.

The drug will be required to demonstrate symptomatic benefits (pain and/or function) coupled with structural modifications to meet regulatory requirements as a disease-modifying agent.19,20 To date, no agent has been approved by the regulatory agencies.17 Some argue that the improvements in structural change (in the absence of any meaningful symptomatic benefits) should be a meaningful target for approval, in and of itself. However, this is unlikely to meet consumers needs as their primary reason for clinical presentation relates to symptomatic complaints.30

On the other hand, OA is a slowly progressive disease and only 14% of patients with incident OA have measurable disease progression over a 1-year period (Figure 2).157 Therefore, structure-modifying effects using targeted therapy would be optimal to delay or even avoid disease worsening and joint replacement. In OA, symptom-structure discordance is often described.158 Analysis of data from the Osteoarthritis Initiative revealed that changes in bone structure over 2 years do not translate into pain worsening until 4 years,159 suggesting that a structure-modifying drug may need longer follow-up to detect symptomatic benefit. In addition, a variety of disease outcomes using different OA subtypes (genotypes, phenotypes and endotypes) are needed to demonstrate the ability of a structure-modifying drug to directly predict for symptomatic benefits to overcome the regulatory hurdles.18

In addition, FDAs formal recognition of OA as a serious disease paves the way for using surrogate outcome measures for regulatory approval of DMOADs under accelerated approval regulations. However, two challenges need to be addressed: 1) selection/qualification of appropriate surrogate outcome measures, and 2) appropriate designs for post-marketing confirmatory studies. To overcome the first challenge, the Foundation for NIH (FNIH) OA Biomarkers Consortium initiative was established.160 For addressing the second challenge, Kraus et al proposed two major study design scenarios: 1) prospective trial continuation which continue all patients on initial drug allocation into the post-marketing approval trial until a failure threshold is achieved; and 2) separate post-marketing approval study which use different study population administered with active treatment only.161

The imaging standard in OA clinical trials has been radiographically measured mJSW which is notoriously unresponsive to change as well as possessing several other drawbacks such as issues with alignment, positioning and assuming JSW as the composite contribution of changes in other structures in this heterogeneous OA with multiple-tissue involvement.162,163 Therefore, utilization of this insensitive-to-change measure may limit our opportunity to detect any modification in what oftentimes is a slow-moving disease.

In 2015 OARSI published recommendations related to the applications of knee imaging in knee OA trials to set standards and improve quality assurance.164 Although a range of different MRI approaches have been developed to evaluate changes in overall joint structure,165167 further validation studies and evaluation of their clinimetrics are required to gain acceptance by regulatory authorities as a suitable surrogate endpoint which is the focus of the FNIH OA Biomarkers Consortium.160

In addition, the emergence of approved surrogate outcomes would allow pharmaceutical companies to examine the efficacy of the DMOADs in a shorter duration of clinical trials and reduce drug development costs. In this way, there is a possibility of instituting accelerated approval based on surrogate imaging endpoints and post-marketing approval studies to prove the longitudinal benefit-to-harm profile and the durability of the potential new therapies.161

In the study design for post-marketing approval which uses observational outcomes such as time-to-event of joint replacement surgery, considerable barriers exist in terms of need for large sample sizes due to low annual incidence rates (1.611.9%),14 long study follow-ups (>5 years at least),46 and the impact of non-disease and other subjective factors on the outcome (ie, comorbidities and/or age of the patient, costs, insurance cover, etc.).168,169 There is a lack of universal consensus criteria for guiding patient recommendations regarding joint replacement surgery, leading to differences even among treatment centres within the same region. These issues need to be adequately addressed by study design.161 There is a need for developing a criteria set to define appropriateness for total knee replacement or a virtual total knee replacement.170

Instead of utilizing the systemic route of administration which may produce undesirable systemic toxicity and off-target effects, many of the agents in the development pipeline are focused on an intra-articular route for drug delivery. This can also potentially enhance the local bioavailability, thereby maximizing therapeutic effects locally in the joint with a higher safety profile compared to systemic exposure.171 On the other hand, the marked placebo effect generated by local intraarticular administration is well-documented in the literature,172 making the assessment of symptom efficacy more challenging.30

Another issue related with the intra-articular therapy is that drugs have a short residence time within the joint.171,173 To overcome this barrier, a variety of drug delivery systems were proposed to prolong drug residence time while providing a stable concentration within the therapeutic window, leading to a reduction of side effects and better patient compliance.174 It remains unclear how long particular drugs have to remain in the joint for a meaningful symptomatic relief and/or structure-modification after an intra-articular administration. An ideal drug delivery system should comply with adequate disease modification, biocompatibility, and biodegradability while responding to its physiological environment.175

In the randomized clinical trials for IA drugs, saline is commonly used as the placebo in the control group. A recent meta-analysis examining the effects of IA saline in 50 clinical trials (n=4076) revealed significant improvement of pain severity on 0100 VAS up to 6 months [13.4 (21.7/5.1)] and WOMAC function sub-score [10.1 (12.2,-8.0)]. The pooled responder rate after saline injections using the OMERACT-OARSI criteria is 48% at 3 months and 56% at 6 months,47 challenging the concept of saline being a mere placebo.176 However, there is no evidence supporting hypotheses advocating the disease-modifying role of saline injection. Future scientifically robust studies which examined the effects of sham injections compared with saline injections are required to shed new light on this issue.

The IA therapies show a considerably larger therapeutic effect after the adjustment for the effects of IA saline, suggesting an inappropriate underestimating of the true effect of the active medication.177 Further research is required to determine the underlying mechanisms and the factors influencing the placebo response and ways to overcome it. In addition, the mechanisms of pain genesis in OA are poorly understood and thought to involve a complex interaction among local pathological processes in the OA joint and neuronal mechanisms and alterations of pain processing (ie central sensitization, especially in advanced OA).178 Further studies should focus on the effects of these interactions on the outcomes in the placebo-controlled clinical trials. It is also necessary to strictly report in each clinical trial what placebo has been used as well as the presence or absence of any additional blinded clinical evaluator, even more, if considering clinical trials with intra-articular therapies.

As OA is a heterogeneous disease with a combination of different endotypes in varying degree at different stages of the disease process, a one size fits all approach using a single therapeutic agent targeting a single target within a single endotype may be unlikely to succeed in the management of OA.179 Therefore, as in the oncology therapeutic area, combinations of drugs targeting different hallmarks of OA pathogenic process should be considered. Further research examining the potential synergistic action of combining anabolic therapies with those that downregulate catabolic factors will be required.

OA is well known for marked variations of disease expression,180 involves a variety of tissue pathologies as a whole joint disease16 and presents with different pathobiological manifestations,181 suggesting the potential value of personalised and precision medicine from the treatment perspective. Personalized medicine is used for treatment focusing on the patient based on their individual clinical characterization, considering the diversity of symptoms, severity, and genetic traits.182 In precision medicine, the molecular information maximizes the accuracy with which the patients are categorized and treated, typically applying large amounts of data for identification of patient subtypes which possess sharing specific relevant characteristics to predict diagnosis, progression, or treatment response, and to utilize appropriate therapeutic targets.183 The use of precision medicine in OA remains limited.

The implementation of private/ public initiatives, such as the Osteoarthritis Initiative, the FNIH biomarkers consortium, the European APPROACH ((Applied Public-Private Research enabling OsteoArthritis Clinical Headway)) project have contributed greatly to moving the field forward. Clinical phenotypes, endotypes, and molecular and imaging biomarkers are being identified, but the exact interplay among them and underlying mechanisms of each remain to be elucidated.24 While these biomarkers may have potential benefits in detecting those patients with the greatest risk for structural progression, their use still needs to be translated into more efficient clinical trial design and widespread clinical application.184

There remains an immense unmet need for effective and safe targeted interventions to inhibit both pain and disease progression. The complex overlapping interplay among the pathobiological OA processes and heterogeneity of clinical presentations of patients with OA, call for a universally accepted classification of phenotypes and endotypes for developing targeted disease-modifying therapy and providing the appropriate treatment in clinical setting. Although challenges exist towards the eventual management of OA by applying the concepts of personalized and precision medicine, the lessons learned through failed clinical trials, the ongoing developments of more advanced imaging and sophisticated biomarkers tools and effective drug delivery systems are leading to substantial progress in our field.

WMO is supported by the Presidential Scholarship of Myanmar for his PhD course. DJH is supported by the NHMRC Investigator Grant. VD is supported by a University of Sydney Postgraduate Award scholarship.

DJH provides consulting advice on scientific advisory boards for Pfizer, Lilly, TLCBio, Novartis, Tissuegene, Biobone. CL has provided consulting advice for Merck Serono and Galapagos Pharmaceuticals, and receives research funding from numerous pharmaceutical companies (Fidia Farmaceutici, Inter-K Peptide Therapeutics Ltd, Taisho Pharmaceutical Co. Ltd, Concentric Analgesics Inc, Cynata Therapeutics, CEVA Animal Health, Regeneus) through specific services/testing contract research agreements between and managed by The University of Sydney or the NSLHD. The authors report no other conflicts of interest in this work.

1. Hawker GA. Osteoarthritis is a serious disease. Clin Exp Rheumatol. 2019;37 Suppl 120(5):36.

2. OARSI TP-cCfOPo. OARSI White Paper- OA as a Serious Disease; 2016.

3. Safiri S, Kolahi AA, Smith E, et al. Global, regional and national burden of osteoarthritis 19902017: a systematic analysis of the Global Burden of Disease Study 2017. Ann Rheum Dis. 2020;79(6):819828. doi:10.1136/annrheumdis-2019-216515

4. Neogi T. The epidemiology and impact of pain in osteoarthritis. Osteoarthritis Cartilage. 2013;21(9):11451153. doi:10.1016/j.joca.2013.03.018

5. Murphy L, Schwartz TA, Helmick CG, et al. Lifetime risk of symptomatic knee osteoarthritis. Arthritis Rheum. 2008;59(9):12071213. doi:10.1002/art.24021

6. Hootman JM, Helmick CG, Barbour KE, Theis KA, Boring MA. Updated projected prevalence of self-reported doctor-diagnosed arthritis and arthritis-attributable activity limitation among US Adults, 20152040. Arthritis Rheumatol. 2016;68(7):15821587. doi:10.1002/art.39692

7. WHO. Chronic rheumatic conditions; Published 2021. https://www.who.int/chp/topics/rheumatic/en/. Accessed June 7, 2021.

8. Osteoarthritis and Allied Disorders. In: The Burden of Musculoskeletal Diseases in the United States (BMUS). Third ed. 2014.

9. Lo J, Chan L, Flynn S. A systematic review of the incidence, prevalence, costs, and activity and work limitations of amputation, osteoarthritis, rheumatoid arthritis, back pain, multiple sclerosis, spinal cord injury, stroke, and traumatic brain injury in the United States: a 2019 Update. Arch Phys Med Rehabil. 2021;102(1):115131. doi:10.1016/j.apmr.2020.04.001

10. Puig-Junoy J, Ruiz Zamora A. Socio-economic costs of osteoarthritis: a systematic review of cost-of-illness studies. Semin Arthritis Rheum. 2015;44(5):531541. doi:10.1016/j.semarthrit.2014.10.012

11. Leyland KM, Gates LS, Sanchez-Santos MT, et al. Knee osteoarthritis and time-to all-cause mortality in six community-based cohorts: an international meta-analysis of individual participant-level data. Aging Clin Exp Res. 2021;33(3):529545. doi:10.1007/s40520-020-01762-2

12. Bannuru RR, Osani MC, Vaysbrot EE, et al. OARSI guidelines for the non-surgical management of knee, hip, and polyarticular osteoarthritis. Osteoarthritis Cartilage. 2019;27(11):15781589. doi:10.1016/j.joca.2019.06.011

13. Kolasinski SL, Neogi T, Hochberg MC, et al. 2019 American college of rheumatology/arthritis foundation guideline for the management of osteoarthritis of the hand, hip, and knee. Arthritis Care Res. 2020;72(2):149162. doi:10.1002/acr.24131

14. Weinstein AM, Rome BN, Reichmann WM, et al. Estimating the burden of total knee replacement in the United States. J Bone Joint Surg Am. 2013;95(5):385392.

15. Shane Anderson A, Loeser RF. Why is osteoarthritis an age-related disease? Best Pract Res Clin Rheumatol. 2010;24(1):1526. doi:10.1016/j.berh.2009.08.006

16. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 2012;64(6):16971707. doi:10.1002/art.34453

17. Oo WM, Yu SP, Daniel MS, Hunter DJ. Disease-modifying drugs in osteoarthritis: current understanding and future therapeutics. Expert Opin Emerg Drugs. 2018;23(4):331347. doi:10.1080/14728214.2018.1547706

18. Food and Drug Administration of the United States. Osteoarthritis: structural Endpoints for the Development of Drugs; 2018.

19. Food and Drug Administration of the United States. Draft guidance for industry: clinical development programs for drugs, devices, and biological products intended for the treatment of osteoarthritis (OA); 1999.

20. European Medicines Agency. Clinical investigation of medicinal products used in the treatment of osteoarthritis; 2010.

21. Felson DT. Identifying different osteoarthritis phenotypes through epidemiology. Osteoarthritis Cartilage. 2010;18(5):601604. doi:10.1016/j.joca.2010.01.007

22. Bierma-Zeinstra SM, Verhagen AP. Osteoarthritis subpopulations and implications for clinical trial design. Arthritis Res Ther. 2011;13(2):213. doi:10.1186/ar3299

23. Karsdal MA, Michaelis M, Ladel C, et al. Disease-modifying treatments for osteoarthritis (DMOADs) of the knee and hip: lessons learned from failures and opportunities for the future. Osteoarthritis Cartilage. 2016;24(12):20132021. doi:10.1016/j.joca.2016.07.017

24. Mobasheri A, Saarakkala S, Finnil M, Karsdal MA, Bay-Jensen A-C, van Spil WE. Recent advances in understanding the phenotypes of osteoarthritis. F1000Res. 2019;8:F1000Faculty Rev2091. doi:10.12688/f1000research.20575.1

25. DellIsola A, Allan R, Smith SL, Marreiros SS, Steultjens M. Identification of clinical phenotypes in knee osteoarthritis: a systematic review of the literature. BMC Musculoskelet Disord. 2016;17(1):425. doi:10.1186/s12891-016-1286-2

26. Van Spil WE, Kubassova O, Boesen M, Bay-Jensen AC, Mobasheri A. Osteoarthritis phenotypes and novel therapeutic targets. Biochem Pharmacol. 2019;165:4148. doi:10.1016/j.bcp.2019.02.037

27. Jameson JL, Longo DL. Precision medicine--personalized, problematic, and promising. N Engl J Med. 2015;372(23):22292234. doi:10.1056/NEJMsb1503104

28. Deveza LA, Nelson AE, Loeser RF. Phenotypes of osteoarthritis: current state and future implications. Clin Exp Rheumatol. 2019;37 Suppl 120(5):6472.

29. Mobasheri A, van Spil WE, Budd E, et al. Molecular taxonomy of osteoarthritis for patient stratification, disease management and drug development: biochemical markers associated with emerging clinical phenotypes and molecular endotypes. Curr Opin Rheumatol. 2019;31(1):8089. doi:10.1097/BOR.0000000000000567

30. Oo WM, Hunter DJ. Disease modification in osteoarthritis: are we there yet? Clin Exp Rheumatol. 2019;37 Suppl 120(5):135140.

31. Troeberg L, Nagase H. Proteases involved in cartilage matrix degradation in osteoarthritis. Biochim Biophys Acta. 2012;1824(1):133145.

32. Wang M, Sampson ER, Jin H, et al. MMP13 is a critical target gene during the progression of osteoarthritis. Arthritis Res Ther. 2013;15(1):R5R5. doi:10.1186/ar4133

33. Glasson SS, Askew R, Sheppard B, et al. Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 2005;434(7033):644648. doi:10.1038/nature03369

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Univ. of Washington and Sana researchers use gene editing to prep stem cells for heart repair – GeekWire

By daniellenierenberg

Heart muscle regeneration researchers (left to right) Naoto Muraoka, Elaheh Karbassi, and Chuck Murry. (University of Washington Photo)

Human stem cell scientists have long dreamed of repairing damaged hearts, but have been stymied by researchshowing that the cells yield irregular heartbeats in laboratory animals. A new genetic engineering approach overcomes this barrier, according to a report at the annual meeting of the International Society for Stem Cell Research by scientists at the University of Washington and Sana Biotechnology, a Seattle-based company.

A heart attack typically kills about one billion cells, said Charles Murry, director of the Institute for Stem Cell and Regenerative Medicine at the UW, who presented the data Monday. Such massive cell death can lead to downstream effects such as heart failure, an often-debilitating condition that affects about 6.2 million people in the U.S. Using stem cells to repair the damage after a heart attack has long been a goal in his lab.

One major challenge in the field is that implanting cells into the hearts of laboratory animals can nudge the whole heart into beating rapidly, a condition called engraftment arrhythmia, said Murry, who is also a senior vice president and head of cardiometabolic cell therapy at Sana, which went public earlier this year.

This engraftment arrhythmia, where the heart races too quickly, has been one of the major hurdles weve been trying to overcome en route to clinical trials, said Murry in a press release.

In their study, Murry and his colleagues quelled engraftment arrhythmia using a genetic engineering strategy in cells implanted into pig hearts. Their next step is to see if the cells can repair heart damage in macaques if those studies work, the researchers will initiate clinical trials in people, he said.

To quell the arrhythmia, Murry and his colleagues turned to CRISPR, the Nobel Prize-winning technique to knock out genes. They knocked out three genes in stem cells encoding different ion channels, molecules embedded in the cell membrane that mediate impulses that propagate heart beats. They also added DNA for another ion channel, KCNJ2, which mediates the movement of potassium across the membrane, Its a chill out channel, Murry told GeekWire, It tells the heart cell not to be so excitable.

The engineered stem cells, derived from human embryonic stem cells, were coaxed in a petri dish to produce heart muscle cells, which were then implanted into pigs via open heart surgery or a catheter. The result was an even heartbeat the genetically altered cells did not cause engraftment arrhythmia.

The researchers landed on this strategy after years of effort, assessing which channels were present in the cells during arrhythmia, and knocking out multiple types of channels until they hit the right combination.

In their next set of experiments in macaques, We want to make sure these cells are still effective, said Murry, They look good beating in culture, so I think they are going to be OK. Moving forward, the researchers will also use induced human pluripotent stem cells, obtainable from adults and more amenable longer-term for clinical use.

In another recent study, published in Cell Systems, scientists at the Allen Institute for Cell Science took a close look at cardiac muscle cells derived from stem cells. They found that they could classify the state of the cells, such as how mature they were, by assessing both cell structure and which genes were turned on.

This paints a broader picture of our cells. If someone wants to really understand and characterize a cells state, we found that having both of these types of information can be complementary, said Kaytlyn Gerbin, a scientist at the Allen Institute for Cell Science in a statement. The findings provide a fine-tooth analysis of cell state, which may guide future experiments on cardiac muscle and other cell types.

Murrys research was conducted primarily at the UW, with financial support from Sana. In addition to its cardiac program, Sana has cell and gene therapy programs in diabetes, blood disorders, immunotherapy and other areas.

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Autologous Stem Cell Based Therapies Market to Eyewitness Huge Growth by 2027 with Covid-19 Impact The Manomet Current – The Manomet Current

By daniellenierenberg

This Autologous Stem Cell Based Therapies market report provides vital info on survey data and the present market place situation of each sector. The purview of this Autologous Stem Cell Based Therapies market report is also expected to involve detailed pricing, profits, main market players, and trading price for a specific business district, along with the market constraints. This anticipated market research will benefit enterprises in making better judgments.

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This type of comprehensive and specialized market investigation also ponders the effect of these modernizations on the markets future development. Several innovative businesses are bouncing up in the business that are executing original innovations, unique approaches, and forthcoming contracts in order to govern the worldwide market and build their footprint. It is clear that market participants are making progress to combine the most cutting-edge technology in order to stay competitive. This is achievable since innovative products are introduced into the market on a frequent basis. The range of this Autologous Stem Cell Based Therapies market report extends outside market settings to comprise analogous pricing, gains, vital players, and market value for a major market areas. This foreseeable marketing plan will help firms make more up-to-date decisions.

Key global participants in the Autologous Stem Cell Based Therapies market include:Med cell Europe US STEM CELL, INC. Tigenix Mesoblast Pluristem Therapeutics Inc Brainstorm Cell Therapeutics Regeneus

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Segmentation on the Basis of Application:Neurodegenerative Disorders Autoimmune Diseases Cardiovascular Diseases

Market Segments by TypeEmbryonic Stem Cell Resident Cardiac Stem Cells Umbilical Cord Blood Stem Cells

Table of Content1 Report Overview1.1 Product Definition and Scope1.2 PEST (Political, Economic, Social and Technological) Analysis of Autologous Stem Cell Based Therapies Market2 Market Trends and Competitive Landscape3 Segmentation of Autologous Stem Cell Based Therapies Market by Types4 Segmentation of Autologous Stem Cell Based Therapies Market by End-Users5 Market Analysis by Major Regions6 Product Commodity of Autologous Stem Cell Based Therapies Market in Major Countries7 North America Autologous Stem Cell Based Therapies Landscape Analysis8 Europe Autologous Stem Cell Based Therapies Landscape Analysis9 Asia Pacific Autologous Stem Cell Based Therapies Landscape Analysis10 Latin America, Middle East & Africa Autologous Stem Cell Based Therapies Landscape Analysis 11 Major Players Profile

This market study also includes a geographical analysis of the world market, which includes North America, Europe, Asia Pacific, the Middle East, and Africa, as well as several other important regions that dominate the world market. The Market study highlights some of the most important resources that can assist in achieving high profits in the firm. This Autologous Stem Cell Based Therapies market report also identifies market opportunities, which will aid stakeholders in making investments in the competitive landscape and a few product launches by industry players at the regional, global, and company levels. As numerous successful ways are offered in the study, it becomes possible to expand your firm. By referring to this one-of-a-kind market study, one can achieve business stability. With the help of this Market Research Study, you may achieve crucial positions in the whole market. It does a thorough market analysis for the forecast period of 2021-2027.

Autologous Stem Cell Based Therapies Market Intended Audience: Autologous Stem Cell Based Therapies manufacturers Autologous Stem Cell Based Therapies traders, distributors, and suppliers Autologous Stem Cell Based Therapies industry associations Product managers, Autologous Stem Cell Based Therapies industry administrator, C-level executives of the industries Market Research and consulting firms

This comprehensive Autologous Stem Cell Based Therapies market report offers a practical perspective to the current market situation. It also compiles pertinent data that will undoubtedly aid readers in comprehending particular aspects and their interactions in the current market environment. The material offered in this Market research report is discussed in detail on numerous levels, including technological advancements, effective methods, and market penetration factors. The reports recommendations are mostly employed by existing industry participants. It provides sufficient statistical data to comprehend its operation. It also outlines the changes that must be made in order for current businesses to grow and adapt to market developments in the future.

About Global Market MonitorGlobal Market Monitor is a professional modern consulting company, engaged in three major business categories such as market research services, business advisory, technology consulting.We always maintain the win-win spirit, reliable quality and the vision of keeping pace with The Times, to help enterprises achieve revenue growth, cost reduction, and efficiency improvement, and significantly avoid operational risks, to achieve lean growth. Global Market Monitor has provided professional market research, investment consulting, and competitive intelligence services to thousands of organizations, including start-ups, government agencies, banks, research institutes, industry associations, consulting firms, and investment firms.ContactGlobal Market MonitorOne Pierrepont Plaza, 300 Cadman Plaza W, Brooklyn,NY 11201, USAName: Rebecca HallPhone: + 1 (347) 467 7721Email: info@globalmarketmonitor.comWeb Site: https://www.globalmarketmonitor.com

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Autologous Stem Cell Based Therapies Market to Eyewitness Huge Growth by 2027 with Covid-19 Impact The Manomet Current - The Manomet Current

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Stopping, blocking and dampening how Aussie drugs in the pipeline could treat COVID-19 – The Conversation AU

By daniellenierenberg

While widespread vaccination is key in our fight against COVID-19, people who are infected still need better treatment to improve their chance of survival and making a full recovery.

Early on, the world had high hopes for a range of repurposed medications which had previously been approved to treat other conditions including hydroxychloroquine, remdesivir and ivermectin to treat COVID-19. But the results have been disappointing.

Diseases caused by viruses are among the most difficult to treat, due to their ability to invade and repurpose infected cells. This limits the ability for drugs to directly act on the virus.

Read more: Developing antiviral drugs is not easy here's why

Yet researchers around the world are finding ways to overcome these barriers and directly target the coronavirus, including in Australia. So whats being developed here and how do they work?

Researchers at Queenslands Menzies Health Institute, in collaboration with scientists from the United States, have developed a novel treatment which targets key genes of the coronavirus, stopping the viruss ability to replicate.

The treatment uses an engineered particle called a small interfering RNA (si-RNA), which detects and binds to areas of the hosts genome, where the virus resides.

The si-RNA is encased in a nanoparticle to protect it as it travels through the bloodstream. It enters all cells in the hosts body, but will only act on the cells infected by the virus.

Read more: Have Australian researchers developed an effective COVID-19 treatment? Potentially, but we need to wait for human trials

Studies in mice showed the treatment reduced the amount of the virus in the lungs by more than 90%.

Its unclear if the results will translate to humans, but if they do, it could potentially protect infected people from severe disease and make them less likely to transmit the illness to others.

If it is successful, the researchers estimate the treatment could be available in 2023.

Another strategy is to block the virus from invading all together.

A number of research teams across Australia are working on engineered antibody treatments, which hunt out and bind to the virus before it enters a cell, effectively blocking it out.

Researchers at the Garvan and Kirby institutes in New South Wales are building on research developed after the 2003 SARS outbreak to create treatments using monoclonal antibodies. These antibodies are generated in the lab and mimic the immune system response to infection.

Once these monoclonal antibodies are injected into an infected person, they bind to the virus and stop it from invading host cells. They also mark it for destruction by the other immune cells.

While this research is in the pre-clinical (lab testing) phase, the researchers at Garvan are already working with clinicians at the Kirby institute to identify the best antibodies and move them through to human clinical trials.

As monoclonal antibody treatments are widely used in a range of diseases, these could potentially be deployed quickly for patients with COVID-19, or to protect people who have been exposed to the virus, to stop them getting sick and becoming infectious.

Another team at the Walter and Elizabeth Hall Institute in Melbourne is harnessing unique nanobodies, which are significantly smaller than human antibodies, derived from the immune system of alpacas.

These nanobodies have powerful and specific binding capacity. By vaccinating the alpacas with a synthetic component of the SARS-CoV-2 virus, nanobodies targeting the virus can be identified and synthesised for human use.

While these treatments are in the very early stages of development, they could prove revolutionary for all kinds of infectious and non-infectious diseases.

While some treatments aim to neutralise the virus, others are being developed to protect patients from the consequences of COVID-19.

One of the most severe reactions to an infection with the coronavirus is a widespread inflammatory reaction known as a cytokine storm, causing severe damage to the lungs.

While potent anti-inflammatory drugs such as hydrocortisone can help to prevent this response, they also can have severe side-effects such as bone weakening, immune system weakening, psychiatric symptoms and insomnia.

Researchers at the Victor Chang Cardiac Institute and St Vincents hospital in Sydney are proposing to trial a novel stem cell therapy, in an attempt to counteract this inflammatory storm.

While they havent disclosed specifically which cells they are planning to use, human studies show stem cell treatments can suppress inflammatory responses from the immune system.

The researchers are seeking approval for clinical studies and are using a stem cell that has been used in humans previously potentially speeding the pathway to clinical use.

Read more: Could a simple pill beat COVID-19? Pfizer is giving it a go

Another anti-inflammatory drug to control the damaging levels of immune response to the viral infection is being developed by Implicit Bioscience.

Its drug has already shown promising preliminary results in small trials for acute lung injury and amyotrophic lateral sclerosis (AML, a rare neurological disease also known as motor neurone disease), with phase 2 trials for AML due to be completed in 2021.

Two trials at major medical centres, one led by the US National Institutes of Health and the other by Quantum Leap Healthcare Collaborative, are now underway to test whether the drug is effective in patients with severe COVID-19.

Australian biotech company Ena Respiratory is developing a nasal spray to fight COVID-19.

These nasal sprays contain a compound designed to trigger a rapid immune response in the upper airways. This allows the immune system to destroy the virus and infected cells before serious disease can occur.

Ena Respiratorys product, called INNA-051, has produced promising results in animal models, with up to a 96% decrease in SARS-CoV-2 virus replication.

The next step is to see if these results translate to humans.

Australia has a long history of strong performance on the world stage in research. Fortunately, this has continued through the COVID-19 pandemic, with a number of key developments and innovations as described, which show promise for translation to human clinical trials.

Developments are continuing, including research by Vasso and her team into novel re-purposed and experimental drugs aimed at stopping coronavirus replication. This is a collaboration between Victoria University and researchers from the United States and Greece, and the team hopes to be able to report on its progress soon.

Read more: I'm a lung doctor testing the blood plasma from COVID-19 survivors as a treatment for the sick a century-old idea that could be a fast track to treatment

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Stopping, blocking and dampening how Aussie drugs in the pipeline could treat COVID-19 - The Conversation AU

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Taking on Harmful Cells That Contribute to Age-Related Diseases – Tufts Now

By daniellenierenberg

Its not the fountain of youth, but a fast-emerging class of drugs could bring us closer to achieving the age-old quest for longer life, better health, and greater vitality.

The drugs, called senolytics, carry out search-and-destroy missions against senescent cells, which are linked to aging. Early in life, senescent cells support crucial functions such as embryonic tissue development and later wound repair. They also send signals that cause women to go into labor and initiate live birth.

But senescent cells stop dividing over timethat is how they function. They accumulate in the body and release harmful molecules that contribute to arthritis, osteoporosis, glaucoma, Parkinsons disease, Alzheimers disease, and many other age-related conditions and afflictions. They were recently shown to be a major mediator of fatalities in coronavirus-infected mice, possibly explaining the increased susceptibility of older people to COVID-19.

To find out more about senolytics and their potential to prolong both the quality and length of life, Tufts Now talked with Christopher Wiley, a researcher on the Basic Biology of Aging Team at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts.

Tufts Now: What is cellular agingor senescenceand how does it contribute to aging?

Chris Wiley: Senescent cells are those that have been dividing, but stop doing so and go into permanent lockdown. If the cells are stem cells or other forms of progenitor cells, they are not able to contribute in a meaningful, positive way to that tissue ever again. If you have too many of these cells, you can easily imagine a situation in which your body is unable to regenerate after illness or injury.

The more problematic part of senescence is that these cells dont just sit there after their positive contributions are over. Instead, they release a blend of factors called the senescence-associated secretory phenotype, or SASP. This is a combination of molecules that can cause disease by promoting inflammation and disrupting the environment around the cell.

Senescent cells show up in virtually every vertebrate, from fish to humans. If you live long enough, they appear in nearly every tissue in the body. We cant keep up with the number of diseases that they seem to drive. Its almost as if theres a new one discovered every other month.

Can you provide some examples of senolytics?

One of the first that was discovered is fisetin, a flavonoid found in strawberries, apples, onions, and cucumbers. Flavonoids are compounds, often found in plants, that have many properties. For example, vitamin B2, or riboflavin, is a well-known dietary flavonoid.

Fisetin is one of the most prolific senolytics tested on mice so far, and has even entered clinical trials in humans. But this is not something where you eat a couple of strawberries every day and get a dose that would kill senescent cells. Youd have to consume an extremely large number, which no one should try. It is currently being sold to the public as a dietary supplement.

Another senolytic, quercetin, is the most abundant flavonoid in food. It is found in green tea, coffee, various berries, apples, onions, broccoli, grapes, citrus fruits, and red wine. Like fisetin, it is available as a dietary supplement.

What have studies shown about the effects of senolytics?

Studies in mice suggest that by destroying senescent cells, senolytics extended life by as much as 27 percent, which is pretty considerable. I want to be careful about extrapolating, but for illustrative purposes, life expectancy in the US before COVID was almost 79 years. If the mouse results were to apply in humans, that would boost life expectancy to 100 years.

Its not just that the mice lived longer, since if they were unhealthy, that wouldnt be good. Encouragingly, results from senolytic studies include better cardiac function, less dementia, fewer cataracts, and reduced muscle loss.

Early studies with human volunteers, which are designed to first test for safety, offer grounds for optimism. In one three-week trial, 14 patients with pulmonary fibrosis walked further, faster, and rose more quickly from their chairs after receiving a handful of doses of senolytics. I want to be cautious and note that there was not a control group for this early-stage study, the participants took additional medications, and many aspects of the disease did not improve.

The field is undergoing explosive growth, with as many as 100 companies exploring senolytics. Academic researchers are just as active. For example, theres a clinical trial for senolytics with diabetic kidney disease and another for addressing frailty. There are many others. The FDA process emphasizes drugs for specific diseases, so researchers are testing senolytics for individual conditions, even if they might have broader implications for aging.

I presume we shouldnt leap to the conclusion that these are miracle drugs. What caution would you offer about their efficacy and possible side effects?

Senolytics are only now being tested on humans, and while their effect on mice is often dramatic, we know that results from mice dont always translate to humans. Were also at the earliest stages of understanding efficacy, which will likely take years. There are at least 20 clinical trials taking place right now.

To date, side effects of senolytics have been things such as cough, shortness of breath, and gastrointestinal discomfort or heartburn. As we develop new senolytics, we should be able to improve both the efficiency of senescent cell elimination and the incidence of side effects.

Should people be taking these supplements based on these early findings?

Im a researcher, not a health-care provider, and people should consult their health-care provider before taking supplements.

Heres what I think: People should not look at early positive test results from studies in mice and start taking senolytic supplements. First, supplements are poorly regulated. At the basic level, there is no guarantee that youre going to get what it says on the bottle.

Second, you dont know what else has been added to the supplement.

Third, even if something works in mice, it is far from certain that it will work in humans.

Fourth, taking supplements may be harmful in some cases. If you take a senolytic supplement and have surgery, or a wound, senolytics could weaken the capacity of the body to respond properly.

And in light of the importance of senescent cells in embryo formulation, most definitely dont take them if you are or could be pregnant. This field is in its infancy; we have so much more work to do with safety and efficacy.

What does your senolytic research focus on?

There is a specific fatty acid made in small amounts in the body called dihomo-gamma-linoleic acid or DGLA. Its also present in tiny amounts in the diet. When I gave aged mice larger amounts of DGLA, they went from having quite a few senescent cells to having significantly fewer.

This presents a new therapeutic target. I identified a candidate compound using the DGLA metabolic pathway that works at a dose that is over 1,000 times lower than fisetin, so you can imagine were quite excited by these results.

Like many biomedical discoveries, it was accidental. DGLA makes anti-inflammatory lipids, which help alleviate conditions such as rheumatoid arthritis. I was studying this aspect of DGLA when I was surprised to discover that it killed senescent cells.

My work is in its very early stages, and weve only studied a small number of mice, so its too early for even tentative conclusions, although Im obviously pleased that weve seen the elimination of a meaningful number of senescent cells in old mice. Well be closely monitoring DGLAs positive effects as well as any negative effects on the mice.

How would DGLA be given to people?

We are several years away from that, because everything has to be perfect with mice before we even think about trials with people.

First, we have to figure out how DGLA is killing senescent cells in mice. Again, not all studies with mice yield similar results in humans, so we are very careful about how we convey our findings and possible future actions.

But being at the HNRCA, I have met USDA researchers and nutrition scientists, and discovered that some of those folks were developing DGLA-enriched soybeans. In one scenario, you might go out for sushi and get a little bowl of DGLA-enriched edamame as a side. By the time youre done eating, youve helped reduce the odds of getting some age-related pathology.

I dont know if it will play out that way, but its an idea were working toward. I also am working on therapies that elevate the amount of naturally occurring DGLA in senescent cells that I am very excited about, so this would be an alternative approach.

You are also studying ways to test senolytic therapies beyond such measures as improvement in distance walked, right?

Yes, I am developing a quick and easy test to tell if senolytic therapy is working. Testing for senolytic effectiveness is not really being done nowyou just look for improvement in symptoms or functioning and essentially conclude that its due to the therapy.

But we cant say that with full confidence. Currently, researchers obtain skin or fat samples from patients in these trials before and after senolytic treatment to look for senescent cells. But this is an invasive procedure and its especially challenging for older people to undergo this testing.

One way to solve this dilemma is to identify a biomarker, a measurable compound that consistently and reliably can confirm an interventions effectiveness. For example, we know that a certain lipid, dihomo-15d-PGJ2, accumulates in large amounts inside of senescent cells.

When we give a senolytic therapy that kills these cells in mice or human cells, this lipid is liberated. Detecting it in blood and urine is far less invasive, so thats what Im working on now. Our aim is to be able to test people receiving senolytic therapy for the presence of dihomo-15d-PGJ2 in their blood and urine by the end of the summer.

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Novo Nordisk partners with Heartseed on heart failure cell therapy – PMLiVE

By daniellenierenberg

Danish pharma company Novo Nordisk has announced a new collaboration and licence agreement with Japans Heartseed to develop the companys investigational cell therapy HS-001 for heart failure.

HS-001, Heartseeds lead asset, is an investigational cell therapy using purified cardiomyocytes derived from induced pluripotent stem cells (iPSC). The therapy is currently being developed as a treatment for heart failure.

Heartseed is already planning to launch a phase 1/2 study of HS-001 in Japan in the second half of 2021, which will evaluate the safety and efficacy of the therapy for the treatment of heart failure caused by ischaemic heart disease.

Under the terms of their agreement, Novo Nordisk will gain exclusive rights to develop, manufacture and commercialise HS-001 globally, excluding Japan where Heartseed will retain the rights to solely develop the therapy.

However, Novo Nordisk has the rights to co-commercialise HS-001 with Heartseed in Japan, with equal profit and cost sharing.

In return, Heartseed is eligible to receive up to a total $598m, with $55m earmarked in upfront and near-term milestone payments.

The Japanese biotech company is also eligible to receive tiered high single-digit to low double-digit royalties of annual net sales on the product outside Japan.

"We are delighted to have a company with the expertise and resources of Novo Nordisk as our partner for development and commercialisation of HS-001, and are also honoured that Novo Nordisk has recognised the innovativeness and high potential of our technology," said Keiichi Fukuda, chief executive officer of Heartseed.

"We believe that the partnership with Novo Nordisk is very valuable as we seek to disseminate our Japan-origin innovation globally as early as possible, he added.

Through this important collaboration with Heartseed, we aim to pioneer novel treatment solutions for people with cardiovascular disease, said Marcus Schindler, chief scientific officer, EVP research and early development at Novo Nordisk.

We [will] gain access to an innovative clinical asset, underlying technology and deep expertise within the field of iPSC biology and cardiac cell transplantation, which can be combined with our knowledge and capabilities in stem cell biology and manufacturing, he added.

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Global Cardiovascular Drug Delivery Markets Report 2021: Cell and Gene Therapies, Including Antisense and RNA Interference are Described in Detail -…

By daniellenierenberg

DUBLIN, May 21, 2021 /PRNewswire/ -- The "Cardiovascular Drug Delivery - Technologies, Markets & Companies" report from Jain PharmaBiotech has been added to ResearchAndMarkets.com's offering.

The cardiovascular drug delivery markets are estimated for the years 2018 to 2028 on the basis of epidemiology and total markets for cardiovascular therapeutics.

The estimates take into consideration the anticipated advances and availability of various technologies, particularly drug delivery devices in the future. Markets for drug-eluting stents are calculated separately. The role of drug delivery in developing cardiovascular markets is defined and unmet needs in cardiovascular drug delivery technologies are identified.

Drug delivery to the cardiovascular system is approached at three levels: (1) routes of drug delivery; (2) formulations; and finally (3) applications to various diseases.

Formulations for drug delivery to the cardiovascular system range from controlled release preparations to delivery of proteins and peptides. Cell and gene therapies, including antisense and RNA interference, are described in full chapters as they are the most innovative methods of delivery of therapeutics. Various methods of improving the systemic administration of drugs for cardiovascular disorders are described including the use of nanotechnology.

Cell-selective targeted drug delivery has emerged as one of the most significant areas of biomedical engineering research, to optimize the therapeutic efficacy of a drug by strictly localizing its pharmacological activity to a pathophysiologically relevant tissue system. These concepts have been applied to targeted drug delivery to the cardiovascular system. Devices for drug delivery to the cardiovascular system are also described.

The role of drug delivery in various cardiovascular disorders such as myocardial ischemia, hypertension, and hypercholesterolemia is discussed. Cardioprotection is also discussed. Some of the preparations and technologies are also applicable to peripheral arterial diseases. Controlled release systems are based on chronopharmacology, which deals with the effects of circadian biological rhythms on drug actions. A full chapter is devoted to drug-eluting stents as treatment for restenosis following stenting of coronary arteries.Fifteen companies are involved in drug-eluting stents.

New cell-based therapeutic strategies are being developed in response to the shortcomings of available treatments for heart disease. Potential repair by cell grafting or mobilizing endogenous cells holds particular attraction in heart disease, where the meager capacity for cardiomyocyte proliferation likely contributes to the irreversibility of heart failure.

Cell therapy approaches include attempts to reinitiate cardiomyocyte proliferation in the adult, conversion of fibroblasts to contractile myocytes, conversion of bone marrow stem cells into cardiomyocytes, and transplantation of myocytes or other cells into injured myocardium.

Advances in the molecular pathophysiology of cardiovascular diseases have brought gene therapy within the realm of possibility as a novel approach to the treatment of these diseases. It is hoped that gene therapy will be less expensive and affordable because the techniques involved are simpler than those involved in cardiac bypass surgery, heart transplantation and stent implantation.

Gene therapy would be a more physiologic approach to deliver vasoprotective molecules to the site of vascular lesions. Gene therapy is not only a sophisticated method of drug delivery; it may at times need drug delivery devices such as catheters for transfer of genes to various parts of the cardiovascular system.

Selected 83 companies that either develop technologies for drug delivery to the cardiovascular system or products using these technologies are profiled and 80 collaborations between companies are tabulated. The bibliography includes 200 selected references from recent literature on this topic.

Key Markets

Key Topics Covered:

Executive Summary

1. Cardiovascular Diseases

2. Methods for Drug Delivery to the Cardiovascular System

3. Cell Therapy for Cardiovascular Disorders

4. Gene Therapy for Cardiovascular Disorders

5. Drug-Eluting Stents

6. Markets for Cardiovascular Drug Delivery

7. Companies involved in Cardiovascular Drug Delivery

8. References

For more information about this report visit https://www.researchandmarkets.com/r/qqxmpd

Media Contact:

Research and Markets Laura Wood, Senior Manager [emailprotected]

For E.S.T Office Hours Call +1-917-300-0470 For U.S./CAN Toll Free Call +1-800-526-8630 For GMT Office Hours Call +353-1-416-8900

U.S. Fax: 646-607-1907 Fax (outside U.S.): +353-1-481-1716

SOURCE Research and Markets

http://www.researchandmarkets.com

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Global Cell Therapy Markets, Technologies, and Competitive Landscape Report 2020-2030: Applications, Cardiovascular Disorders, Cancer, Neurological…

By daniellenierenberg

DUBLIN, May 21, 2021 /PRNewswire/ -- The "Cell Therapy - Technologies, Markets and Companies" report from Jain PharmaBiotech has been added to ResearchAndMarkets.com's offering.

This report describes and evaluates cell therapy technologies and methods, which have already started to play an important role in the practice of medicine. Hematopoietic stem cell transplantation is replacing the old fashioned bone marrow transplants. The role of cells in drug discovery is also described. Cell therapy is bound to become a part of medical practice.

The cell-based markets was analyzed for 2020, and projected to 2030. The markets are analyzed according to therapeutic categories, technologies and geographical areas. The largest expansion will be in diseases of the central nervous system, cancer and cardiovascular disorders. Skin and soft tissue repair, as well as diabetes mellitus, will be other major markets.

The number of companies involved in cell therapy has increased remarkably during the past few years. More than 500 companies have been identified to be involved in cell therapy and 316 of these are profiled in part II of the report along with tabulation of 306 alliances. Of these companies, 171 are involved in stem cells.

Profiles of 73 academic institutions in the US involved in cell therapy are also included in part II along with their commercial collaborations. The text is supplemented with 67 Tables and 26 Figures. The bibliography contains 1,200 selected references, which are cited in the text.

Stem cells are discussed in detail in one chapter. Some light is thrown on the current controversy of embryonic sources of stem cells and comparison with adult sources. Other sources of stem cells such as the placenta, cord blood and fat removed by liposuction are also discussed. Stem cells can also be genetically modified prior to transplantation.

Cell therapy technologies overlap with those of gene therapy, cancer vaccines, drug delivery, tissue engineering, and regenerative medicine. Pharmaceutical applications of stem cells including those in drug discovery are also described. Various types of cells used, methods of preparation and culture, encapsulation, and genetic engineering of cells are discussed. Sources of cells, both human and animal (xenotransplantation) are discussed. Methods of delivery of cell therapy range from injections to surgical implantation using special devices.

Cell therapy has applications in a large number of disorders. The most important are diseases of the nervous system and cancer which are the topics for separate chapters. Other applications include cardiac disorders (myocardial infarction and heart failure), diabetes mellitus, diseases of bones and joints, genetic disorders, and wounds of the skin and soft tissues.

Regulatory and ethical issues involving cell therapy are important and are discussed. The current political debate on the use of stem cells from embryonic sources (hESCs) is also presented. Safety is an essential consideration of any new therapy and regulations for cell therapy are those for biological preparations.

Key Topics Covered:

Part One: Technologies, Ethics & Regulations

Executive Summary

1. Introduction to Cell Therapy

2. Cell Therapy Technologies

3. Stem Cells

4. Clinical Applications of Cell Therapy

5. Cell Therapy for Cardiovascular Disorders

6. Cell Therapy for Cancer

7. Cell Therapy for Neurological Disorders

8. Ethical, Legal and Political Aspects of Cell therapy

9. Safety and Regulatory Aspects of Cell Therapy

Part II: Markets, Companies & Academic Institutions

10. Markets and Future Prospects for Cell Therapy

11. Companies Involved in Cell Therapy

12. Academic Institutions

13. References

For more information about this report visit https://www.researchandmarkets.com/r/oletip

Media Contact:

Research and Markets Laura Wood, Senior Manager [emailprotected]

For E.S.T Office Hours Call +1-917-300-0470 For U.S./CAN Toll Free Call +1-800-526-8630 For GMT Office Hours Call +353-1-416-8900

U.S. Fax: 646-607-1907 Fax (outside U.S.): +353-1-481-1716

SOURCE Research and Markets

http://www.researchandmarkets.com

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Global Cell Therapy Markets, Technologies, and Competitive Landscape Report 2020-2030: Applications, Cardiovascular Disorders, Cancer, Neurological...

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Obesity-Related Inflammation and Endothelial Dysfunction in COVID-19: | JIR – Dove Medical Press

By daniellenierenberg

Obesity, COVID-19 and Inflammation

The coronavirus disease 2019 (COVID-19) pandemic has put into evidence another pandemic obesity, an increasing threat to societies around the world.1 The first studies of COVID-19 did not provide body mass index (BMI) data,2 and the association between disease severity and obesity was not perceived initially. Subsequent data from several countries, however, cast light on this association,3,4 and several studies have documented the association between obesity and COVID-19 severity.47 Currently, obesity may be considered a true independent risk factor for COVID-19 mortality.8

The mechanisms underlying the increased risk of complications and mortality in obese patients with COVID-19 are many, and of diverse nature (Figure 1). Obesity is associated with several disorders, related to defective homeostasis of the dysfunctional adipose tissue, in which local and systemic chronic inflammation, oxidative stress, altered release of cytokines, and impaired immune response play important roles911; all of these have been demonstrated to be associated with higher risk and worse prognosis of infectious diseases in this patient population.1214

Figure 1 The mechanisms underlying the increased risk of complications and mortality in obese patients with COVID-19 based on the association of low-grade inflammation, adipose tissue dysfunction and endothelial dysfunction: In obese patients with COVID-19 or SARS-CoV-2, as well as, the bacterial endotoxins (LPS) of the intestinal bacterial translocation promote the activation of TLR4 in favor of the MyD88-dependent pro-inflammatory pathway. The activation of NF-B is linked to the production of TNF-, IL-1, IL-6, IL-12 and other cytokines, contributing to the activation of NLRP3 inflammasomes and increased expression of ECA2. In the adipose tissue of patients with COVID-19, there is an increase in the expression of ECA2, promoting greater entry of SARS-CoV-2, making this tissue a viral reservoir. Metabolic inflammation in obese patients is characterized by dysfunctional adipose tissue, with mitochondrial dysfunction and decreased fatty acid oxidation, causing an amount of inflammatory cells showing an increase in the influx of M1 macrophages and chemotactic signaling, via MCP-1 and release of IL-8 by adipocytes, associated with an increase in reactive oxygen species. Associated with this process of immune activation, obese patients with COVID-19 have systemic microvascular dysfunction and a predisposition to thrombus formation that is exacerbated by higher levels of circulating inflammatory cytokines, such as TNF-, IL-1 and IL-6, worsening the outcomes in COVID-19.

Inflammation plays a central role in obesity.15 Obesity promotes profound changes in the structure and function of adipose tissue, as adipocytes undergo hypertrophy and hyperplasia, increasing oxygen need, which remains unmet due to the insufficient vascularization relative to the enlarged adipose tissue. This leads to tissue hypoxia and immune cell infiltration that perpetuates local inflammation.1618 Insulin resistance is also a link between obesity-related metabolic disorders and inflammation, as the remodeling of the adipose tissue leads to activation of NLRP3-inflammasome, which ultimately impairs of the insulin-signaling pathway and insulin resistance, a key factor in the development of the metabolic syndrome.19

Additionally, mitochondrial dysfunction in adipocytes may be a cause of adipose tissue inflammation and insulin resistance. The defective mitochondrial function and decreased fatty acid oxidation in adipocytes increase triglyceride accumulation, adipocyte enlargement and consequent adipose tissue hypoxia; this, in its turn, leads to accumulation of hypoxia-inducible factor-1 (HIF-1), which promotes adipose tissue inflammation and fibrosis.20 This continuous inflammatory cycle also contributes to a neuro-immuno-endocrine dysregulation in the context of the metabolic syndrome.21 The inflammatory state affecting obese individuals is called metabolic inflammation or metainflammation, in which there is also an increased influx of M1 macrophages occurring, as well as decreased M2 macrophages and Treg cells in the visceral adipose tissue22 through chemotactic signaling, via MCP-1 and IL-8 released by adipocytes.23

The excessive intake of carbohydrates is an important trigger for these processes.24 In addition, peripheral inflammation and various pro-inflammatory signals in the nucleus accumbens, including reactive gliosis, increased expression of cytokines, antigen-presenting markers and transcriptional activity of NFB25 contribute to the activation of the innate immune response, mainly through activation of Toll-type receptors (TLR), specifically TLR-4, considered an intersection of dysfunctional metabolism and activated immunity in obesity.26 NF-B is a molecular hub for pro-inflammatory gene induction both in innate and adaptive immune responses since it is highly regulated and regulates the expression of a vast array of genes.27 Among many different immune effects, NF-B activation is linked to the production of TNF-, IL-1, IL-6, IL-12 and other cytokines, and is also involved in NLRP3 inflammasome regulation and activation of CD4+ T-helper cells.28 It is noteworthy that there is evidence that the virus can bind and activate TLR4 signaling in favor of the proinflammatory MyD88-dependent and contributing to increased expression of ACE2 and promoting greater viral entry.29

The chronic impairment of systemic vascular endothelial function in patients with cardiovascular and metabolic disorders, including hypertension, obesity, diabetes mellitus, coronary artery disease and heart failure, when intensified by the detrimental effects of the severe acute respiratory syndrome coronavirus (SARS-CoV-2) over the endothelium, may explain their worse outcomes in COVID-19.3033 Regarding obesity, a community-based clinical trial (n=521; mean follow-up of 8.5 years) showed that increases in weight, body mass index, waist circumference and body-fat percentage over time were associated with worsening of microvascular endothelial function, assessed by flow-mediated dilation in the brachial artery.34 Most subjects (84%) were overweight or obese at baseline; those who lost weight over time had improved vascular endothelial function.34

In fact, vascular endothelial dysfunction and increased arterial stiffness are thought to contribute to a unfavorable response of the endothelium to the infection by SARS-CoV-2, whereas alterations in cardiac structure and function and the prothrombotic environment in obesity could provide a link for the augmented cardiovascular events in these patients.35 Moreover, fast increasing evidence from basic science, imaging and clinical observations suggest that COVID-19 could be considered as a vascular disease.36,37

Obesity is accompanied by functional and structural systemic microvascular dysfunction,38 and endothelial-dependent microvascular vasodilation is severely impaired in obesity.3941 Endothelial-dependent capillary recruitment, induced either by reactive hyperemia or by shear stress, is blunted in obese subjects, compared to non-obese counterparts.42,43 In the clinical setting, endothelial function and reactivity can be assessed using different technologies that evaluate microvascular flow and tissue perfusion coupled to physiological or pharmacological stimuli,44,45 to activate different vasodilator pathways resulting in increased microvascular conductance. The most commonly used provocations are the administration of endothelial-dependent vasodilators by transdermal iontophoresis,4648 thermal hyperemia49,50 and post-occlusive reactive hyperemia.5153 In this context, the cutaneous microcirculation is now considered as an accessible and representative vascular bed for the assessment of systemic microcirculatory reactivity.45,5456 A reduced vasodilation response to these different stimuli is indicative of microvascular endothelial dysfunction and is also considered to be predictive for cardiovascular and metabolic diseases and clinical prognosis.5760

In patients with established cardiovascular disease, the reduction of microvascular endothelial-dependent vasodilation (ie, endothelial dysfunction) is associated with increasing BMI, even after adjustment for treated diabetes mellitus, hypertension, hypercholesterolemia, and smoking.61 In that study, BMI was classified in three different intervals: <25, 25-to 30 and >30 kg/m2.61 Moreover, Csipo et al showed that weight loss (reduction of BMI from 31.8 to 27.5 kg/m2, accompanied by a reduction of serum cholesterol, LDL, triglycerides, and increased HDL) after a low-carbohydrate, low-calorie diet, resulted in improvement of microvascular endothelial function in geriatric obese (class 1) patients,62 assessed by laser speckle contrast imaging in the skin, after post-occlusive reactive hyperemia. Additionally, endothelial function of resistance arterioles of the gluteal subcutaneous tissue is impaired in non-diabetic subjects with moderate levels of obesity (BMI 34.7 4.0 kg/m2), in association with systemic inflammation. In women, BMI was significantly associated with high-sensitivity C-reactive protein.63

Regarding mechanisms of microvascular dysfunction, using a new methodology of microdialysis in the skeletal muscle, La Favor et al showed a significant increase in superoxide anions, as well as in NADPH oxidase subunit expression, associated with microvascular endothelial dysfunction in obese subjects relative to lean and overweight/mildly obese subjects.64 Interestingly, 8 weeks of aerobic exercise training resulted in decreased H2O2 levels and improved microvascular endothelial function in the muscle tissue of obese subjects.64 The study therefore linked NADPH oxidase, as a source of reactive oxygen species, to microvascular endothelial dysfunction in obese individuals, with amelioration induced by aerobic exercise.

Microvascular dysfunction has been considered to be a pathophysiological link between overweight/obesity and cardiometabolic diseases, including arterial hypertension, insulin resistance, and glucose intolerance.43,6569 Acknowledged mechanisms include changes in the secretion of adipokines, leading to increased levels of free fatty acids and inflammatory mediators, and decreased levels of adiponectin, all of which may impair endothelial insulin signaling.7073 It is also of note that there are changes at the level of the microvascular network in obesity, involving a reduction in the number of arterioles or capillaries within vascular beds of various tissues (such as the skeletal muscle and skin), which is defined as vascular (capillary) rarefaction.7477 In fact, obese individuals have both structural and functional alterations in skin microcirculation that are proportional to the increase in the degree of global and central obesity, arterial pressure levels and with the degree of insulin resistance.42 In non-diabetic, untreated hypertensive patients, reduced capillary density has also been related to obesity and other cardiometabolic risk factors.78 In addition, in adults and also in prepubertal children, visceral adiposity measured with magnetic resonance imaging is inversely associated with endothelial-dependent skin capillary recruitment, and is accompanied by increased plasma levels of inflammatory markers.79

Impaired left ventricular diastolic function and higher risk of heart failure in obese individuals has been suggested to be associated with myocardial microvascular dysfunction.80 In obese patients undergoing coronary artery bypass graft surgery, coronary microvascular density is significantly lower, compared to non-obese patients, and accompanied by increased body mass index and percent body fat together with increased left ventricular filling pressures.80 Moreover, in patients with suspected coronary artery disease, increasing body mass index is associated with reduced microvascular endothelial function, even after adjustment for treated diabetes mellitus, hypertension, hypercholesterolemia, and smoking.61 Interestingly, the study evaluated microvascular endothelial function three different technologies, including peripheral arterial tonometry, laser Doppler flowmetry and digital thermal monitoring.61

Reduced skeletal muscle capillary density and microvascular reactivity in obese subjects improved after 4 weeks of either sprint interval training, or moderateintensity continuous training, together with increased endothelial eNOS content.81

It has also been shown that bariatric surgery improves microvascular dysfunction in obese patients who were free of metabolic syndrome after surgery, in association with postoperative increases in HDL-cholesterol levels and decreases in oxidized LDL levels.82

Another clinical study investigated microvascular endothelial function using flow-mediated dilation in arterioles isolated from subcutaneous adipose tissue in young women presenting with obesity (age: 33 2 years, body mass index: 33.0 0.6 kg/m2).83 The results showed that a 6-week low-carbohydrate diet, associated or not with caloric restriction, improve endothelial-dependent microvascular function through increases in nitric oxide bioavailability.83 On the other hand, this nutritional intervention did not affect macrovascular endothelial function, evaluated using brachial artery flow-mediated dilation.83

Regarding putative pathophysiological mechanisms, a study by Dimassi et al84 in young individuals with obesity (BMI >30 kg/m2, n = 69), compared with controls with normal weight, suggested that the expression of circulating microparticles containing endothelial nitric oxide synthase (eNOS) is significantly reduced in obesity individuals with endothelial-dependent microvascular dysfunction characterized using cutaneous laser Doppler flowmetry.84

Low-grade inflammation is the common feature that encompasses all the high-risk patients for developing severe COVID-19. Obesity is associated with a fivefold increased risk of developing SARS in SARS-CoV-2 infected individuals, and the well-documented increased susceptibility of obese patients to develop severe forms of COVID-19 may be linked to the elevated systemic metabolic inflammation in these patients.19 Metabolic alterations seen in obese and in diabetic patients are related to an inflammatory response,85,86 and several studies report elevated levels of circulating inflammatory cytokines such as TNF-, IL-1 and IL-6 in obese patients.87 Furthermore, visceral fat shows significant univariate association with the need for intensive care in COVID-19 patients,15 and deregulated expression of adipokines, such as leptin and resistin, increases the expression of vascular adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) that contribute to increased vascular leukocyte adhesiveness and additional oxidative stress.88 To further complicate the scenario, adipose-derived mesenchymal stem cell (ASCs), a specialized cell population in adipose tissue, are functionally compromised in obesity and changes its regulatory protective activity to a pro-inflammatory profile increasing its ability to secrete TNF-, IL-8, IL-6 and MCP-1.89,90 Therefore, ASCs from obese patients may not be able to modulate the immune response and tissue repair in SARS-CoV-2 infection contributing to more severe tissue injury.10

SARS-CoV-2 uses its viral spike (S) protein to invade target cells, such as epithelial cells, through binding to angiotensin-converting enzyme 2 (ACE2) after proteolytic activation by transmembrane protease serine 2 (TMPSS2).91 Others enzymes like furin, trypsin and elastase may also activate the S protein and facilitate cellular entry by the virus.9294 Interestingly, adipose tissue highly expresses ACE2 and the expression is even higher in visceral adipose tissue.95 Of relevance, ACE2 expression is upregulated in obesity.96 Also, another suggested receptor for SARS-CoV-2, dipeptidyl peptidase 4 (DPP4), is expressed in adipose tissue and is upregulated in obesity.97,98 Finally, CD147, the alternative receptor for SARS-CoV-2, is positively correlated with an increase in body mass index.99 Taken together, the evidence of high expression of different SARS-CoV-2 receptors in adipose tissue may be the basis for increased severity of COVID-19 in obese patients involving at least two different possibilities: First, infection of adipocytes with SARS-CoV-2 may exacerbate the innate immune response through pathogen recognition receptors in an already inflammation-primed tissue, increasing the magnitude of the response. Second, adipocytes may function as a reservoir for the SARS-CoV-2 and therefore may fuel the inflammatory response in adipose tissue and elsewhere in the organism by releasing viral NA and antigens that, by reaching the circulation generate ripple inflammatory effects across the organism. Importantly, these two possibilities are not mutually exclusive and may well combine their pathophysiological potential towards a deregulate systemic inflammatory response with widespread tissue injury and consequent organ dysfunction. It is important to add that as the pandemic evolves, new mechanistic interactions may unravel. For instance, new virus variants with mutations at the receptor-binding domain of the S protein may change the infectivity of the virus by changing its interactions with cellular receptors. In Brazil, a variant designated as P1, with multiple mutations in the S protein, was recently identified and is seemingly more infective than previous lineages of the virus.100 How this variant may interact with adipocytes increasing infectivity to these cells or potentiating the formation of an adipocyte reservoir of the virus causing a more severe disease in obese individuals is yet unknown. What is known is that a second wave caused by this new P1 variant is promoting devastating effects in Brazil with apparently higher mortality and a faster progression of the disease.

Severe COVID-19 is characterized by a massive production of pro-inflammatory mediators, in special cytokines. Frequently, the term cytokine storm is called up to describe the massive production of cytokines that occurs in viral infections (including SARS-CoV and MERS-CoV), in sepsis and more recently, in severe COVID-19.101 Increased levels of IL-6, TNF-, IP10 are commonly found in patients with severe COVID-19.102 It is reasonable to propose that obese patients who already have an underlying chronic inflammation when infected with SARS-CoV-2 are prone to develop a more intense and deregulated response, and in doing so, developing a severe presentation of the disease. In addition, dysfunctional metabolism, endothelium, and overall immune response would further contribute to an unfavorable evolution of the disease in the obese patients. The questions about the molecular mechanisms behind this disproportional response remain unanswered, but our knowledge about this disease is growing in an unprecedented velocity and we may soon have the answer. However, a few possibilities may be put forward (Figure 1).

As stated above, obesity is characterized by the induction of a low-grade chronic proinflammatory state and NF-B is described as a key factor in the low-grade inflammation state in atherosclerosis and hypertension.103,104 Also, the NF-B pathway is involved in insulin resistance, a condition frequently seen in obese patients, and in -cell dysfunction.105 In addition, free fatty acids can also promote inflammation and activate the NF-B and JNK1 pathways.106 All those pieces put together may point to NF-B being a key player in obese patients with COVID-19. Importantly, cell culture experiments combined with system biology approach showed that overexpression of Nsp1 during infection with SARS-CoV-2 strongly increases signaling through the nuclear factor of activated T cells (NFAT) and increases cytokine production and immune-dependent pathogenesis. Both NF-B and NFAT pathways share common regulation signals, such as Foxp3 and Foxd1, and a similar mechanism of activation against infection.107

We must also consider that binding of SARS-CoV-2 to ACE2 leads to receptor internalization and high cytosolic levels of angiotensin II, which is a recognized activator of NLP3 inflammasome in the lung108 and other tissues. The NLRP3 inflammasome regulates pyroptosis through gasdermin D, along with the release of cytosolic contents into the extracellular spaces. The release of alarmins, ATP, ROS, cytokines, chemokines, LDH and viral particles elicits an immediate reaction from surrounding immune cells, inducing a pyroptotic triggered reaction further fueling inflammation. Interestingly, different studies have reported elevated levels of LDH, a cytosolic enzyme that is measured for monitoring pyroptosis in patients with the severe form of COVID-19.109 On the other hand, diet-induced alterations in the gut leading to increased gut permeability to bacterial endotoxins are known to promote activation of NLRP3 inflammasomes via Toll-like receptors (TLRs). This event is followed by the accumulation of IL-1 family cytokines, which modulate insulin production by pancreatic beta cells.110 Importantly and at the same time, a decrease in endogenous protective mechanisms occurs.111 NLRP3 inflammasome activation is involved in endothelial lysosome membrane permeabilization, cathepsin B release, and impaired glycocalyx thickness,112 thus further contributing to the endothelial cell dysfunction, enhanced susceptibility to cardiovascular injury and thrombotic events, a common complication in severe COVID-19 patients.

In fact, thrombotic events are now recognized as a common feature in COVID-19 patients, and COVID-19 has recently been suggested to be a thrombotic viral fever.113 Obese patients are prone to thrombotic events for many different reasons,113 and COVID-19 may contribute even further to this complication. The imbalance of the ACE/ACE2 system caused by internalization of ACE2 after binding to virus S protein causes a switch towards pro-thrombotic activity by decreasing Ang-(1-7)-Mas axis (antithrombotic) and increasing angiotensin II (prothrombotic). This mechanism may be of central pathogenic relevance explaining the poor outcome of obese patients with COVID-19.113

In summary, there are many different ways by which low-grade inflammation caused by metabolic changes in obesity may contribute to the worse prognosis of obese patients infected by SARS-CoV-2, in a combination of factors and mechanisms leading to a subversion of the defensive responses of the organism against the virus.

The authors report no conflicts of interest in this work.

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Adaptation to mitochondrial stress requires CHOP-directed tuning of ISR – Science Advances

By daniellenierenberg

INTRODUCTION

Mitochondrial diseases are a heterogeneous group of devastating disorders characterized by respiratory chain dysfunction (1). Although mitochondrial disorders have distinct tissue and organ presentation, they seem to activate common stress responses evolved to mitigate the negative impact of respiratory deficiency on cellular and organismal metabolism (1). It appears that mitochondrial stress responses precede respiratory chain deficiency, thereby suggesting that they constitute an early event in pathogenesis of mitochondria-related diseases (2). This suggests that monitoring the activation and/or alteration of mitochondrial stress responses may provide early diagnostic markers in these conditions. Moreover, manipulation of mitochondrial stress responses may be beneficial for patients with mitochondrial disease and thus therapeutically exploited (3, 4).

Initially, the mitochondrial unfolded protein response (UPRmt) was postulated to be a common stress response to respiratory chain dysfunction (5). UPRmt constitutes a transcriptional program that up-regulates mitochondrial chaperones and proteases aimed to restore the loss of organelle proteostasis. Notwithstanding that UPRmt was first described to be triggered by the accumulation of misfolded proteins within the mitochondrial matrix in mammalian cells (5), most of the subsequent mechanistic studies were performed in Caenorhabditis elegans (6). In contrast, many aspects of the mammalian UPRmt signaling are less well understood. In mammalian cells, it is thought that mitochondrial proteotoxic stress leads to CHOP [CCAAT/enhancer binding protein (C/EBP) homology protein] up-regulation resulting in up-regulated transcription of UPRmt-responsive genes (5, 7). The CHOP-binding sites in the UPRmt gene promoters are presumably flanked by two conserved regions named the mitochondrial UPR elements 1 and 2 (MURE1 and MURE2) (7, 8). The role of CHOP in governing transcription of UPRmt genes is however controversial as the transcription factors that bind to MURE1 and MURE2 elements have not been identified (7, 9). Nevertheless, multiple studies confirmed up-regulation of the CHOP mRNA in cells derived from patients with various mitochondrial disorders, as well as mitochondrial disease models (2, 1012). This illustrates that although CHOP plays a pivotal role in mammalian mitochondrial stress responses, the underpinning mechanisms of its actions in the context of mitochondrial dysfunction are still obscure.

Recently, it became clear that unlike in C. elegans, mammalian UPRmt may not be the primary response to mitochondrial dysfunction but rather function as a part of more complex mitochondrial stress response (1114). Mammalian cells treated with mitochondrial toxins exhibit transcriptional reprogramming mimicking the integrated stress response (ISR) arm of the UPR, which is centered on the activating transcription factor 4 (ATF4) (13, 14). Consistent with this, studies carried out in models with defects in different steps of mitochondrial DNA (mtDNA) expression and protein synthesis revealed activation of ISR transcriptional signatures (11, 12). ISR hallmarks are increased eIF2 phosphorylation, reduction in ternary eIF2:tRNAiMet:guanosine 5-triphosphate (GTP) complex levels, and subsequent inhibition of global protein synthesis that is paralleled by selectively induced translation of a subset of inhibitory upstream open reading frame (uORF) containing stress-responsive mRNAs, including ATF4, CHOP, and GADD34 (15). CHOP induction during ISR is thought to lead to cell death via induction of Growth Arrest and DNA Damage-Inducible Protein 34 (GADD34)mediated eIF2a dephosphorylation and activation of Endoplasmic Reticulum Oxidoreductase 1 Alpha (ERO1A) endoplasmic reticulum (ER) oxidase (16).

CHOP is a multifunctional transcription factor that dimerizes with members of the C/EBP and ATF/cyclic adenosine 3,5-monophosphate response element binding protein families (17). Although up-regulated in response to a wide variety of stresses such as growth arrest and DNA damage, amino acid and glucose deprivation, hypoxia, and ER stress, the role of CHOP in cellular physiology is incompletely understood. CHOP is considered to induce apoptosis, but its transcriptional targets largely overlap with those of ATF4, including genes promoting cell survival and growth (16, 18). These findings highlight the intricate interaction partnerdependent roles of CHOP under different stresses and in various tissues. They also point out the importance of understanding the context-dependent role of CHOP under different physiological conditions. In the context of mitochondrial respiratory chain dysfunction, the role of CHOP is particularly important as CHOP was proposed to be the main transcription factor that conveys specificity of the mitochondrial stress response (5).

Here, we aimed to decipher the role of CHOP in the regulation of the mitochondrial stress response. As a model for the most common cause of mitochondrial diseases, namely, loss of mitochondrial translation, we used mice deficient in the mitochondrial aspartyl transfer RNA (tRNA) synthase DARS2 specifically in heart and skeletal muscle (DARS2 KO) (2). We demonstrate a beneficial role of CHOP in mitochondrial mutants as its loss leads to a marked shortening of life span in DARS2/CHOP double knockout (DKO) as compared to DARS2 KO animals. The beneficial effects of CHOP appear to be independent of UPRmt activation but rather mediated by attenuation of harmful overactivation of the ISR and a consequent metabolic imbalance. We also provide mechanistic evidence that these effects stem from the interplay between CHOP, ATF4, and C/EBP in regulation of mitochondrial ISR targets.

To determine the in vivo function of CHOP in the context of mammalian mitochondrial dysfunction, we intercrossed whole-body Chop/ mice (CHOP KO) with heart and skeletal muscle-specific DARS2-deficient mice (Dars2fl/fl; Ckmm-Cre+/tg; DARS2 KO) (fig. S1, A and B) (2). The resulting animals deficient in both CHOP (whole body) and DARS2 (heart and skeletal muscle) (Dars2fl/fl; Ckmm-Cre+/tg; Chop/ and DKO) were born in Mendelian ratios (fig. S1C). We previously showed that DARS2 depletion mediated by Ckmm-Cre expression induces dilated cardiomyopathy preceding any pathological phenotypes in skeletal muscle (2). Hence, we monitored the effects of CHOP loss on pathologies caused by DARS2 abrogation in the heart.

Approximately from 2 weeks of age, a large number of DKO mice became increasingly susceptible to sudden death during a routine ear-clipping handling for genotyping. This procedure was tolerated well up to postnatal day 13 (P13) by mice of all four genotypes; hence P13 (1) was defined as the early stage of heart dysfunction in DKO animals (DKOE). It appeared that the deterioration of the health status of DKO mice characterized by lower spontaneous cage activity, piloerection, unsteady gait, and overall droopiness is a very rapid process as the interval from the first apparent symptoms to death of the mice at around P17 (2) was between 24 and 48 hours. This interval was defined as the late/terminal stage in DKO mice (DKOL). Consequently, the life expectancy of DKOs was severely reduced (>60%) compared to DARS2 KOs, signifying the essential role for CHOP in adaptation to impaired mitochondrial protein synthesis in heart (Fig. 1A). CHOP deficiency in the absence of DARS2 resulted in dilated cardiomyopathy (Fig. 1B and fig. S1, D to F) characterized by increased expression of mRNAs encoding cardiac hypertrophy markers Nppa and Nppb (Fig. 1C). Although no gross morphological changes were observed upon hematoxylin and eosin (H&E) staining, ultrastructural analyses suggested a disrupted myocardial organization, characterized by severely disorganized sarcomeric structures, expected to cause disturbances in contractile function of DKOL hearts (Fig. 1, D and E). Therefore, DKOL animals display very similar pathological changes, as compared to the terminal stage DARS2 KO mice (2), whereby the onset of these pathologies is markedly accelerated upon CHOP loss.

(A) Kaplan-Meyer survival curves for wild-type (WT; n = 36), CHOP KO (n = 35), DARS2 KO (n = 47), and DKO animals (n = 60). The life span of DKO in comparison to DARS2 KO mice is significantly decreased (P < 0.0001; log-rank test and Gehan-Breslow-Wilcoxon test). The viability of CHOP KO mice was WT-like in a 12-month follow-up. (B) Heart gross morphology. (C) Fold changes of the cardiac hypertrophy markers Nppa and Nppb obtained from the RNA sequencing dataset at P17 (2) (n = 4). Bars represent means SD [multivariate analysis of variance (MANOVA) followed by one-way ANOVA and Tukeys multiple comparisons test, **P < 0.05, ***P < 0.001, and ****P < 0.0001]. (D) H&E staining; (n = 3) at P17 (2). Scale bars, 50 m. (E) Transmission electron microscopybased analyses of cardiac tissue biopsies; (n = 1) at P17 (2). Scale bars, 2 m.

We next sought to identify pathways that are affected by the CHOP deficiency in the context of DARS2 KO. To this end, we compared global changes in mRNA levels to corresponding changes in the proteome in CHOP KO, DARS2 KO, and DKOL versus control hearts using the anota2seq algorithm (19). Scatter plots comparing mRNA and protein changes in DARS2 KO hearts revealed alterations in protein levels that were mainly independent of the mRNA levels, thus arguing for a prevalent impact of translational and/or protein stability changes on the proteome (Fig. 2A, fig. S2A, and table S1). In contrast, DKOL animals primarily showed congruent changes in mRNA and protein levels, which accounted for ~75% of detected alterations in protein levels (Fig. 2A, fig. S2A, and table S1).

(A) Scatter plots of total mRNA and protein fold changes (FC) comparing CHOP KO, DARS2 KO, or DKOL to WT. The numbers of significantly regulated genes are indicated for translation/protein stability (red), and mRNA abundance (green). RNA sequencing and quantitative proteomics were performed on hearts of animals at P17 (2) (n = 4). (B) A GO network of overrepresented terms among genes regulated via changes in translation/protein stability (up-regulated, light red; down-regulated, dark red) and mRNA abundance (up-regulated, light green; down-regulated, dark green) in DKO versus WT. Nodes represent identified GO terms, while the pie chart within each node indicates the proportion of genes regulated. (C and D) Heatmap of protein expression (P) and total mRNA (T) log2 fold changes of (C) the OXPHOS subunits grouped in respective complexes and (D) OXPHOS assembly factors (n = 4). (E) In organello translation assay (left) of cardiac mitochondria at P17 (2). De novo protein synthesis was determined after 1 hour of 35S-methionine pulse labeling; protein turnover was assessed after 3 hours of the cold chase. Coomassie brilliant bluestained gel was used as a loading control. Relative protein synthesis and turnover rates (right) (n = 3). (F) Oxygen consumption of intact cardiac mitochondria at P17 (2). State 3: adenosine 5-diphosphate (ADP)stimulated respiration using CI or CI + CII substrates. State 4: Respiration upon addition of oligomycin. ETS, maximum respiration upon mitochondrial uncoupling (CI) and after addition of rotenone (CII) (n = 3 to 4). Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, and ***P < 0.001).

Gene Ontology (GO) analysis performed using ClueGO (20) and annotation from the GO consortium (21) on genes whose expression was reduced indicated that oxidative phosphorylation, electron transport, complex I assembly, adenosine 5-triphosphate (ATP) biosynthesis, fatty acid oxidation, and heart contraction are predominantly disrupted in DKO hearts (Fig. 2B). This is consistent with the impairment of mitochondrial energy production and heart failure in DKO animals and similar to other models of mitochondrial cardiomyopathy (11). In contrast, translation, tRNA metabolism, mitochondrial RNA, and glutathione metabolism were primarily up-regulated pathways (Fig. 2B). We observed further perturbations in apoptotic pathways, amino acid catabolism, and purine nucleotide metabolism that contained a combination of up- and down-regulated gene expression changes (Fig. 2B).

A general down-regulation of steady-state levels of individual oxidative phosphorylation (OXPHOS) subunits detected in DARS2 KO hearts was further decreased in DKOL animals (Fig. 2C and fig. S2B). Intriguingly, while in DARS2 KO animals, most of the changes in the levels of OXPHOS subunits were not accompanied by alterations in mRNA abundance, numerous OXPHOS subunit-encoding genes exhibited congruent changes in mRNA and protein levels in DKOL animals (Fig. 2C). These include three of four subunits of succinate dehydrogenase (SDH; complex II), a complex fully encoded by nuclear DNA, usually up-regulated upon mitochondrial translation defects. This was further confirmed using an enzyme-histochemical assay, showing that substantial cyclooxygenase (COX) deficiency observed in DKOL animals is not accompanied by a compensatory SDH up-regulation (fig. S2C), as observed in DARS2 and other mitochondrial mutants (2, 22). Furthermore, while we detected a general compensatory up-regulation of OXPHOS assembly factors in DARS2 KO hearts, many were either unaltered or down-regulated in DKOL samples (Fig. 2D).

Although Dars2 deletion primarily interferes with mitochondrial protein synthesis, at P17, only a moderately dysbalanced mitochondrial translation was observed in DARS2 KO (Fig. 2E). In contrast, mitochondrial de novo protein synthesis in DKOL mice was significantly decreased and severely dysregulated, whereas the protein turnover remained unaffected (Fig. 2E). The exaggerated translation defect observed in DKOL animals was not caused by a decrease in mtDNA or mtRNA levels (fig. S2, D and E). Some mtRNAs were up-regulated (e.g., mt-COX3 and mt-ND1) in both DARS2 KO and DKO hearts, possibly as a compensatory response to defective protein synthesis (Fig. 2E and fig. S2E).

Severe dysregulation of mitochondrial translation in DKOL was accompanied with a strong decrease in the respiration capacity of all inducible states in mitochondria isolated from DKOL hearts (Fig. 2F). In contrast, no major defects in DARS2 KO heart mitochondria respiration were observed, thus suggesting compensation for the mitochondrial protein synthesis defect (Fig. 2F).

Unexpectedly, a comparable defect at the level of assembled respiratory chain complexes and supercomplexes was detected in DARS2 KO and DKOL mice despite higher levels of individual OXPHOS subunits in DARS2 KO (Fig. 2C and fig. S2F). These data suggest that, at early stages of DARS2 deficiency, nascent nuclear-encoded OXPHOS subunits are not efficiently incorporated in respiratory chain complexes in DARS2 KO hearts and are likely turned over at higher rates. Although DKOL and DARS2 KO mitochondria have comparable levels of respiratory chain supercomplexes (fig. S2F), DKOL mitochondria fail to sustain normal respiration (Fig. 2F). This suggests that the OXPHOS activity is further indirectly affected by CHOP deficiency that might lead to disruption of mitochondrial integrity or supply of critical metabolites.

CHOP deficiency in the context of mitochondrial dysfunction is expected to blunt the mitochondrial stress response (5). Therefore, by analyzing changes in the transcriptome, we compared pathways that are affected in DARS2-deficient hearts before and after CHOP depletion (table S2).

In DARS2 KO heart, relatively few mRNAs changed their expression, and most were up-regulated. Notably, using Cytoscape plug-in iRegulon, we demonstrated that two-thirds of these transcripts overlapped with an ISR signature activated by ATF4, which was also identified as the most prominent regulator of gene expression in DARS2 KO hearts (Fig. 3A and tables S2 and S3) (18, 23, 24). The most up-regulated transcripts in DARS2 KO hearts encoded enzymes involved in one-carbon metabolism, serine biosynthesis, and trans-sulfuration, as well as Gdf15 and Fgf21 (Fig. 3, A and B, and table S2), the two cytokines shown to be excreted from tissues upon OXPHOS deficiency (25, 26). Similar changes (Fig. 3A) were previously described in other cellular and in vivo models for mitochondrial OXPHOS defects, confirming that DARS2 deficiency causes a stress response relevant for many mitochondrial disease states (1114).

(A) Heatmap of total mRNA fold changes (log2) of significantly changed ATF4 target genes [as predicted by Cytoscape plug-in iRegulon (23, 24)], in DARS2 KO animals compared to WT controls. Black boxes above DKO and below CHOP KO rows indicate their respective significantly changed transcripts as compared to WT controls (n = 4). (B) Fgf21 log2 raw expression counts (sequenced reads, +0.5) as this gene was not detected in multiple WT and CHOP KO samples and hence was excluded during data filtering. Of note, these samples will obtain negative log2 values (n = 4). (C) Western blot analysis (left) and quantification of ISR markers (right). HSC70 was used as a loading control. Antibodies used were raised against proteins indicated in panels. Experiments were performed on cardiac lysates of mice at P17 (2) (n = 3). (D) p-eIF2/eIF2 ratio quantified from (C). (B to D) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (E) Western blot analysis and quantification of UPRmt markers in WT, CHOP KO, DARS2 KO, and DKO animals at P17 (2). Antibodies used were raised against proteins indicated in panels. HSC70 was used as a loading control. Bars represent means SD; no significant differences were detected (MANOVA: Wilks test, P = 0.176; Hotelling-Lawleys test, P = 0.183; Pollais test, P = 0.232) (n = 3). (F) Heatmap of total mRNA fold changes (log2) for the selected alleged CHOP target genes involved in apoptosis (n = 4).

The ISR activation in DARS2 KO hearts was confirmed by increased eIF2 phosphorylation, accompanied by up-regulation of ATF4 (Fig. 3, C and D). These effects were further potentiated by CHOP loss, whereby induction of both eIF2 phosphorylation and ATF4 was more pronounced in DKOL relative to DARS2 KO hearts (Fig. 3, C and D). Transcript and protein levels of almost all ATF4 targets were highly up-regulated in DKOL as compared to DARS2 KO animals (Fig. 3, A to C). Consistently, further analysis of binding motifs in genes up-regulated in DKOL hearts established ATF4 as the most prominent signature (table S3) (23, 24). The most up-regulated transcripts in DARS2 KO and DKOL showed a notable overlap. To this end, of the top 11 most up-regulated transcripts, 8 overlapped, despite the 40-fold difference in the number of overall changes between the two models (table S2). The only difference was that these transcripts were, on average, more than 10-fold more up-regulated in DKOL than in DARS2 KO hearts (table S2). In contrast, UPRmt markers were not significantly changed in DARS2 KO or DKOL animals, adding evidence that UPRmt is neither an early nor prominent stress response in mammalian cells and tissues upon mitochondrial OXPHOS dysfunction (Fig. 3E). Instead, our data suggest a central role for ISR and ATF4-dependent regulation in the context of mitochondrial dysfunction in vivo and point to an unexpected role of CHOP in the suppression of the transcriptional overactivation of ATF4 targets.

CHOP is proposed to be involved in the regulation of apoptosis upon ER stress, although the exact mechanism remains controversial, as exogenously expressed CHOP has also been reported to positively regulate genes involved in protein synthesis and not apoptosis (16, 18). Henceforth, we analyzed changes in the expression levels of various apoptotic genes reported to be CHOP targets (27). Notably, proapoptotic members of the B-cell lymphoma 2 (BCL-2) family (Puma/Bbc3, Bid, Bax, and Bim/Bcl2l11) and genes encoding proteins involved in the activation or execution of apoptosis (Dr5/Tnfrsf10b, Casp3, and Ero1l) were not suppressed but often further up-regulated upon loss of CHOP in DARS2-deficient animal (Fig. 3F). Similarly, the steady-state level of proapoptotic protein BCL2-associated X protein (BAX) was up-regulated, and we observed a higher cleavage of caspase 3 in DKOL hearts as compared to control animals (fig. S3A). These results suggest that, unexpectedly, apoptosis may be up-regulated in DARS2-deficient hearts upon CHOP depletion and thus contribute to the detrimental phenotype observed in DKOL mice.

As we observed major changes in the abundance of proteins involved in amino acid metabolism, we next measured amino acid levels by liquid chromatographytandem mass spectrometry. While only minor perturbations in amino acid levels were observed in DARS2 KO hearts, most amino acids were significantly up-regulated in DKO mice (fig. S3B). Of note, serine, glutamine, glutamate, and aspartate levels were not significantly changed in either DARS2 KO or DKOL relative to control hearts (fig. S3B). The unaltered serine levels, despite the increased levels of enzymes involved in serine synthesis [Phosphoglycerate dehydrogenase (PHGDH), Phosphoserine Aminotransferase 1 (PSAT1) , and Phosphoserine Phosphatase (PSPH)], suggest an increased flux of serine-derived one-carbon units for further methylation reactions into the one-carbon cycle. Similarly, glutamine and glutamate are likely used to replenish tricarboxylic acid cycle intermediates and aspartate production that is essential for nucleotide synthesis and cell proliferation (28, 29). Increased levels of citrate and isocitrate in DKOL, but not DARS2 KO, hearts suggest that glutamine primarily undergoes reductive metabolism (fig. S3C), as seen in the patient-derived cell lines harboring mtDNA mutations (30). Increased citrate levels can propagate intracellular acidosis, leading to hypocalcemia caused by reduced availability of Ca2+, further contributing to reduced contractility of the heart through a vicious circle of the excitation-contraction-metabolism impairment (31). Additional effects of elevated citrate levels on the regulation of metabolic enzyme and/or chromatin dynamics by acetylation may further contribute to accelerated pathological phenotypes observed in DKOL hearts.

Next, we tested whether mitochondrial stressinduced ISR has a beneficial or detrimental role in conditions of mitochondrial dysfunction. For these analyses, we took advantage of two cell models for mitochondrial respiratory chain dysfunction: (i) mouse skin fibroblasts with severe mitochondrial dysfunction caused by the loss of COX10 (COX10 KO), an early assembly factor of the respiratory cytochrome c oxidase (32); and (ii) mouse embryonic fibroblasts (MEFs) treated with actinonin, an inhibitor of mitochondrial peptide deformylase causing impairment in mitochondrial translation (33).

In the COX10 KO cells, a robust activation of the ISR was detected as evidenced by increased levels of phosphorylated eIF2, ATF4, and ATF4 targets (Fig. 4A and fig. S4A). To test whether increased ATF4 levels are a direct result of ISR activation, we incubated COX10 KO cells with the ISR inhibitor (ISRIB) (34). This treatment abrogated ATF4 induction and attenuated up-regulation of its downstream targets at both transcript and protein levels (Fig. 4A and fig. S4A). The phosphorylation of eIF2 remained unchanged (Fig. 4A), which was expected as ISRIB bolsters guanine-nucleoside exchange activity of eIF2B without affecting on phospho-eIF2 levels (34). Similarly, increased ATF4 levels induced by actinonin treatment were suppressed by ISRIB (Fig. 4B). Mirroring the results from DKOL mice, loss of CHOP combined with mitochondrial dysfunction induced by actinonin treatment greatly increased ATF4 protein and transcript levels, and expression of ATF4 targets Shmt2, Pycr1, and Mthfd2 (Fig. 4, B and C).

(A) Western blot analysis (left) and relative protein levels (right) of ISR markers and ATF4 downstream targets in immortalized COX10 KO and WT fibroblasts upon 48-hour treatment with DMSO () or ISRIB (+). (B) Western blot analysis of WT and CHOP KO MEFs treated for 48 hours with DMSO () or actinonin (+) in the presence (+) or absence () of ISRIB during the last 4 hours before protein isolation. (C) Relative transcript levels in WT and CHOP KO MEFs treated for 48 hours with DMSO (control) or actinonin. Tbp expression was used for normalization (n = 3). (D) Growth curves of respective exponential growth phases of WT and CHOP KO MEFs treated with DMSO (control), actinonin, and +/ISRIB, respectively. Curves were determined using linear regression (n = 3). Bars represent means SD. (E) Western blot analysis of heart lysates from 4-week-old WT and DARS2 KO animals treated with control (DMSO) and ISRIB, according to the experimental setup presented in the schematic illustration (top). Animals are treated with daily injections of saline (control) or ISRIB solution for 7 days (blue boxes), starting at P19, and euthanized at P27 (red line) (n = 3). (F) Quantification of ISR markers (top), OXPHOS subunits (bottom), and p-eIF2/eIF2 ratio (right) from the Western blot analysis at (E). (A, B, and F) Antibodies used were raised against proteins indicated in panels. HSC70 was used as a loading control. (A, C, and F) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Prevention of ISR overactivation in CHOP KO MEFs by ISRIB treatment resulted in a partial rescue of the proliferation defect induced by actinonin (Fig. 4D). In turn, wild-type (WT) cells treated with actinonin and CHOP KO cells grown under control conditions showed minor growth defects, which were not further affected by ISRIB (Fig. 4D). Therefore, CHOP deficiency, only in conditions of mitochondrial dysfunction, results in a detrimental ISR activation, which can be partially rescued by ISRIB treatment.

To assess the effect of ISRIB treatment in vivo, DKOL mice and respective controls were injected with ISRIB (5 g/g) for up to 7 or 12 days, starting from 1 week of age (fig. S4B). Unfortunately, neither protocol resulted in the suppression of ATF4 levels or downstream targets in either DKO or DARS2 KO animals nor did it affect steady-state levels of OXPHOS subunits (fig. S4, C and D). However, this is not unexpected given the fact that ISRIB inhibits low-level ISR activity but does not affect strong ISR signaling (35), as observed in DKO mice. In contrast, a 7-day treatment of DARS2 KO animals with ISRIB, starting from P20, resulted in an apparent reduction of ISR markers (Fig. 4, E and F). Nevertheless, ISRIB-mediated suppression of ISR in DARS2 KO animals up to 4 weeks of age was not beneficial as it also prevented compensatory complex II (CII) up-regulation.

One of the hallmarks of the acute ISR is suppression of global protein synthesis, accompanied by translational activation of some uORF-containing mRNAs (15). To further understand the consequences of ISR activation in our model, we measured the global protein synthesis rate at P6, P13, and P17 in vivo in DKO and control hearts (36). At P6, cytoplasmic translation of all four genotypes was similar, in agreement with no phenotypes observed at this time point (fig. S5A). Coinciding with increased eIF2 phosphorylation, a 70% decrease in general protein synthesis was detected in mice at P13 (DKOE; Fig. 5A and fig. S5B). Within a few days, this effect seems to be reversed as we detected fully recovered protein synthesis rates in DKOL hearts at P17 (Fig. 5B and fig. S5C). This was despite unaltered eIF2 phosphorylation levels and activation of ATF4 and its targets that were comparable between DKOE and DKOL hearts (fig. S5D). These findings suggested a transition from acute to prolonged ISR, characterized by recovery of global protein synthesis and ongoing translation of ISR-sensitive mRNAs (37). These distinctions in global protein synthesis levels reflected different phenotypes of DKOE and DKOL mice. In the acute ISR, when global translation is strongly down-regulated, DKOE (P13 1) animals cope better with the mitochondrial translation defect when compared to DARS2 KO animals (Fig. 5C). This is illustrated by the unaffected levels of OXPHOS complexes and supercomplexes in DKOE animals (Fig. 5D and fig. S5E). However, these effects are reversed when DKO animals reach the prolonged ISR stage, which is characterized by partial recovery of global mRNA translation and sustained ATF4-mediated transcriptional reprograming (fig. S5D). This reactivation of normal translation is likely to result in ER stress, and further energy crisis as protein synthesis is highly energy demanding (38). Consistently, we detected increased levels of the ER-chaperone binding immunoglobulin protein (BIP) in P17 DKOL hearts, which mirrored findings in DARS2-deficient hearts at the terminal state of 6 weeks of age (Fig. 5E). Levels of several ER Ca2+ transporter proteins were also profoundly disturbed [Ryanodine receptors (RyR), Sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), and The inositol 1,4,5-trisphosphate receptor type 2 (IP3RII)], which may explain defects in the conductive system of the heart (Fig. 5F). Perturbed Ca2+ homeostasis due to the dysregulation of the ER Ca2+ transporters and increased Ca2+ release by ERO1-stimulated IP3R activation may also contribute to ER stress leading to the development of fatal cardiomyopathy (Fig. 5, E and F). Therefore, although strong activation of ISR, as seen in DKOE animals, brings brief protection from the mitochondrial dysfunction, it cannot be sustained over prolonged period of time and results in a detrimental switch to a prolonged ISR program leading to additional ER stress, loss of Ca2+ homeostasis, and premature death.

(A and B) The relative protein synthesis rate of animals injected with puromycin at (A) P13 and (B) P17. Bars represent means SD (one-way ANOVA and Tukeys multiple comparisons test, **P < 0.01 and ***P < 0.001) (n = 4). (C) De novo synthesis in mitochondria isolated from WT, CHOP KO, DARS2 KO, and DKOE and DKOL animals after 1 hour of 35S-methionine pulse labeling followed by SDS-PAGE. Coomassie bluestained gel was used as a loading control. (D) Blue native polyacrylamide gel electrophoresis (BN-PAGE) and subsequent Western blot analysis of OXPHOS complexes and supercomplexes in mitochondria isolated from WT, CHOP DO, DARS2 KO, and early (DKOE) and late-stage (DKOL) DKO animals. Subunit-specific antibodies (left) were used to detect respective complexes and supercomplexes (right) (n = 3). (E) Western blot analysis of BIP levels in WT, CHOP KO, DARS2 KO, and DKO at P17 (2) (top) and WT and DARS2 KO at 6 weeks (bottom) (n = 3). (F) Western blot analysis proteins involved in the Ca2+ metabolism in WT, CHOP KO, DARS2 KO, and DKOL at P17 (2) (n = 3). (E and F) HSC70 was used as a loading control (n = 3).

The prolonged activation of ISR in DKOL hearts may have adverse effects on cellular and organismal fate. GADD34, a regulatory subunit of the enzyme dephosphorylating eIF2, is thought to function as ISR rheostat acting to restore protein synthesis and block excessive ATF4 activation (15). Unexpectedly, although CHOP was proposed to be a primary Gadd34 transcriptional activator (16), DKOL animals at P17 showed a significant up-regulation of Gadd34 transcripts to similar levels as those observed in terminal, 6-week-old DARS2 KO animals (Fig. 6A). This result suggests that CHOP may play a GADD34-independent role in the suppression of the overactivation of ATF4 induction and ATF4-mediated transcriptional reprogramming.

(A) Relative Gadd34 transcript levels at P17 (2) WT, CHOP KO, DARS2 KO, and DKO animals, as well as in 6-week-old WT and DARS2 KO mice. Bars represent means SD, samples were normalized to WT mice of the respective age (P17: one-way ANOVA, *P < 0.05, **P < 0.01, and ***P < 0.001; 6 weeks: unpaired Students t test) (n = 4). (B) Coimmunoprecipitation (co-IP) of CHOP from WT, CHOP KO, DARS2 KO, and DKOL hearts. The CHOP and C/EBP interaction was monitored with Western blotting using an antibody against C/EBP. One percent of the input fractions was used as loading controls. Asterisks indicate the immunoglobulin G heavy and light chains. (C) Western blot analysis (left) and quantification (right) of the three CEBP isoforms LAP1, LAP2, and LIP in CHOP KO MEFs treated for 48 hours with actinonin along with the respective control (n = 3). (D) Western blot analysis (left) and quantification (right) of steady-state levels of ISR markers in actinonin-treated (48 hours) CHOP KO MEFs expressing the CEBP LIPL120T mutant variant along with the respective controls (n = 3). (E) Western blot analysis of the ATF4 and three CEBP isoforms in actinonin-treated (48 hours) CHOP KO MEFs expressing the CEBP LIP WT and CEBP LIPL120T mutant variant along with the WT cells and respective controls (n = 4). (C to E) Antibodies used were raised against proteins indicated in the panels. HSC70 was used as a loading control. (A, C, and D) Bars represent means SD (MANOVA followed by one-way ANOVA and Tukeys multiple comparisons test, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001) (n = 3).

As a prerequisite for DNA binding, CHOP needs to heterodimerize with other transcription factors (17). To this end, to identify CHOP-interacting partners that may play a role in mitochondrial stress responses, we immunoprecipitated CHOP from DARS2 KO heart extracts, followed by mass spectrometry (table S4). Notably, besides CHOP, only six proteins were identified. Among those, the most enriched protein and the only transcription factor was C/EBP (table S4). These results were confirmed by Western blot analysis following coimmunoprecipitation (co-IP) against CHOP (Fig. 6B). Notably, CHOP and C/EBP appear to interact only upon mitochondrial dysfunction (i.e., DARS2 KO), despite similar levels of C/EBP in WT and DARS2 KO hearts (Fig. 6B and fig. S6A). The mass spectrometry analysis of C/EBP immunoprecipitates corroborated these results (table S5). In DKO hearts, C/EBP instead interacted with ATF4 and ATF3 (table S5). Previously, the induction of Atf3 was detected in the terminal stages mitochondrial stress responses along with UPRmt (12).

Further interplay of the three proteins is illustrated by the fact that mitochondrial dysfunction in C/EBP-deficient cells exacerbated the ISR stress and led to ATF4 activation similar to CHOP KO (fig. S6B). Interaction of CHOP with C/EBP was previously proposed in the context of mitochondrial dysfunction, wherein CHOP/C/EBP dimers are thought to bind and activate the promoters of UPRmt-responsive genes (5). Consistent with these results, we propose that C/EBP-CHOP heterodimers might act as suppressors of ATF4 overactivation upon mitochondrial dysfunction.

C/EBP is primarily regulated at the translational level and exists in three different isoforms, two activating [Liver-enriched activator protein (LAP1) and LAP2], and one inhibitory [Liver-enriched inhibitor protein (LIP)] (39). The C/EBP target genes are presumably positively regulated by LAP1/2 proteins, whereas LIP binding is thought to repress the transcription of respective promoter (39), although recently more complex functions have been proposed for C/EBP LIP in vivo (40). To further dissect the interplay between CHOP and C/EBP, we assessed the levels of all three C/EBP isoforms in different models of mitochondrial dysfunction. COX10 KO cells with strong chronic mitochondrial dysfunction presented an increase of all C/EBP isoforms (fig. S6C). Acute mitochondrial dysfunction caused by actinonin treatment in MEFs or DARS2 deficiency in heart had a milder effect on the levels of LAP isoforms (Fig. 6C and fig. S6D). Still, C/EBP LIP levels were strongly increased by actinonin treatment in WT cells (Fig. 6C). Notably, this effect was strongly blunted in CHOP-deficient cells and DKOL mice, indicating that an increase in C/EBP LIP levels is dependent on the CHOP presence (Fig. 6C and fig. S6D). In general, the CHOP presence seems to have a positive effect on the C/EBP levels in MEFs, indicating a regulation opposite to that of ATF4.

Under ER stress, CHOP and C/EBP LIP are shown to act in concert to exert their respective functions in the nucleus (41). According to the proposed model, CHOP depends on the interaction with C/EBP LIP to enter the nucleus, while the interaction with CHOP is thought to mask the nuclear export signal (NES) of C/EBP LIP, thereby reducing its exclusion from the nucleus and subsequent proteasomal degradation (41). To test whether C/EBP LIP plays a role in the direct regulation of the mitochondrial dysfunctioninduced ISR, we expressed mutant LIPL120T, carrying a leucine-to-threonine substitution predicted to disrupt NES (42), in CHOP KO cells treated with actinonin (Fig. 6D). The expression of LIPL120T in CHOP KO cells resulted in intense ablation of basal and actinonin-induced ATF4 mRNA and protein levels and a marked decrease in the mRNA and protein levels of its downstream targets, even in the absence of mitochondrial insult (Fig. 6D and fig. S6E). Moreover, expression of LIPL120T mutant resulted in decreased expression of the endogenous C/ebp gene (fig. S6E). Intriguingly, moderate overexpression of WT C/EBP LIP in CHOP KO cells resulted in a mild further increase of ATF4 levels upon mitochondrial dysfunction (Fig. 6E). In contrast, C/EBP LIPL120T mutant suppresses ATF4 while also decreasing endogenous C/EBP levels (Fig. 6E). These results also suggest that mutant C/EBP LIPL120T does not require CHOP for its action.

It has been shown that ER stress leads to interdependent translocation and retention of C/EBP and CHOP inside the nucleus (41). Therefore, we next investigated the effects of mitochondrial stress on subcellular localization of C/EBP, CHOP, and ATF4. In both WT and CHOP KO cells, C/EBP and ATF4 were detected mainly in the nucleus (fig. S6F). The expression of either WT or mutant C/EBP LIP did not affect the subcellular localization of ATF4 in CHOP KO cells (Fig. 7A). Therefore, the ATF4 levels in CHOP-deficient cells appear not to be regulated through alterations in subcellular localization of LIP. Alternatively, in the absence of CHOP, C/EBP LIPL120T may bind ATF4 and prevent its translocation to the nucleus, thus promoting its degradation. To test this hypothesis, we incubated WT and CHOP KO cells in the presence or absence of the proteasome inhibitor MG132. In control conditions, both ATF4 and C/EBP were rapidly degraded, and only a modest fraction was retained and transported to the nucleus (Fig. 7B and fig. S6G). The rate of turnover, however, appeared not to be affected by mitochondrial function or CHOP deficiency (Fig. 7B and fig. S6G). In turn, mitochondrial dysfunction, induced by actinonin treatment, induced translocation of ATF4 to the nucleus and promoted activation of ISR. Of note, the expression of LIPL120T mutant resulted in lower levels of ATF4 in all fractions (Fig. 7B and fig. S6G). Overall, these results suggest that fine-tuning of mitochondrial stress responses is dependent on CHOP:C/EBP LIP interaction but not their subcellular localization nor their potential effects on the nuclear translocation of ATF4.

(A) Cell fractionation followed by the Western blot analysis of the ATF4 and three C/EBP isoforms in actinonin-treated (48 hours) WT or CHOP KO MEFs expressing WT C/EBP LIP or C/EBP LIPL120T mutant. (B) Cell fractionation followed by the Western blot analysis of the ATF4 and three C/EBP isoforms in actinonin-treated (48 hours) CHOP KO MEFs expressing WT C/EBP LIP and C/EBP LIPL120T mutant along with the WT cells and respective controls. The MG132 (15 M) was applied in the last 6 hours of the actinonin treatment. Elevated protein ubiquitination reflects proteasome inhibition. (A and B) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and H3K4me3 were used as loading controls and to determine quality of fractionation (n = 3). (C) CHOP levels increase early upon mitochondrial dysfunction leading to its association with C/EBP. The interaction with C/EBP likely promotes translocation of CHOP to the nucleus where it negatively regulates Atf4 levels and transcription of downstream ISR targets. Abrogation of CHOP results in increased ATF4:C/EBP association and transcription of ISR-regulated genes, created with BioRender.com.

Understanding of the mitochondrial stress response in mammals remains incomplete. In the present study, we uncovered an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBP, and ATF4. Contrary to its previously proposed role as a transcriptional activator of UPRmt, we present strong evidence that CHOP, through its interaction with C/EBP, attenuates prolonged ISR and mitochondrial cardiomyopathy through regulation of ATF4 levels (Fig. 7). Our results argue that upon mitochondrial dysfunction, the interaction of CHOP with C/EBP is needed for the adjustment of an ATF4-regulated transcriptional program. Very early upon DARS2 depletion, Chop is increasingly expressed (2) and forms a complex with C/EBP, which might facilitate the translocation of CHOP:C/EBP heterodimers to the nucleus. Regulation of ATF4 levels by C/EBP isoform LIP inhibition was proposed during ultraviolet (UV) stress, but CHOP was shown not to play a role in this context (43).

Similar to CHOP, C/EBP is a pleiotropic transcription factor that contributes to the regulation of homeostasis in several tissues, including bone, skin, and fat (40). We showed that in the context of mitochondrial dysfunction, the C/EBP accumulates in the cell (in particular, LIP isoform) and dimerizes with CHOP to presumably prevent overactivation of an ATF4-mediated response. In the absence of CHOP, C/EBP dimerizes with ATF4, which correlates with further induction of ISR. Our data suggest that C/EBP also dimerizes with ATF3 when CHOP is absent in DKO animals. ATF3 is shown to be activated during the second stage of ISR (12, 44). Once expressed, ATF3 binds promoters of ISR-responsive genes, leading to a subsequent suppression of transcription back toward the basal level (44). It is possible that also in the DKO animals, ATF3:C/EBP interaction is part of the feedback loop intended to suppress the ATF4 overactivation. In contrast, the interaction of ATF4 with C/EBP positively activates targeted genes under different conditions (45), which might have a deleterious outcome leading to, e.g., skeletal muscle atrophy (46). In contrast, we show that a dominant-negative C/EBP LIPL120T fully suppresses Atf4 and C/ebp overexpression upon mitochondrial dysfunction and down-regulates even basal levels of these transcription factors. Our findings thus suggest that C/EBP acts as a promiscuous transcription factor in the context of mitochondrial dysfunction, whereby differential transcriptional activity and associated functional outcomes are determined via interactions with CHOP and ATF4 (Fig. 7C). Further work is however required to dissect precise mechanisms of the observed interplay between CHOP, ATF4, and C/EBP.

CHOP is a transcription factor that is ubiquitously expressed at very low levels but quickly activated by a variety of insults such as ER stress, amino acid deprivation, glucose starvation, and UV irradiation (47). To date, CHOP was mostly studied in the context of ER stress, where it was proposed to regulate many pro- and anti-apoptotic genes in the late phase of ISR (47, 48). While numerous functions related to cell proliferation, differentiation, and development have been described for this transcription factor, in unstressed conditions, CHOP-deficient mice do not present any conspicuous phenotype (48, 49). Nevertheless, these mice seem to be protected from transient renal insufficiency caused by acute tubular necrosis (49). CHOP depletion seems to be beneficial in various other conditions, e.g., by delaying the onset of metabolic disease in several diabetic models (50), protecting livers from diet-induced hepatosteatosis (51), or delaying the onset of brain ischemia-induced neuronal cell death (52). Collectively, these studies suggest that loss of CHOP often leads to beneficial effects by delaying apoptosis in vivo. Unexpectedly, in mitochondrial mutants, CHOP depletion does not seem to decrease levels of proteins involved in the activation of apoptosis, as even the proposed bona fide CHOP targets BH3 interacting-domain death agonist (BID), Bcl-2-like protein 11 (BIM), ERO1A, and Tribbles homolog 3 (TRIB3) further increase their levels in DKO mutants.

We also provide evidence that CHOP loss is detrimental in mitochondrial mutants as it leads to early-onset fatal mitochondrial cardiomyopathy. This is, at least in part, mediated by the overactivation of ISR that is paralleled by inhibition of global protein synthesis and appears to be beneficial for a short time as DKOE animals maintain higher levels of OXPHOS complexes and balanced mitochondrial translation. However, loss of CHOP mitigates sustained suppression of protein synthesis in vivo that results in rapid loss of OXPHOS complexes and mitochondrial respiration. This is likely to affect mitochondrial import capacity leading to vicious cycle of damaging events. Simultaneously, mRNA translation rates are restored in DKOL around P17, coinciding with a detrimental phenotype. This is partly reminiscent of a transition from the acute to prolonged ISR in the cellular model of ER stress (37). During the acute ISR phase, global translation is reduced, and only a subset of stress-responsive mRNAs are translated, whereas the prolonged ISR is characterized by recovery of global translation while still allowing execution of acute ISR translational programs (37). While the prolonged ISR appears to have a beneficial effect in vitro by preventing cell death under conditions of ER stress (37), we show that in vivo, mitochondrial dysfunction in the heart impedes a sustained chronic ISR program. To this end, recovery of protein synthesis escalates ER stress possibly by increasing ER load. Recovery of global translation is also expected to significantly increase the energy demand and thereby result in energy depletion caused by massively reduced respiratory capacity due to DARS2 loss. According to the energy starvation hypothesis, suboptimal ATP supply predisposes for the contractile dysfunction observed during heart failure (53). It was shown that even very few cardiomyocytes with severe mitochondrial dysfunction are sufficient to promote ventricular arrhythmias, which lead to heart failure (54). Considering the severe impairment of electron transport chain (ETC) function in DKO mice, the occurrence of cardiac arrhythmias in those animals, contributing to the pathology, seems likely.

The pathology observed in DKOL animals is not a DARS2-specific phenomenon but a prevalent cardiac phenotype in mutants affecting mitochondrial gene expression and translation, as shown by a comparative study of five different models (11). At the molecular level, we demonstrated markedly increased serine synthesis and remodeling of the one-carbon cycle in hearts of DARS2 KO, DKOL mice, and cell culture models, attributable to OXPHOS deficiency and not to the loss of DARS2 in particular. Moreover, similar changes are described in other models and different tissues (11, 13, 14, 55). The vast majority of these alterations have been attributed to ATF4, which has been identified as a major regulator of amino acid metabolism feeding into the folate cycle during ISR induced by different stress signals including mitochondrial dysfunction (13, 14, 56). Although ATF4 may be activated by several different pathways, such as nuclear respiratory factor 2 (NRF2) stabilization or mechanistic (previously mammalian) target of rapamycin (mTOR) signaling (57, 58), we showed that ATF4 up-regulation caused by mitochondrial OXPHOS deficiency could be successfully prevented by suppression of the ISR.

In conclusion, we found a regulatory mechanism that fine-tunes the activation of the ISR upon mitochondrial dysfunction. We showed that CHOP is needed to prevent excessive activation of the ATF4-mediated stress response that results in cardiotoxic effects. This is mediated by CHOP interaction with C/EBP, which likely promotes CHOP:C/EBP heterodimer translocation to the nucleus. Our results also highlight an unforeseen opportunity of exploring a therapeutic intervention targeting ATF4 activity in various mitochondrial diseases.

DARS2 KO (Dars2fl/fl; Ckmm-Cre+/tg) mice were generated as previously described (2). WT control animals (Dars2fl/fl; Ckmm-Cre+/+ and Dars2+/fl; Ckmm-Cre+/+) were also obtained from this breeding. CHOP KO [B6.129S(Cg)-Ddit3tm2.1Dron/J] mice were obtained from the Jackson laboratory. Those mice are characterized by a Chop::LacZ KO allele, resulting in the whole-body KO of Chop (Chop/) (49).

Conditional Dars2-floxed mice (Dars2fl/fl) were crossed to CHOP KO mice (Chop/) to obtain CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/). Triple transgenic mice were generated by intercrossing of CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/), with transgenic mice harboring one copy of the Cre recombinase under control of the striated muscle creatine kinase (Ckmm) promoter (Ckmm-Cre+/tg). Resulting heterozygous triple transgenic mice (Dars2+/fl; Ckmm-Cre+/tg; Chop+/) and CHOP-deficient animals with floxed Dars2 alleles (Dars2fl/fl; Chop/) were used to lastly generate CHOP KO (Dars2+/fl; Ckmm-Cre+/+; Chop/ and Dars2fl/fl; Ckmm-Cre+/+; Chop/) and DKO (Dars2fl/fl; Ckmm-Cre+/tg; Chop/) mice. Genotyping for the Dars2 allele was performed as previously described (2). Genotyping for the Ckmm-Cre and Chop alleles was performed following the instructions of the Jackson laboratory using the protocol 22415 along with the primers oIMR3884, oIMR3885, and oIMR3886 for the Chop allele and the protocol Tg(Ckmm-Cre)5Khn along with the primers oIMR1085, oIMR6754, oIMR8744, and oIMR8745 for the Ckmm-Cre allele, respectively (www.jax.org). One- to 6-week-old animals were used in experiments approved and authorized by the Animal Ethics Committee of North-Rhein Westphalia (Landesamt fr Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen) following the German and European Union regulations. Animal work was performed in conformity with the recommendations and guidelines of the Federation of European Laboratory Animal Science Associations.

Immortalized MEFs and fibroblasts were cultured in standard conditions, at 37C and 5% CO2. The cell culture medium was composed of Dulbeccos modified Eagles medium [glucose (4.5 g/liter), GlutaMAX, and sodium pyruvate; Gibco Life Technologies] supplemented with 10% Fetal Bovine Serum Premium, South American Origin (Biowest) and penicillin-streptomycin (Pen-Strep) (Gibco Life Technologies). In conditions of mitochondrial dysfunction (induced either genetically or by treatment), the medium was additionally supplemented with uridine (50 g/ml). At 90% confluency, cells were split cell typedependently in ratios ranging from 1:4 to 1:20.

Generation of immortalized MEF lines. Embryos from embryonic day 13.5 of intercrossed CHOP KO (Chop/) mice were used to isolate primary MEFs (59). Immortalization was achieved by transformation with the SV40 T antigen.

Drug treatments. For induction of mitochondrial dysfunction by actinonin treatment, 80% confluent cells were treated for 48 hours with 100 M actinonin (Sigma-Aldrich). Proteasome was inhibited with 15 M MG132 for the last 6 to 8 hours of treatment as indicated. Inhibition of the ISR was achieved by 4- or 48-hour 1 M ISRIB (Sigma-Aldrich) treatments of 90% confluent cells. All compounds were solubilized in dimethyl sulfoxide (DMSO). Untreated cells were supplemented with corresponding amounts of the solvent. Treatments were renewed on a daily basis.

Transfection. Transfection of plasmids conferring hygromycin resistance (pTK-Hyg LIP, pTK-Hyg LIPwestern, pTK-Hyg LAP, and pTK-Hyg C/EBP) was performed with Lipofectamine 2000 or Lipofectamine LTX (Invitrogen) according to the manufacturers instructions using the forward transfection procedure. Seventy-two hours after transfection, the culture medium was replaced by hygromycin-supplemented (100 g/ml) medium for negative selection of untransfected cells. Transfected cells were maintained in hygromycin-supplemented (100 g/ml) medium.

Cell growth estimation. To estimate differences in cell growth caused by CHOP deficiency and/or mitochondrial dysfunction, an equal number of cells were seeded and treated as indicated. The numbers of cells were determined at the indicated time points using the Countess Automatic Cell Counter (Invitrogen) combined with trypan blue staining.

Freshly collected hearts were immediately transferred into 10 ml of prechilled mito-isolation buffer (MIB) [100 mM sucrose, 50 mM KCl, 1 mM EDTA, 20 mM N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid, and 0.2% bovine serum albumin (BSA) free from fatty acids (pH adjusted to 7.2)] supplemented with 1 g of subtilisin (Sigma-Aldrich) per mg of tissue. Approximately 20 long strokes of a Potter S (Sartorius) homogenizer at 1000 rpm were required for homogenization. After centrifugation (800g, 5 min, 4C), the mitochondria-containing supernatant was transferred into a fresh tube. Pelleted mitochondria (8500g, 5 min, 4C) were resuspended in 30 ml of MIB and subjected to a third centrifugation step (700g, 5 min, 4C). Last, mitochondria were pelleted (8500g, 5 min, 4C) and resuspended in 100 l of macrophage inflammatory protein without BSA. Protein concentration of mitochondria was determined using Bradford reagent (Sigma-Aldrich) according to the manufacturers instructions. Mitochondria were either immediately used (respirometry or in organello translation) or snap-frozen and stored at 80C.

High-resolution respirometry using an Oxygraph-2k (OROBOROS Instruments) and a carbohydrate substrate-uncoupler-inhibitor titration protocol was conducted to determine mitochondrial oxygen consumption rates. First, the respiration medium (120 mM sucrose, 50 mM KCl, 20 mM tris-HCl, 1 mM EGTA, 4 mM KH2PO4, 2 mM MgCl2, and 0.1% BSA) was added to the Oxygraph chamber, and air equilibration was performed. Next, 25 g of freshly isolated cardiac mitochondria was added. The respiration medium was supplemented with 2 mM pyruvate, 0.8 mM malate, 2 mM glutamate, and 2 mM adenosine 5-diphosphate (ADP) to assess CI-dependent respiration. By providing additional 4 mM succinate, convergent CI- and CII-dependent respiration was determined. Inhibition of ATP-synthase-complex V (CV) by addition of oligomycin (1.5 g/ml) allowed evaluating the coupling efficiency. The maximal capacity of the electron transfer system (ETS) was assessed by titration of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (0.5 M increments). Maximal capacity of the ETS of CII solely could be determined by inhibition of CI through addition of 0.5 M rotenone. Last, inhibition of CIII by supplementation of 2.5 M antimycin A allowed the determination of the residual oxygen consumption.

De novo mitochondrial translation was assessed by incubation (1 hour, 37C, on rotating wheel) of 1.5 mg of freshly isolated mitochondria in 1 ml of 35S-translation buffer [100 mM mannitol, 10 mM Na-succinate, 80 mM KCl, 5 mM MgCl2, 1 mM KH2PO4, 25 mM Hepes (pH 7.4), 5 mM ATP, 200 M GTP, 6 mM creatine phosphate, creatine kinase (60 g/ml), cysteine (60 g/ml), tyrosine (60 g/ml), amino acids (60 g/ml) (Ala, Arg, Asp, Asn, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, and Val), 35S-methionine (7 l/ml)]. Subsequently, mitochondria were pelleted (12,000g, 2 min) and resuspended in 1 ml of nonradioactive translation buffer containing methionine instead of 35S-methionine. Half of the sample (pulse fraction) was pelleted again, resuspended in 100 l of SDSpolyacrylamide gel electrophoresis (PAGE) loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% -mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue], and lysed (30 min, room temperature) before transfer at 20C. For the cold chase allowing to estimate the protein turnover, the remaining 500 l of resuspended mitochondria was incubated for 3 hours at 37C on a rotating wheel. Subsequently, the chase fraction was pelleted, resuspended in 100 l of SDS-PAGE loading buffer, and lysed as the pulse sample before.

Separation of mitochondrial proteins was achieved by SDS-PAGE. Ten microliters per sample was loaded on a 15-cm-long, 15% polyacrylamide gel and run in a SE600X Chroma Deluxe Dual Cooled Vertical Protein Electrophoresis Unit (Hoefer) overnight at 80 V continuously. After fixing (50% methanol and 10% acetic acid) for 30 min, staining in Coomassie solution, and destaining (20% methanol and 10% acetic acid) of the polyacrylamide gel, the latter one was placed on Whatman paper (GE Healthcare) and dried (2 hours, 80C) in a gel dryer. For detection of radioactive signals of de novo synthetized proteins, Amersham Hyperfilm MP (GE Healthcare) was exposed to the dried polyacrylamide gel.

Cellular protein lysates. Washed cell pellets were resuspended in cold radioimmunoprecipitation assay buffer [150 mM NaCl, 1% Triton X-100 (v/v), 0.5% Na-deoxycholate (w/v), 0.1% SDS (w/v), 50 mM tris-HCl (pH 7.4), 50 mM NaF, and 2 mM EDTA] supplemented with 1 protease inhibitor cocktail (Sigma-Aldrich) and 1 PhosSTOP phosphatase inhibitor cocktail (Roche). Next, cells were incubated 30 min on ice with brief vortexing every 10 min. Following 2 45-s sonication, the lysates were cleared (10 min, 20,000g, 4C) and transferred into fresh tubes.

Cardiac tissue protein lysates. Homogenization of 25 mg of cardiac tissue samples in 400 l of cold organ lysis buffer [50 mM Hepes (pH 7.4), 50 mM NaCl, 1% Triton X-100 (v/v), 0.1 M NaF, 10 mM EDTA, 0.1% SDS (w/v), 10 mM Na-orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 1 protease inhibitor cocktail (Sigma-Aldrich), and 1 PhosSTOP phosphatase inhibitor cocktail (Roche)] was performed with the Precellys CK 14 (Bertin Technologies) (5000 rpm, 30 s). Cleared protein lysates (45 min, 20,000g, 4C) were transferred into fresh tubes. Determination of protein concentration was performed with Bradford reagent (Sigma-Aldrich) according to the manufacturers instructions. Protein lysates were stored at 80C.

SDSpolyacrylamide gel electrophoresis. Protein samples were dissolved in SDS-PAGE loading buffer [50 mM tris-HCl (pH 6.8), 2% SDS (w/v), 10% glycerol (v/v), 1% -mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue] before denaturation. Depending on the required range of protein sizes, the proteins were separated on 8 to 15% acrylamide gels [stacking gel: 5% acrylamide-bisacrylamide (37.5:1), 12.5 mM tris-HCl, 0.1% SDS (w/v), 0.25% Ammonium persulfate (APS), and 0.25% Tetramethylethylenediamine (TEMED) (pH 6.8); separating gel: 8 to 15% acrylamide-bisacrylamide (37.5:1), 37.5 mM tris-HCl, 0.1% SDS (w/v), 0.1% APS, and 0.1% TEMED (pH 8.8)] in running buffer [25 mM tris-HCl, 250 mM glycine, and 0.1% SDS (w/v) (pH 8.3)].

Western blot. Transfer of proteins on a nitrocellulose membrane by Western blot was conducted in transfer buffer (30 mM tris-HCl, 240 mM glycine, 0.037% SDS, and 20% methanol) at 400 mA for 2 hours at 4C. For a first evaluation of the transfer, shortly washed membranes (dH2O) were stained with Ponceau S solution (Sigma-Aldrich). Depending on the antibody requirements, destaining and blocking of membranes were performed for 1 hour either in 5% milk-PBST (Phosphate-Buffered Saline/Tween) or 3% BSA-TBST (Tris-Buffered Saline/Tween) on a gently shaking platform before subsequent immunodecoration with the indicated antibodies according to the manufacturers instructions. Secondary horseradish peroxidasecoupled antibodies (1:5000) were incubated for 1 hour before detection by Pierce ECL Western blotting substrate (Thermo Fisher Scientific). Densitometry-based quantification of Western blots was performed with ImageJ and Image Studio Lite Software.

Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed on the basis of the NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to the manufacturers instructions. For analysis of mitochondrial supercomplexes, 10 g of mitochondria was lysed with 4% of digitonin. Analysis of individual mitochondrial complexes was conducted after lysis of 10 g of mitochondria in 1% n-dodecyl--D-maltoside (DDM). After completion of lysis (15 min on ice), lysates were cleared (30 min, 20,000g, 4C), and the resulting supernatant was loaded on a 4 to 16% bis-tris gradient gel. Subsequently, proteins were transferred to an Amersham Hybond polyvinylidene difluoride membrane (GE Healthcare) by Western blot and subsequently immunodecorated with indicated antibodies.

Independently normalized label-free proteomics and RNA sequencing data were scaled before analysis using the anota2seq algorithm (version 1.4.2) (19). Furthermore, datasets were reduced to genes identified on both platforms resulting in a total of 2556 mRNAs for analysis. Analysis of changes in protein levels and total mRNA was performed using the anota2seqAnalyze function to identify differences between CHOP KO, DARS2 KO, and DKO compared to WT. Changes were considered significant when passing the following parameters within the anota2seqSelSigGenes function: maxPAdj = 0.15, minSlopeTranslation = 1, maxSlopeTranslation = 2, selDeltaPT = log2(1.2), selDeltaP = 0, and selDeltaT = 0. Changes in translation or protein stability, as well as changes in mRNA abundance, were characterized using the anota2seqRegModes() function. GO analysis (60) was performed in Cytoscape (v 3.8.0) (23) using the ClueGO (v 2.5.7) app (20). Within ClueGO, four gene lists were provided corresponding to the identified modes for regulation of gene expression using anota2seq (i.e., translation/protein stability and mRNA abundance) divided into up- and down-regulated mRNAs. GO term inclusion parameter was set to a 5 gene overlap and <4% of total genes present in the GO term. For the resulting network, GO term grouping and fusion parameters were enabled, and only GO terms with a false discovery rate of <5% were displayed. Furthermore, anota2seq was applied on the full RNA sequencing dataset (14,174 protein coding transcripts) following the same approach as above. Master regulators among significantly up-regulated total mRNAs in the DARS2 KO versus WT comparison were detected using iRegulon (v1.3) with default settings (24).

The Q5 Site-Directed Mutagenesis Kit (New England Biolabs) was used to introduce a point mutation (L120T) in the pTK-Hyg LIP plasmid (41). For primer design, the New England Biolabs (NEB) online design software NEBaseChanger was used. All three steps described in the protocol [exponential amplification, Kinase, Ligase & DpnI treatment (KLD) reaction, and transformation] were performed as indicated in the manual.

Protein synthesis was determined using the nonradioactive technique called surface sensing of translation described in (61). This assay is based on the incorporation of the structural analogue of tyrosyl-tRNA puromycin in nascent polypeptide chains and subsequent detection of puromycylated proteins using an anti-puromycinspecific antibody.

Briefly, mice were injected at the indicated time points intraperitoneally with 0.04 mol of puromycin dissolved in phosphate-buffered saline (PBS) per gram of body weight. Thirty minutes after injection, the animals were euthanized, and collected tissues were snap-frozen in liquid nitrogen. Subsequently, protein lysates of the collected tissues were prepared and processed by SDS-PAGE and Western blot. The relative signal intensity of the anti-puromycinspecific antibody is proportional to the relative protein synthesis rates at the time point of puromycin injection.

Briefly, mice were injected intraperitoneally with 5 g of ISRIB (stock solution: 5 mg/ml in DMSO, dissolved in PBS up to the weight-dependent injection volume of 30 to 50 l) per gram of body weight or the corresponding amount of PBS-dissolved solvent (DMSO) on a daily basis for the indicated time periods. One day after the last injection, the animals were euthanized, and collected tissues were snap-frozen in liquid nitrogen. Subsequently, protein lysates of the collected tissues were prepared and processed by SDS-PAGE and Western blot.

Numerical data are expressed as means SD. Statistical analysis was performed using the indicated statistical tests. If not indicated differently, statistical significance was considered for P < 0.05. With exception of multivariate analysis of variance (MANOVA) and omics analyses, all statistical tests were performed, and graphs were plotted using GraphPad Prism 8.0 software. MANOVA was performed with XLSTAT version 2020.3 software.

Acknowledgments: We wish to thank the CECAD Imaging and Proteomics Core Facilities for excellent support. Funding: The work was supported by Aleksandra Trifunovics grants of the Deutsche Forschungsgemeinschaft [DFG; German Research Foundation (SFB 1218)Projektnummer 269925409 and TR 1018/8-1] and the Center for Molecular Medicine Cologne, University of Cologne. S.K. received scholarship from the Cologne Graduate School of Ageing Research (CGA). I.T. acknowledges Senior Scholar Award from Le Fonds de recherche du QubecSant (FRQS) and support from Canadian Institutes for Health Research (MOP-363027) and Joint Canada-Israel Health Research Program (JCIHRP) (108589-001) to I.T. and O.L. O.L.s lab was supported by grants from the Swedish Research Council (2016-02891), the Swedish Cancer Society (19 0314), and the Wallenberg Academy Fellows program (2013.0181). M.H.s laboratory is supported by NIH R01 DK060596 grant. Author contributions: Conceptualization: A.T., S.K., C.O., K.Sz., O.L., I.T., and M.H. Data curation: S.K., C.O., A.T., S.B., O.L., and K.Sz. Formal analysis: S.K., C.O., A.T., S.B., O.L., and K.Sz. Funding acquisition: A.T., S.K., O.L., I.T., and M.H. Investigation: S.K., C.O., A.K., K.Se., K.Sz., C.L., S.B., and O.L. Visualization: S.K., C.O., A.T., O.L., I.T., and M.H. Writing: A.T., S.K., C.O., O.L., and I.T. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Further information and requests for resources and reagents should be addressed to and will be fulfilled by A.T. Mouse and cell lines requests include signing of material transfer agreement.

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Clinical Application of Cytokines in Cancer Immunotherapy | DDDT – Dove Medical Press

By daniellenierenberg

Introduction

Cancer is a disease characterized by the abnormalities in the regulation of cell proliferation and differentiation. Many factors contribute to cancer development including genetics,1 lifestyle, and environmental carcinogens, among others.2 Cancer is the second leading cause of death worldwide after cardiovascular disease, accounting for 9.6 million deaths in 2018 according to data from the International Agency for Research on Cancer. Lung cancer is the leading cause of cancer death (18.4%), followed by breast cancer (11.6%), and prostate cancer (7.1%).3 Clinical manifestations include pain, bleeding, lumps and ulcers at the site of disease, along with systemic symptoms such as weight loss and fatigue leading to cachexia. Traditional treatment modalities including surgery, radiotherapy, and chemotherapy have various disadvantages and cause side effects that are in some cases severe. Immunotherapies such as blockade of programmed death (PD)-1 and programmed death ligand 1 (PD-L1) immune checkpoints; chimeric antigen receptor T cell immunotherapy (CAR-T); using the monoclonal antibody against cancer antigen; and cytokine therapy offer a promising alternative to the conventional treatment approaches for cancer.4 In particular, cytokine therapy has shown encouraging results in both basic and clinical research settings.5

Cytokines are small proteins produced by various cells (immunocytes and non-immunocytes) as molecular messengers to communicate with each other or with other cells. Cytokines have versatile roles in several steps of the cancer immunity cycle including cancer antigen presentation, T cell priming and activation, effector T cell infiltration in cancer site, and cancer cell death, as shown in Figure 1. More importantly, cytokine-mediated signaling pathways control the direction of nave CD4+ T cell differentiation and thus determine the effects of anticancer immunity (Figure 2 and Table 1). Briefly, transforming growth factor (TGF-) signaling in nave CD4+ T cells is required for the differentiation of regulatory T cells (Tregs) and T helper type 17 (Th17) cells, both of which promote tumor progression. Additionally, Th17 cell differentiation and clonal expansion require a cocktail of cytokines (IL-6, IL-21, IL-23, IL-1, and TGF-).610 IL-17 secreted by Th17 cells guides macrophages and neutrophils to cancer sites and induces cancer-promoting inflammation. Th17 cells themselves also exert antitumor effects in the melanoma microenvironment by potentiating the functions of CD8+ T cells and T helper type 1 cells (Th1 cells).11 IL-10, IL-11, IL-4, and IL-13 are critical for the differentiation and development of T helper type 2 cells (Th2 cells),1217 whereas IL-12, IL-18, IL-1, and interferon (IFN)- promote Th1 cell development and activity.1823 Th1 cells modulate tumor-suppressing pathways by stimulating IFN- secretion and enhancing the cytotoxicity of natural killer (NK) cells and CD8+ T cells, while Th2 cells inhibit the anticancer immune responses by blocking Th1 cell differentiation and the release of IFN-. Vascular endothelial growth factor (VEGF) and tumor necrosis factor (TNF)- promote cancer progression by directly facilitating angiogenesis, although recombinant TNF- has been shown to enhance the effect of combined chemotherapy regimens by increasing the permeability of tumor blood vessels.2427

Figure 1 Cytokines in the cancer immunity cycle.6 1) Antigens from dead cancer cells are captured by APCs, mainly by DCs. 23) DCs present cancer antigens to T cells to prime the adaptive immune response. 45) Activated effector T cells infiltrate cancer cells and then 6) kill cancer cells. Dead cancer cells release cancer antigens to continue the immune cycle. Cytokines that have been shown to promote or inhibit the anticancer immune responses are highlighted.

Abbreviations: IFN, interferon; IL, interleukin; TGF-, transforming growth factor ; TNF, tumor necrosis factor.

Figure 2 Varied roles of cytokines involved in anticancer immunity. Different cytokines determine nave CD4+ T cell fate to Tregs, Th17, Th1 or Th2, and further regulate anticancer immunity. IL-12, IL-18, IL-1, IL-10 and IL-11 secreted by dendritic cells (DCs) drive Th1 or Th2 cell differentiation. TGF-, IL-11, IL-6, and IL-21 are important signals for Treg and Th17 cell differentiation. IFN-, IL-2, IL-15, and IL-7 secreted by Th1 cells enhance the anticancer effects of cytotoxic T lymphocytes, NK cells, B cells, and macrophages, which can be suppressed by IL-4, IL-13, and IL-10 secreted by Th2 and Treg cells. IL-17 secreted by Th17 cells play a role in the induction of cancer-promoting anticancer inflammation by MDSCs. VEGF and TNF- promote cancer progression by facilitating angiogenesis. Cytokines functions are shown in text boxes; those that promote anticancer immunity are in red while those that inhibit anticancer immunity are in black.

Because the roles of cytokines are diverse and precise applications of cytokines are greatly needed, it is urgent to update the progresses of cancer immunotherapy with cytokines. Here, we review total 2339 clinical trials using or by targeting cytokines for precise treatment of cancers registered with ClinicalTrials.gov; summarize the therapeutic efficacy of typical cytokines based on clinical data; and highlight progress in the development and application of nanomaterials for cytokine-based therapy.

In order to review clinical application of cytokines in cancer therapy, we have searched all the known cytokines in ClinicalTrials.gov. In the advanced search page of ClinicalTrials.gov, we entered cytokine name, such as IL-2, in other term section and chose completed in the recruitment status section. Then, we download the search results and screen the trials item by item to make sure that the intervention of the trial includes cytokine-based drugs and the condition of the trial is cancer. As a result, we got 25 cytokines with clinical trials that had completed recruitment in ClinicalTrials.gov, and we also checked 2 cytokines (IL-10 and IL-17) without published clinical studies for cancers because of their crucial roles in anticancer immunity. Finally, we screen out 2630 clinical trials using cytokines as either therapeutic agents or targets in treating cancers registered with ClinicalTrials.gov that had completed recruitment up to January 2021.

It is interesting that G-CSF, GM-CSF, VEGF, IL-2 and IFN- are the five most studied cytokines (Figure 3A and Supplementary Table 1), which could be explained by the fact that they have been discovered and clinically studied very early (Figure 4A and B) and they play very important roles in cancer treatment. VEGF is the most studied target for the treatment of most types of cancer because the role of VEGF in angiogenesis induction, cell proliferation and promoting vascular permeability is extremely important for cancer growth, migration and infiltration. CSF can promote proliferation and differentiation of multiple immune cells such as macrophage, granulocytes, and mononuclear phagocytes, and thus is widely used as medication to stimulate the production of white blood cells following chemotherapy. Similarly, IL-2 is used to stimulate T cell production for enhancing anti-cancer immunity. IFN- can directly inhibit tumor cell proliferation and augment anti-tumor immunity by promoting MHC expression, antigen presentation, and the function of tumor-infiltrating Th1 cells, CTLs and macrophages. The clinical trials of cytokines cover nearly all cancer types (Figure 3B) but most of the clinical trials are done on melanoma and hematological malignancies because the two cancer types have better responses and outcomes than other cancers in the immune therapy.28,29

Figure 3 Clinical research status of cytokines. Number of cancer clinical trials using cytokine-based drugs treating all cancer types (A) or each cancer type (B) registered with ClinicalTrials.gov as of January 2021.

Figure 4 Historical timelines of cytokine research. (A) Timeline of cytokine discovery. The time point is the year in which the cytokines, EPO,69 IFNs,70,71 EGF,72 G-CSF,73,74 FGF,75 IL-1,76 IL-2,77 IGF,78 TNF,79 GM-CSF,80 TGF-,81 IL-3,82 IL-4,83 IL-6,84 IL-7,85 IL-10,86 IL-12,87 IL-13,88 VEGF,89 IL-11,90 IL-15,91 IL-17,92 IL-18,93 IL-21,94,95 and CCL21,96,97 were first described. (B) Timeline of the first clinical trials of cytokines for cancer treatment. The time point is the year that the trial was first registered with ClinicalTrials.gov. Clinical trial registry (NCT) numbers are shown.

The year of discovery of each cytokine and the year of the first clinical trial with the cytokine for cancer treatment are shown, respectively, in Figure 4A and B, which gives a visualized understanding of research progresses of cytokines in certain years. The main cytokines were discovered in the last 3 decades of last century and the clinical trials were carried out intensively between 1998 and 2008. The interval time from the discovery to the first clinical trial of certain cytokine is varied with the maximum of 95 years (EPO) and minimum of 7 years (IL-21). The cytokine-based drugs could be grouped into two types: cytokine drugs and drugs targeting cytokines.

IL-2, type I IFN, IL-12, chemokine (C-C motif) ligand (CCL) 21, and colony-stimulating factors (CSF) family cytokines are known to promote anticancer immunity. Although IFN-, TNF-, and IL-1 families play a dual role in the cancer immunity cycle, they are widely studied for their anticancer activity. In this section, we present the efficacy of these cytokine-based drugs in cancer treatment.

There are 268 trials registered with ClinicalTrials.gov using IL-2 for cancer treatment. Of the 52 trials for which results are available, 7 treated cancer with IL-2 alone, including 3 trials using IL-2 and 4 using IL-2 derivatives (hu14.18-IL12, denileukin diftitox [ONTAK], and ALT-801) for the treatment of melanoma, breast cancer, metastatic renal cell carcinoma (mRCC), and neuroblastoma. There were 45 trials investigating the effects of IL-2 combined with other therapies. In general, melanoma and leukemia responded better than other types of cancer to IL-2 treatment and IL-2 performed more outstanding when combined with other therapies in cancer treatment. The objectives of clinical studies using IL-2 in cancer treatment are summarized in Figure 5.

Figure 5 Application of IL-2 in 52 clinical trials for cancer therapy. IL-2 has been used in combination with lymphocytes, NK cells, genetically engineered cells, monoclonal antibodies, and tumor antigens as well as with radiotherapy, chemotherapy, and chemoradiotherapy. Dose finding, selected studies, and effects of recombinant IL-2 are shown. Numbers in parentheses are the number of clinical trials.

In 1992, high-dose aldesleukin became the first cytokine approved by the US Food and Drug Administration (FDA) for the treatment of mRCC based on an objective response rate (ORR) of 14% in 255 patients.30 In 2006, a new trial using high-dose aldesleukin for the treatment of mRCC was conducted by the Cytokine Working Group to evaluate the clinical utility of PD-L1, B7 homolog 3 protein, carbonic anhydrase 9, plasma VEGF, and fibronectin levels as biomarkers for therapeutic response monitoring. PD-L1 and B7 homolog 3 protein were identified as candidate markers but require independent validation.31 The IL-2 derivative hu14.18-IL-2, which consists of 2 molecules of IL-2 covalently linked via the Fc region, has demonstrated long-term tumor control in animal models.32 In Phase I and II trials, hu14.18-IL-2 prolonged the tumor-free survival period in some patients with recurrent stage III or stage IV melanoma following resection.33

The anticancer efficacy of IL-2 may be enhanced when it is used in combination with other immunotherapies and chemotherapy agents. In one trial, 6 of 11 patients with non-Hodgkin lymphoma treated with IL-2 plus rituximab achieved complete or at least partial remission (NCT00994643). A Phase III trial reported that IL-2 combined with other immunotherapeutic reagents, including dinutuximab and granulocyte/macrophage (GM)-CSF, enhanced the efficacy of isotretinoin in the treatment of neuroblastoma after stem cell transplantation; the 3-year event-free survival rates for isotretinoin with and without immunotherapy is 62.9% against 48.1%, respectively (NCT00026312). Results from 3 other trials supported the survival benefits of combination treatment (NCT01334515, NCT01592045, and NCT01041638). In addition to immunotherapy, data from 27 trials suggest that chemotherapy drugs such as ONTAK, etoposide, cyclophosphamide can increase the antitumor activity of IL-2.

Given the therapeutic effects of IL-2, other members of the IL-2 family including IL-7, IL-15, and IL-21, that are known to act independently or synergistically with IL-2 in the anticancer immune response have been investigated for the treatment of breast cancer, renal cell cancer, melanoma, and leukemia. However, in a trial of IL-7 in patients with metastatic castration-resistant prostate cancer (NCT01881867), the number of T cells per 300,000 peripheral blood mononuclear cell was not higher than in the comparator group. In trials investigating the efficacy of intravenous (NCT01385423 [phase I]) or subcutaneous (NCT02395822 [phase II]) recombinant human IL-15 in enhancing the effects of NK cell therapy in patients with acute myelogenous leukemia, 32% of patients in the phase I trial and 40% of those in the Phase II trial achieved complete remission.34 In a phase II trial evaluating the efficacy and safety of IL-21 in the treatment of malignant melanoma (NCT01152788), IL-21 did not demonstrate a clinical benefit over dacarbazine, with a progression-free survival (PFS) of 1.87 vs 2.04 years, although IL-21 was associated with fewer adverse events.

Type I IFNs including IFN- and IFN- play an essential role in the presentation of cancer antigens by mediating the maturation and activation of dendritic cells (DCs) and inducing the expression of major histocompatibility complex I molecules on tumor cells.35,36 Since 1996, there have been 248 trials investigating the therapeutic potential of IFN- in the treatment of cancers including melanoma and leukemia, with results for 76 available on ClinicalTrials.gov. Although there is in vitro evidence that IFN- more potently inhibits tumor cell proliferation than IFN-, there have been no clinical trials demonstrating its efficacy in cancer therapy.

A study conducted from 1988 to 2010 evaluating the efficacy of high-dose IFN--2b in 1150 patients who had undergone resection for stage II or III melanoma (NCT00003641) found no improvements in 5-year relapse-free survival and overall survival (OS) rates. In addition to treating melanoma, IFN- has been used as first-line treatment for mRCC, but was found to be less effective than the tyrosine kinase inhibitor su011248 in a phase III trial (NCT00083889). Various forms of IFN- including pegylated (PEG)-IFN- and recombinant adenovirus (rAd)-IFN (encoding IFN-2b) have been evaluated in clinical studies. Two trials compared the efficacy of PEG-IFN- and IFN- in different types of cancer; in patients with melanoma, the median OS was 25.63 months with PEG-IFN- vs 20.67 months with IFN- (NCT03552549), whereas in chronic myelogenous leukemia, the 12-month survival rate was slightly higher in the IFN- group than in the PEG-IFN- group (91.3% [158/173] vs 90.1% [154/171]) (NCT03547154). In both trials, more severe adverse effects were reported in patients receiving PEG-IFN- treatment. In another phase II study (NCT01687244), rAd-IFN showed promising results in patients with Bacillus Calmette-Gurin-refractory or relapsed bladder cancer.

The antitumor activity of IFN- can be dramatically enhanced by including other types of immunotherapy in the treatment regimen. In a phase III trial initiated in 2004 (NCT00738530), 649 patients with mRCC received IFN- alone or with bevacizumab; PFS was 5.5 and 10.2 months, respectively, and ORR was 12.5% and 32.4%, respectively. When the chemotherapy drug vinblastine was added to the regimen, the PFS was increased to 274 days (NCT00520403). Results from 5 other trials supported the effects of IFN- in combination with bevacizumab. A trial assessing the efficacy of pembrolizumab (anti-PD-1) plus sylatron (PEGIFN--2b) for the treatment of advanced cholangiocarcinoma was initiated in 2017, but no patients completed the study due to adverse effects (NCT02982720).

As the sole type II IFN, IFN- is a typical pro-inflammatory cytokine that exerts antitumor effects by suppressing proliferation and promoting apoptosis in tumor cells and inducing necrotic death and inhibiting angiogenesis in tumors. However, IFN- was shown to upregulate PD-L1 expression on tumor cells, which suppressed anticancer immunity through the binding of PD-L1 to its receptor PD-1 on lymphocytes.23 Despite these conflicting roles in cancer, the therapeutic potential of recombinant or adenovirus-delivered IFN- is being investigated in 22 trials, although only 2 have posted results. In a phase II trial (NCT00501644), 59 patients with ovarian or fallopian tube cancer or primary peritoneal cancer were treated with subcutaneous GM-CSF and IFN- before and after intravenous carboplatin; the ORR was 56% and median time to progression was 6 months. However, there was no control group in this trial. Another phase II trial assessed the efficacy of IFN- combined with 5-fluorouracil (FU), leucovorin, and bevacizumab in patients with metastatic colorectal cancer (CRC) (NCT00786643), but the specific contribution of IFN- to the treatment effect was not investigated. In summary, the efficacy of IFN- in cancer therapy has yet to be established.

IL-12, which is mainly produced by antigen-presenting cells, plays an important role in regulating innate and adaptive immune responses. There are 47 registered Phase IIII trials evaluating the efficacy and safety of intratumoral IL-12 administration either alone (22 trials) or with other immunotherapies (eg, DCs, T cells, and vaccines; 17 trials) for the treatment of melanoma, Merkel cell carcinoma, ovarian carcinoma, head and neck squamous cell carcinoma, and other cancers. In most cases a plasmid encoding IL-12 was used. A phase III trial that enrolled 51 patients with melanoma optimized the therapeutic strategy (NCT01502293): patients underwent 5 treatment cycles at 3-month intervals consisting of 3 intratumoral injections of IL-12 plasmid immediately followed by in vivo electroporation, which resulted in an ORR of 32.1% higher than the other two groups (underwent 9 cycles [25.0%] and 2 cycles [25.0%] at 6-week intervals, respectively). On the other hand, tumor-infiltrating CD8+T cells expressing IL-12 showed unsatisfactory results for the treatment of metastatic melanoma in a phase I/II trial (NCT01236573).

TNF was initially recognized as an antitumor cytokine. However, endogenous TNF induces the expression of multiple cytokines that act on M2 macrophages to stimulate the extracellular matrix remodeling as well as the differentiation of myeloid endothelial progenitor cells, which promotes tumor angiogenesis.27 These findings suggest that TNF can serve as either therapeutic target or agent. The first clinical trial of TNF for cancer treatment was initiated in February 1992; to date, there have been 20 trials involving at least 1152 participants in which TNF or related biological agents were used to treat 3 main tumor typesnamely, melanoma, CRC, and head and neck cancer. Only one study has published results. Etanercept, a TNF inhibitor, was investigated for the treatment of idiopathic pneumonia in patients with leukemia and lymphoma after stem cell transplantation (NCT00309907), but the results did not reflect the effect of TNF inhibitor.

The anticancer efficacy of CSF family cytokines including GM-CSF, granulocyte (G)-CSF, erythropoietin (EPO), and IL-3, has been widely studied in clinical settings. To date, 1311 clinical trials enrolling over 200,000 cancer patients treated with GM-CSF and G-CSF alone or in combination have been registered at ClinicalTrials.gov; of these, 96% and 94% studied the effects of GM-CSF and G-CSF in combination therapy, respectively (Figure 6A).

Figure 6 The number and ratio of clinical trials of cytokine combined with other agents in cancer treatment. (A) Relative ratio of clinical trials using GM-CSF, G-CSF and VEGF receptor inhibitors alone or in combination. (B and C) Number of clinical trials using cytokine-based drugs alone or in combination.

EPO exhibits pro-proliferative and anti-apoptotic activities in multiple nonhematopoietic cell types including tumor cells.37 EPO has been used to alleviate cancer- and chemotherapy-related anemia. The first clinical trial of EPO for cancer treatment was initiated in 2003 and to date, 15 trials without results have been published at ClinicalTrials.gov.

IL-3, also known as multi-CSF and hematopoietic cell growth factor, has been the focus of 7 clinical trials. A single-arm trial study evaluating the efficacy of DT388IL3 fusion protein for the treatment of patients with acute myeloid leukemia or myelodysplastic syndromes reported an overall response rate of 81.8% (NCT00397579).

IL-1 and IL-18 are members of the IL-1 family; IL-1 is an important regulator in innate immunity,38 and both cytokines stimulate IFN- production by T cells and NK cells. IL-1 has dual roles in anticancer immune response. Clinically, patients with high IL-1 concentrations in tumors have poor prognoses.39 Anakinra is an IL-1 receptor antagonist that is commonly used to treat rheumatoid arthritis; its antitumor efficacy has been assessed in 8 trials. In a phase II trial, anakinra combined with dexamethasone was used to treat multiple myeloma and plasma cell neoplasm (NCT00635154); the 6-month progression-free rate was 90.7%. 6 clinical trials are investigating a recombinant human IL-18, namely SB-485232, for the treatment of patients with melanoma, lymphoma, and ovarian neoplasms, but no results have been published.

Chemokines and their receptors mediate immunocytes trafficking into the cancer microenvironment, playing roles in promoting or inhibiting cancers. CCL21, together with CCL19, regulates the migration of DCs and T cells to secondary lymphoid organs when binding to their receptor CCR7, thus plays an important role in adaptive immunity and immune tolerance.40 Intratumoral injection of CCL21 enhances the infiltration of T cells and DCs in tumor.41 To date, 3 cancer clinical trials using CCL21 have been registered at ClinicalTrials.gov. In a phase II trial (NCT01433172), CCL21 combined with GM.CD40L vaccine (tumor antigen expressing GM-CSF and CD40L) was used to treat lung adenocarcinoma; the 6-month progression-free survival rate was higher in the combination group than in the GM.CD40L group (15.2% [5/33] vs 9.4% [3/32]).42 For chemokine (C-X-C motif) ligand (CXCL)12, CXCL8, CCL2, CCL3 and CCL5 which are involved in cancer progression and metastasis, few clinical trials studied drugs targeting their receptors, CXCR4, CXCR 1/2 and CCR2, and their effects for cancers have not been verified.43

TGF-, VEGF, epidermal growth factor (EGF), insulin-like growth factor (IGF) and broblast growth factor (FGF), IL-4, IL-13, IL-10, IL-6, IL-11, and IL-17 are known to inhibit anticancer immune response. In this section, we present efficacy of cancer therapy by targeting these cytokines.

TGF- is an oncogenic factor that facilitates evasion of systemic immune surveillance.44 The clinical efficacy of various inhibitors of TGF- signaling including GC1008 (fresolimumab, anti-TGF- monoclonal antibody), TEW-7197 (TGF- receptor activin-like kinase [ALK]4/ALK5), and AP 12009 (TGF-2 antisense oligodeoxynucleotide) has been investigated in metastatic breast cancer, RCC, recurrent or refractory high-grade glioma, and advanced melanoma. In a phase II trial examining the efficacy and safety of combined fresolimumab (1 or 10 mg/kg) and local radiotherapy in the treatment of metastatic breast cancer (NCT01401062), overall response rates were 100% with both low and high drug doses and the rate of serious adverse events was 27% and 25%, respectively. The results of 7 other trials of TGF- inhibitors in cancer treatment have yet to be reported.

Angiogenesis is a vital step in tumor progression and metastasis. Sustained expression of VEGF during tumor development induces the formation of tumor vasculature.45 Various VEGF receptor (VEGFR) inhibitors either alone or in combination with other drugs have been investigated for cancer treatment. These inhibitors include antibodies against VEGFR (eg, bevacizumab, ramucirumab, and ranibizumab); inhibitors of receptor protein kinases (eg, axitinib and vandetanib); soluble decoy receptors containing VEGFR domains (eg, aflibercept); and small molecules that interfere with the binding sites of VEGFR (eg, vatalanib). There are 301 trials registered at ClinicalTrials.gov for the treatment of various cancers (CRC, breast cancer, ovarian cancer, non-small-cell-lung cancer, lymphoma, etc) using VEGFR inhibitors, of which 35% have examined the effects of VEGFR inhibitor monotherapy (Figure 6A).

A large phase III clinical trial that enrolled 1690 participants investigated the efficacy of docetaxel alone or with vandetanib in non-small-cell-lung cancer (NSCLC) (NCT00312377). Median PFS was longer with the combination therapy than with docetaxel alone (17.3 vs 14 weeks), although median OS was comparable between the 2 groups (10.6 vs 10 months). In another phase III trial of 913 patients with NSCLC (NCT00532155), aflibercept increased the median OS of docetaxel from 10.05 to 10.41 months and prolonged median PFS from 4.11 to 5.19 months. In a phase III trial examining the efficacy of aflibercept vs a placebo in 1226 patients with metastatic CRC who had failed to respond to the FOLFIRI regimen (irinotecan, 5-FU, and leucovorin) (NCT00561470), median OS was increased from 12.6 to 13.5 months while median PFS was increased from 4.67 to 6.90 months. Besides combination with chemotherapy, the efficacy of VEGF inhibitors combined with other immunotherapies has been evaluated in 40 clinical trials. As described in the paragraph of IFN-, bevacizumab in conjunction with IFN- showed clinical benefits in mRCC and melanoma patients. Thus, VEGFR inhibition is an effective therapeutic strategy for the treatment of multiple cancers.

In addition to VEGF, growth factors such as EGF, IGF and FGF, have been shown to be crucial for the development and progression of certain cancers. Clinically, human epidermal growth factor receptor 1 and 2 (HER1 and HER2), IGF-1 receptor (IGF-1R) and FGF receptor (FGFR) have been found to be overexpressed in various cancers, particularly in breast and lung cancers.4648 There are 206, 71 and 14 trials for blocking EGFR, IGF-1R and FGFR, respectively, with small molecule inhibitors or monoclonal antibodies in treating cancers registered with ClinicalTrials.gov that had completed recruitment. Unsurprisingly, most of these trials are for lung and breast cancers: 71/206 trials of HER1/2 inhibitors and 16/71 of IGF-1R inhibitors are for treating lung cancers; and 44/206 trials of HER 1/2 inhibitors and 9/71 trials are for treating breast cancers. Their effects in combination of chemotherapeutics have been generally studied. A phase II trial (NCT00986674) demonstrated that carboplatin and paclitaxel are more effective when given with cixutumumab (anti-EGFR antibody) and cetuximab (anti-IGF-1R antibody) than with cetuximab alone in treating advanced non-small cell lung cancer, with overall response rates of 22%, 21.7% and 11%, respectively. However, a phase II trial (NCT00684983) showed that cixutumumab did not enhance the effects of capecitabine and lapatinib ditosylate (EGFR and HER2 inhibitors) in treating HER2-positive stage IIIB-IV breast cancers. Five trials with published results showed limited effects of FGFR inhibitors. A phase II trial studied the effects of dovitinib (a multitargeted inhibitor of FGFR and VEGFR) for patients with advanced lung cancer or CRC who have progressed on anti-VEGF treatment, and the overall response rate was 14.3%. Overall, blockade of growth factor receptors brings considerable therapeutic effects when combining with chemotherapy in treating certain cancers.

IL-4 and IL-13 function as immunosuppressive cytokines that inhibit antitumor immunity by enhancing the Th2 cell response and blocking Th1 cell differentiation.16 Mutated forms of IL-4 and IL-13 receptors highly expressed in multiple human tumor cell lines.49,50 Based on these observations, targeted drugs were developed by linking Pseudomonas exotoxin to IL-4 or IL-13 (IL4-PE38KDEL and IL13-PE38QQR, respectively).

Since 2001, there have been 7 cancer trials of IL-4 registered at ClinicalTrial.gov. It was shown in vitro that IL-4 can inhibit the growth of Kaposi sarcoma cells,51 and one trial assessed the efficacy of IL-4 in the treatment of 48 patients with Kaposi sarcoma (NCT00000769) although no findings have been published. IL-4 was also administered as an adjuvant to enhance the effect of a DC vaccine in the treatment of Wilms tumor (NCT00001564) and Ewing sarcoma (NCT00923910),52 but the outcome of these trials is unknown.

The first clinical trial using IL13-PE38QQR (for the treatment of malignant gliomas) was initiated in 2000. Since then, there have been 10 clinical trials involving over 500 participants with malignant gliomas who were treated with IL-13-PE38QQR. It is difficult to conclude these trials as the results have not been published. In one phase III trial of 300 patients with recurrent malignant gliomas (NCT00064779), IL13-PE38QQR was directly infused into the tumor tissue for 96 hours. After 15 days, patients underwent surgery to excise the recurrent tumors and received another infusion. However, no results have been posted for this or any other trial investigating IL13-PE38QQR.

IL-10 functions as an immune suppressor that inhibits the cancer immunity cycle.53 To date, there have been no reports from ClinicalTrial.gov evaluating the efficacy of IL-10 for cancer treatment, although many trials have examined the use of IL-10 for the treatment of autoimmune disease such as rheumatic arthritis.

The IL-6 cytokine family, which includes IL-6 and IL-11, participates in the activation of oncogenic signal transducer and activator of transcription (STAT)3.54 Twenty trials of IL-6 for cancer treatment (multiple myeloma, lymphoma, mRCC, and prostate cancer) have been registered at ClinicalTrials.gov, mostly involving siltuximab, an IL-6 antagonist approved by the FDA for the treatment of multicentric Castleman disease. In a phase II trial of 88 patients with myeloma (NCT00911859), siltuximab combined with VELCADE (a prescription medication for myeloma) resulted in a higher complete response rate (26.5% vs 22.4%) and overall response rate (87.8% vs 79.6%) than VELCADE alone; in the second part of this trial (286 patients; NCT00401843), PFS of the 2 groups was 245 and 232 days, respectively. Given its role in hematopoiesis, IL-11 has been investigated for its potential to increase platelet counts in patients with chronic myelogenous leukemia in 2 trials.

IL-17 is a pro-oncogenic cytokine that is mainly produced by Th17 cells and induces the production of IL-6 by tumor cells to activate the IL-6/STAT3 signaling pathway.55 Elevated IL-17 expression is related to poor prognosis in patients with invasive ductal carcinoma. But no cancer clinical trial using or targeting IL-17 has been published in ClinicalTrial.gov.

According to review of hundreds of clinical trials, we know that efficacy of cytokines as therapeutic drugs on clinical outcomes are limited. One possible reason for this is that because of the short half-life of cytokines in the blood, frequent high doses are required to achieve lasting therapeutic effects. For example, the effective dosage of IL-2 is 600,000 IU/kg administered every 8 hours for 5 days; moreover, 3 treatment cycles are needed for its activity. Because of this, adverse events generally occur in patients receiving cytokine therapy, include fatigue, chills, fever, chest pain, and musculoskeletal pain.56 More serious adverse events are gastrointestinal disorders (eg, stomachache, diarrhea, and gastritis), cardiac abnormalities (eg, myocardial infarction, nodal tachycardia), and disorders of the immune system (eg, anaphylaxis) and blood and lymphatic systems (eg, anemia and febrile neutropenia).

Nanomaterials used as carriers to deliver cytokines to target tissues can improve the stability of cytokines in blood and reduce their toxicity. At the same time, the unique features of nanomaterials have advantages for the therapeutic application of cytokines including aqueous solubility, prolonged circulation time, and preferential accumulation at tumor sites.57 Recent studies on nanomaterials used for cytokine loading are summarized in Table 2.

Table 2 Nanomaterials for Therapeutic Delivery of Cytokines

Nanomaterials can improve the stability and bioactivity of cytokines. For example, sustained released over a period of 1 month was achieved for IFN- encapsulated in poloxamer-blend microspheres.58 Chitosan coated with pJME/GM-CSF (plasmid DNA) was more effective than naked pJME/GM-CSF in promoting DC recruitment.59 Nanoscale liposomal polymeric gels loaded with TGF- inhibitor and IL-2 delayed tumor growth and increased NK cell activity and the number of tumor-infiltrating T cells.60 Nanomaterials can also reduce the toxic effects of cytokine therapy; for instance, encapsulation in PEG liposomes abrogated the diarrhea induced by TNF in rats with subcutaneous BN175 sarcomas.61 Gold nanoparticles were found to enhance the accumulation of TNF around blood vessels in a mouse model of epithelial carcinoma, leading to a significant decrease in tumor volume.62 Magnetic nanoparticles carrying human IFN-2b were enriched in the liver upon application of a magnetic field and compared to the control group, the volume of human liver cancer cell-derived tumors in nude mice was reduced by about 30%.63 Nanomaterials are good auxiliaries for cytokine gene therapy. Chitosan coated with plasmids encoding the cytokines IL-15 and IL-21 suppressed tumor growth and prolonged survival in mice.64,65 PEGpoly (lactic-co-glycolic acid)PEG nanoparticles were shown to be effective carriers for IL18 gene delivery.66 Magnetic nanoparticles carrying a plasmid encoding a small interfering RNA targeting gene encoding epidermal growth factor receptor reduced endogenous epidermal growth factor receptor expression in U251 glioma cells, resulting in tumor regression in vivo.67 Two clinical trials involving 168 patients have investigated the efficacy of colloidal gold-bound TNF for the treatment of primary or metastatic cancer (NCT00356980 and NCT00436410, respectively), but the results have yet to be reported.

Besides, artificial oncolytic viruses are well-established carriers for cytokine gene therapy. There are 17 cytokines including CCL2, CCL5, CCL19, CXCL11, FGF2, FLT3L, GM-CSF, IFN-/, IFN-, IL-2, IL-7, IL-12, IL-15, IL-18, IL-23, IL-24, and TNF- that are delivered by artificial oncolytic viruses derived from adenovirus, herpesvirus, paramyxovirus, poxvirus, or rhabdovirus. More details could be found in the recent review wrote by Pol et al.68

Although animal studies showed that properties and efficacy of cytokine-based drugs can be improved by nanomaterials, sufficient clinical studies are required to support the conclusion. Two ongoing clinical trials involving 168 patients aim to investigate the efficacy of colloidal gold-bound TNF for the treatment of primary or metastatic cancer (NCT00356980 and NCT00436410, respectively), but the results have yet to be reported.

As important immune regulators, cytokine-based drugs offer many possibilities for cancer treatment. Large amounts of cytokines can be readily produced using eukaryotic or prokaryotic expression systems as the cDNA sequences of most cytokines are available, which makes cytokines attractive to new drug development. However, our statistical results (Supplementary Table 1) indicate that a large number of clinical trials of cytokine-based drugs ended up without published results mainly because of the low efficacy, serious adverse effects, and antagonistic roles in immunoregulation. These problems are partly overcome by delivering with nanomaterials or oncolytic viruses in animal experiments, or combining with immunotherapies or chemotherapeutic agents or both (Figure 6B and C) in clinic. Such strategies would be used and improved in the future clinical trials. Moreover, clarifying the immune-regulatory mechanisms of cytokines can improve their efficacy and safety in cancer therapy.

APC, antigen-presenting cell; B7-H3, B7 homolog 3 protein; CAR-T, chimeric antigen receptor T cell; CCL, C-C motif chemokine ligand; CRC, colorectal cancer; CSF, colony-stimulating factor; CTL, cytotoxic T lymphocyte; CTLA-4, cytotoxic T lymphocyte-associated protein 4; CXCL, chemokine (C-X-C motif) ligand; DC, dendritic cell; EGF, epidermal growth factor; EMT, epithelial-to-mesenchymal transition; EPO, erythropoietin; FDA, US Food and Drug Administration; FGF, broblast growth factor; Fu, fluorouracil; G, granulocyte; GM, granulocyte/macrophage; IFN, interferon; IGF, insulin-like growth factor; IL, interleukin; ILC, innate lymphoid cell; i.v., intravenous injection; IU, international unit; JAK, Janus kinase; MDSC, myeloid-derived suppressor cell; MHC, major histocompatibility complex; mRCC, metastatic renal cell carcinoma; NK, natural killer; NKT, natural killer T; NSCLC, non-small-cell-lung cancer; ORR, objective response rate; OS, overall survival; PD-1, programmed death 1; PD-L1, programmed death ligand 1; PEG, pegylated; PFS, progression-free survival; PGE2, prostaglandin E2; PLGA, poly (lactic-co-glycolic acid); rAd, recombinant adenovirus; STAT, signal transducer and activator of transcription; s.c., subcutaneous injection. TAA, tumor-associated antigen; TAM, tumor-associated macrophage; TGF-, transforming growth factor ; Th, T helper cell; TNF, tumor necrosis factor; Treg, regulatory T cell; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.

This work is supported by the National Natural Science Foundation of China (Grant No. 31800006) to YQ; Natural Science Foundation of Guangdong Province (Grant No. 18zxxt26) to YQ; Guangzhou Basic and Applied Basic Research Foundation (Grant No. 202002030127) to JS; Guangdong Basic and Applied Basic Research Foundation (Grant No. 2021A1515012324) to JS; the Fundamental Research Funds for the Central Universities (Grant No. 20ykzd08) to JS; Natural Science Foundation of Guangdong Province (Grant No. 2018A030313563) to JS; Program for Guangdong Introducing Innovative and Entrepreneurial Teams (Grant No. 2016ZT06S252) to JS; Guangdong Financial Fund for High-Caliber Hospital Construction to JS.

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.

The authors declare they have no competing financial interests and other onflicts of interest in this work.

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Heart attack recovery aided by injecting heart muscle cells that overexpress cyclin D2 – The Mix

By daniellenierenberg

Researchers used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has high clinical relevance to human disease.

Researchers used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has high clinical relevance to human disease.In a large-animal study, researchers have shown that heart attack recovery is aided by injection of heart muscle cells derived from human induced pluripotent stem cell line, or hiPSCs, that overexpress cyclin D2. This research, published in the journal Circulation, used a pig model of heart attacks, which more closely resembles the human heart in size and physiology, and thus has higher clinical relevance to human disease, compared to studies in mice.

An enduring challenge for bioengineering researchers is the failure of the heart to regenerate muscle tissue after a heart attack has killed part of its muscle wall. That dead tissue can strain the surrounding muscle, leading to a lethal heart enlargement.

Heart experts thus have sought to create new tissue applying a patch of heart muscle cells or injecting heart cells to replace damaged muscle. Similarly, they have tried to stimulate division of existing heart muscle cells near the damaged area. This current study, led by researchers at the University of Alabama at Birmingham, shows progress in both goals.

After the experimental heart attack, heart tissue around the infarction site was injected with about 30 million bioengineered human cardiomyocytes that were differentiated from hiPSCs. These cells also overexpress cyclin D2, part of a family of proteins involved in cell division.

Compared to control human cardiomyocytes, the cyclin D2-cardiomyocytes showed enhanced potency to repair the heart. They proliferated after injection, and by four weeks, the hearts had less pathogenic enlargement, reduced size of dead muscle tissue and improved heart function.

Intriguingly, the cyclin D2-cardiomyocytes stimulated not only their own proliferation, but also proliferation of existing heart muscle cells around the infarction site of the pig heart, as well as showing angiogenesis, the development of new blood vessels.

These results suggest that the cyclin D2-cardiomyocyte transplantation may be a potential therapeutic strategy for the repair of infarcted hearts, said study leader Jianyi Jay Zhang, M.D., Ph.D., the chair of Biomedical Engineering, a joint department of the UAB School of Medicine and the UAB School of Engineering.

This ability of the graft cyclin D2-cardiomyocytes to stimulate the proliferation of nearby existing heart cells suggested paracrine signaling, a type of cellular communication where a cell produces a signal that induces changes in nearby cells.

Exosomes small blebs or tiny vesicles that are released by human or animal cells and contain proteins and RNA from the cells that release them are one common form of paracrine signaling.

Zhang and colleagues found that exosomes that they purified from the cyclin D2-cardiomyocyte growth media indeed promoted proliferation of cultured cardiomyocytes. In addition, the treated cardiomyocytes were more resistant to programmed cell death, called apoptosis, induced by low oxygen levels. The exosomes also induced proliferation of various other cell types, including human umbilical vein endothelial cells, human vascular smooth muscle cells and 7-day-old rat cardiomyocytes that have almost undetectable proliferation.

Part of the cargo that exosomes carry are microRNAs, or miRNAs. These short pieces of RNA have the ability to interact with messenger RNA in target cells, and they are robust players of gene regulation in cells. Humans have more than 2,000 miRNAs with different RNA sequences, and these are thought to regulate a third of the genes in the genome.

So, the researchers documented which microRNAs were present in exosomes from the cyclin D2-overexpressing cardiomyocytes and in exosomes from non-overexpressing cardiomyocytes. As expected, they found differences.

Jianyi Jay Zhang, M.D., Ph.D.Together, the exosomes from both types of cells contained 1,072 different miRNAs, and 651 were common to the two exosome groups. However, 332 miRNAs were found only in the cyclin D2-overexpressing cardiomyocytes, and 89 miRNAs were specific for the non-overexpressing cardiomyocytes. In preliminary work of characterizing the effects of specific miRNAs, one particular miRNA from the cyclin D2-overexpressing exosomes was shown to stimulate proliferation when delivered into rat cardiomyocytes.

Thus, as the therapeutic potential of exosomes for improving cardiac function becomes more evident, combining an exosome-mediated delivery of proliferative miRNAs with transplantation of cyclin D2-overexpressing cardiomyocytes, or cell products, could become a new promising strategy for upregulating proliferation of the recipient cardiomyocytes and reducing cardiac fibrosis, Zhang said. Altogether, our data suggest that cardiac cell therapy, involving cardiomyocytes with enhanced proliferation capacity, may become an efficacious future strategy for myocardial repair and prevention of congestive heart failure in patients with acute myocardial infarctions.

UAB Department of Biomedical Engineering co-authors with Zhang, in the study Cyclin D2 overexpression enhances the efficacy of human induced pluripotent stem cell-derived cardiomyocytes for myocardial repair in a swine model of myocardial infarction, are Meng Zhao, Yuji Nakada, Yuhua Wei, Weihua Bian, Anton V. Borovjagin, Yang Zhou and Gregory P. Walcott.

Additional co-authors are Yuxin Chu and Min Xie, Division of Cardiovascular Disease, UAB Department of Medicine; Wuqiang Zhu, Department of Cardiovascular Diseases, Physiology and Biomedical Engineering, Mayo Clinic Arizona, Scottsdale; Thanh Nguyen, UAB Informatics Institute; and Vahid Serpooshan, Emory University and Georgia Institute of Technology, Atlanta.

Support came from National Institutes of Health grants HL114120, HL131017, HL149137 and HL134764.

At UAB, Zhang holds the T. Michael and Gillian Goodrich Endowed Chair of Engineering Leadership.

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Stem Cell Therapy Market Report | Know the Cutting-Edge Innovations and Future Trends of Market – BioSpace

By daniellenierenberg

Of late, there has been an increasing awareness regarding the therapeutic potential of stem cells for management of diseases which is boosting the growth of the stem cell therapy market. The development of advanced genome based cell analysis techniques, identification of new stem cell lines, increasing investments in research and development as well as infrastructure development for the processing and banking of stem cell are encouraging the growth of the global stem cell therapy market.

One of the key factors boosting the growth of this market is the limitations of traditional organ transplantation such as the risk of infection, rejection, and immunosuppression risk. Another drawback of conventional organ transplantation is that doctors have to depend on organ donors completely. All these issues can be eliminated, by the application of stem cell therapy. Another factor which is helping the growth in this market is the growing pipeline and development of drugs for emerging applications. Increased research studies aiming to widen the scope of stem cell will also fuel the growth of the market. Scientists are constantly engaged in trying to find out novel methods for creating human stem cells in response to the growing demand for stem cell production to be used for disease management.

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It is estimated that the dermatology application will contribute significantly the growth of the global stem cell therapy market. This is because stem cell therapy can help decrease the after effects of general treatments for burns such as infections, scars, and adhesion. The increasing number of patients suffering from diabetes and growing cases of trauma surgery will fuel the adoption of stem cell therapy in the dermatology segment.

Global Stem Cell Therapy Market: Overview

Also called regenerative medicine, stem cell therapy encourages the reparative response of damaged, diseased, or dysfunctional tissue via the use of stem cells and their derivatives. Replacing the practice of organ transplantations, stem cell therapies have eliminated the dependence on availability of donors. Bone marrow transplant is perhaps the most commonly employed stem cell therapy.

Osteoarthritis, cerebral palsy, heart failure, multiple sclerosis and even hearing loss could be treated using stem cell therapies. Doctors have successfully performed stem cell transplants that significantly aid patients fight cancers such as leukemia and other blood-related diseases.

Global Stem Cell Therapy Market: Key Trends

The key factors influencing the growth of the global stem cell therapy market are increasing funds in the development of new stem lines, the advent of advanced genomic procedures used in stem cell analysis, and greater emphasis on human embryonic stem cells. As the traditional organ transplantations are associated with limitations such as infection, rejection, and immunosuppression along with high reliance on organ donors, the demand for stem cell therapy is likely to soar. The growing deployment of stem cells in the treatment of wounds and damaged skin, scarring, and grafts is another prominent catalyst of the market.

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On the contrary, inadequate infrastructural facilities coupled with ethical issues related to embryonic stem cells might impede the growth of the market. However, the ongoing research for the manipulation of stem cells from cord blood cells, bone marrow, and skin for the treatment of ailments including cardiovascular and diabetes will open up new doors for the advancement of the market.

Global Stem Cell Therapy Market: Market Potential

A number of new studies, research projects, and development of novel therapies have come forth in the global market for stem cell therapy. Several of these treatments are in the pipeline, while many others have received approvals by regulatory bodies.

In March 2017, Belgian biotech company TiGenix announced that its cardiac stem cell therapy, AlloCSC-01 has successfully reached its phase I/II with positive results. Subsequently, it has been approved by the U.S. FDA. If this therapy is well- received by the market, nearly 1.9 million AMI patients could be treated through this stem cell therapy.

Another significant development is the granting of a patent to Israel-based Kadimastem Ltd. for its novel stem-cell based technology to be used in the treatment of multiple sclerosis (MS) and other similar conditions of the nervous system. The companys technology used for producing supporting cells in the central nervous system, taken from human stem cells such as myelin-producing cells is also covered in the patent.

Global Stem Cell Therapy Market: Regional Outlook

The global market for stem cell therapy can be segmented into Asia Pacific, North America, Latin America, Europe, and the Middle East and Africa. North America emerged as the leading regional market, triggered by the rising incidence of chronic health conditions and government support. Europe also displays significant growth potential, as the benefits of this therapy are increasingly acknowledged.

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Asia Pacific is slated for maximum growth, thanks to the massive patient pool, bulk of investments in stem cell therapy projects, and the increasing recognition of growth opportunities in countries such as China, Japan, and India by the leading market players.

Global Stem Cell Therapy Market: Competitive Analysis

Several firms are adopting strategies such as mergers and acquisitions, collaborations, and partnerships, apart from product development with a view to attain a strong foothold in the global market for stem cell therapy.

Some of the major companies operating in the global market for stem cell therapy are RTI Surgical, Inc., MEDIPOST Co., Ltd., Osiris Therapeutics, Inc., NuVasive, Inc., Pharmicell Co., Ltd., Anterogen Co., Ltd., JCR Pharmaceuticals Co., Ltd., and Holostem Terapie Avanzate S.r.l.

This study provides a particularized anatomy according to the L.E.A.P mechanism

The regional analysis offers market assays across:

The study, prepared through the L.E.A.P mechanism adds a dimension of infallibility and assures precise information on all the growth dynamics.

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Stem Cell Therapy Market Report | Know the Cutting-Edge Innovations and Future Trends of Market - BioSpace

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Allogeneic Mesenchymal Stem Cell Segment Is Expected To Lead In the Global Rheumatoid Arthritis Stem Cell Therapy Market over the Forecast Period,…

By daniellenierenberg

The report on the Rheumatoid Arthritis Stem Cell Therapy market provides a birds eye view of the current proceedings and the impact on the COVID-19 pandemic. Further, the report ponders over the various factors that are likely to impact the overall dynamics of the market once the COVID-19 pandemic subsides. The current trends, growth opportunities, restraining factors, and drivers are discussed in the report in detail.

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The growing prevalence and recurrence of rheumatoid arthritis is expected to be the major factor driving the growth of the rheumatoid arthritis stem cell therapy market over the forecast period. Although doctors do not know the exact cause of rheumatoid arthritis, but certain risk factors are observed to be associated with it.

These risk factors include age (most common between the age of 40 and 60), family history, gender, environment (a toxic chemical in the environment can up the odds), obesity and smoking. Changes in lifestyle and eating habits are contributing to the growing prevalence of rheumatoid arthritis.

Rheumatoid Arthritis Stem Cell Therapy Market: Segmentation

Tentatively, the global rheumatoid arthritis stem cell therapy market can be segmented on the basis of treatment type, application, end user and geography.

Based on treatment type, the global rheumatoid arthritis stem cell therapy market can be segmented into:

Based on application, the global rheumatoid arthritis stem cell therapy market can be segmented into:

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Based on distribution channel, the global rheumatoid arthritis stem cell therapy market can be segmented into:

Based on geography, the global rheumatoid arthritis stem cell therapy market can be segmented into:

Rheumatoid Arthritis Stem Cell Therapy Market: Regional Outlook

Geographically, the global rheumatoid arthritis stem cell therapy market can be segmented into viz. North America, Latin America, Europe, Asia-Pacific excluding Japan (APEJ), Japan and the Middle East and Africa (MEA). North America is expected to be the dominant region in the global rheumatoid arthritis stem cell therapy market, owing to the presence of various key players.

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The rheumatoid arthritis stem cell therapy market in Asia Pacific excluding Japan is expected to grow at a significant CAGR due to the expansion of product offerings by key players. Europe is expected to have the second large share in the global rheumatoid arthritis stem cell therapy market throughout the forecast period.

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Allogeneic Mesenchymal Stem Cell Segment Is Expected To Lead In the Global Rheumatoid Arthritis Stem Cell Therapy Market over the Forecast Period,...

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Pigs can breathe through their butts. Can humans? – Livescience.com

By daniellenierenberg

Mice, rats and pigs all share a secret superpower: They can all use their intestines to breathe, and scientists discovered this by pumping oxygen up the animals' butts.

Why run such experiments, you ask? The research team wanted to find a potential alternative to mechanical ventilation, a medical treatment where a machine pushes air into a patient's lungs through the windpipe. Ventilators deliver oxygen to the lungs and help remove carbon dioxide from the blood, but the machines aren't always available.

Early in the COVID-19 pandemic, for example, hospitals faced a severe shortage of ventilators, The New York Times reported. Although doctors can also use a technique called extracorporeal membrane oxygenation (ECMO), where blood is pumped out of the body and reoxygenated with a machine, the procedure carries inherent risks, such as bleeding and blood clots; and it's often less readily available than ventilators, according to Mayo Clinic.

Related: The 10 weirdest medical cases in the animal kingdom

In search of another solution, the study authors drew inspiration from aquatic animals like sea cucumbers and freshwater fish called loaches (Misgumus anguillicandatus), which use their intestines for respiration. It was unclear whether humans and other mammals have similar capabilities, although some scientists attempted to answer that question in the 1950s and 1960s.

"We initially looked at a mouse model system to see if we could deliver oxygen gas intra-anusly," said senior author Dr. Takanori Takebe, a professor at the Tokyo Medical and Dental University and a director at the Center for Stem Cell and Organoid Research and Medicine at Cincinnati Children's Hospital Medical Center.

"Every time we performed experiments, we were quite surprised," Takebe told Live Science.

Without intestinal ventilation, mice placed in a low-oxygen environment survived for only about 11 minutes; with ventilation into their anuses, 75% survived for 50 minutes, thanks to an infusion of oxygen that reached their hearts. The team then tried using oxygenated liquid, rather than gas, in mice, rats and pigs, and they found similarly promising results. The team noted that more work still needs to be done to see if the approach is safe and effective in humans, according to a paper on their findings published May 14 in the journal Med.

"The pandemic has highlighted the need to expand options for ventilation and oxygenation in critical illness, and this niche will persist even as the pandemic subsides," as there will be times when mechanical ventilation is unavailable or inadequate on its own, Dr. Caleb Kelly, a clinical fellow and physician-scientist at Yale School of Medicine, wrote in a commentary of the study. If, after further evaluation, intestinal ventilation eventually becomes common practice in intensive care units, this new study "will be marked by historians as a key scientific contribution," he wrote.

Before starting their experiments in rodents, the team got very familiar with loach guts. The fish take in oxygen mostly through their gills, but occasionally, when exposed to low-oxygen conditions, loaches instead use a portion of their intestines for gas exchange, Takebe said. In fact, in response to the lack of oxygen, the structure of gut tissues near the anus changes such that the density of nearby blood vessels increases and secretion of fluids related to digestion decreases.

These subtle changes allow loaches to "suck up the oxygen more efficiently," Takebe said. In addition, the outermost lining of the loach gut the epithelium is very thin, meaning oxygen can easily permeate the tissue to reach the blood vessels beneath, he added. To simulate this structure in their mouse models, the team thinned out the gut epithelium of the rodents using chemicals and various mechanical procedures.

They then placed the mice under extremely low-oxygen conditions and used a tube to pump oxygen gas up the animals' bums and into their large intestines.

Related: 8 bizarre animal surprises from 'True or Poo' Can you tell fact from myth?

Compared with mice whose gut epithelium had not been thinned, the mice with thin epitheliums survived significantly longer in the experiment with most surviving 50 minutes as compared with about 18 minutes. Again, mice not given any oxygen only survived for about 11 minutes. In addition to surviving longer, the group with thinned-out gut linings showed signs that they were no longer starved for oxygen; they stopped gasping for air or showing signs of cardiac arrest, and the oxygen pressure in their major blood vessels improved.

Although this initial experiment suggested that oxygen could pass through the intestine and into circulation, thinning out the gut epithelium would likely not be feasible in human patients, Takebe said.

Particularly in critically ill patients, "I think additional damage to the gut would be really dangerous, for the treatment perspective," Takebe said. But "over the course of the experiments, we realized that even the intact gut has some, not really efficient, but some capacity to exchange the gas," he noted, meaning there may be a way to introduce oxygen through the gut without first thinning out the tissues.

So in another experiment, rather than using oxygen gas, the team tried perfluorodecalin (PFD), a liquid fluorocarbon that can be infused with a large amount of oxygen. The liquid is already used in people, such as for use in the lungs of infants with severe respiratory distress, the authors noted in their report.

The liquid also acts as a surfactant a substance that reduces surface tension; since a surfactant lines the air sacs of the lungs and helps boost gas exchange in the organ, PFD may fulfill a similar purpose in the intestines, Takebe said.

Much like in the oxygen-gas experiments, the oxygenated PFD rescued mice from the effects of being placed in a low-oxygen chamber, enabling the rodents to meander about their cage more than mice not given the treatment. After just one injection of 0.03 ounces (1 milliliter) of the liquid, the rodents' improvements persisted for about 60 minutes.

"We are not quite sure why this improvement is persisting much longer than the original expectations," Takebe noted, as the authors expected the effects to wear off in just a couple minutes. "But the observation is really reproducible and very robust."

Related: Gasp! 11 surprising facts about the respiratory system

The team then moved on to a pig model of respiratory failure, where they placed pigs on ventilators and only provided a low level of oxygen and then injected PDF into the pigs' posteriors with a long tube. Compared with pigs not given the PFD treatment, pigs given PFD improved in terms of the oxygen saturation of their blood, and the color and warmth returned to their skin. A 13.5 oz (400 ml) infusion sustained these improvements for about 18 to 19 minutes, and the team found that they could give additional doses to the pigs without noticeable side effects.

The team also tested the safety of repeat dosing in rats and found that, while their oxygen levels rose, the animals showed no notable side effects, markers of organ damage or stray PFD lingering in their cells.

Following this success in animal models, Takebe said that his team hopes to start a clinical trial of the treatment in humans sometime next year. They would likely start by testing the safety of the approach in healthy volunteers and beginning to work out what dose levels would be reasonable, he said. However, to make the jump from animals to human patients, the team will need to address a number of critical questions.

For instance, the treatment could potentially stimulate the vagus nerve a long nerve that connects the gut and brain so trial organizers should likely be on the lookout for side effects like falling blood pressure or fainting, Takebe noted. Also, the lower gut contains relatively little oxygen compared with other organs in the body, he added. The community of bacteria and viruses that live in the gut are adapted to these low-oxygen conditions, and a sudden infusion of oxygen might disrupt those microbes, he said.

"The consequence of reversing this so-called 'physiologic hypoxia' is unknown," Kelly noted in his commentary, echoing Takebe's sentiments. In humans, it will be important to determine how many doses of oxygenated liquid could be safely administered into the gut without causing unintended changes to the intestinal environment, he wrote.

In addition, the animal models in the study don't fully reflect what critically ill patients experience during respiratory failure, a condition that often coincides with infection, inflammation and low blood flow, Kelly noted. So there may be additional factors to consider in critically ill patients that weren't relevant in rodents and pigs. And depending on a given patient's condition, they may need a higher or lower dose of PFD all of these fine details will need to be carefully assessed in future trials, Takebe said.

Originally published on Live Science.

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Pigs can breathe through their butts. Can humans? - Livescience.com

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