Comparing the effects of e-cigarettes and regular smoking on the brain.

An ever accumulating volume of scientific and preclinical data shows new evidence of ways that e-cigarettes are dangerous. Understandably, most of the focus has been on the effects on the lungs, cardiovascular disease, and addiction. But recently, a growing body of scientific studies are starting to show the serious potential negative effects e-cigarette use may have on the brain.

Electronic-cigerettes (e-cigarettes), and more broadly electric vaporizers, have a history that goes back almost 100 years. The modern commercial version of the e-cigarette is usually attributed to the Chinese pharmacist Hon Lik, although numerous patents and related technologies developed by others were prevalent throughout the 1980s and 90s.

The immediate urgency in attempting to understand the health effects of e-cigarettes stems from their increasing rate of use, most concerning among young people. The challenge though is that they are simply too new, and not enough time has passed to understand and really appreciate their potential long term clinical effects due to sustained or chronic use.

Among high school students, the use of tobacco products had been on the decline until 1998, attributed to aggressive anti-smoking campaigns through the 90s. But this changed that year, with an increase in tobacco use due exclusively to the use of e-cigarettes. By 2014 e-cigarettes overtook all other tobacco products among this population. Even more concerning is the rate at which their use is increasing. According to the Centers for Disease Control and Prevention (CDC) e-cigarette use among high schoolers increased 77.8% in 2018 over 2017, with similar trends observed internationally.

And while it is possible to find e-cigarette pods and inserts that do not have nicotine, the vast majority do. Whats worse, the trend has been to increase the concentration of nicotine delivered by these products. In the case of the popular Juul brand, the average concentration of nicotine considerably exceeds the concentration in regular cigarettes.

To be fair, one potential positive use of these devices might be in helping long time smokers reduce the use of regular cigarettes. The CDC has stated that that while e-cigarettes are not safe for people that dont use tobacco, they are dohave potential to benefit adult smokers. By triturating the chemical composition and rate of nicotine delivery, it may offer a new tool to assist these individuals. Getting a long time smoker to reduce their dependency on combustible cigarettes is a meaningful thing.

And a National Academies report concluded, ecigarettes are not without risk, but compared to combustible tobacco cigarettes they contain fewer toxicants and are likely to be far less harmful than combustible tobacco cigarettes. The Federal Drug Administration (FDA) has stated that nicotine is what addicts and keeps people using tobacco products, but it is not what makes tobacco use so deadly. Yet, at the same time, even within the FDA and CDC, they state that they continue to investigate the distressing incidents of severe respiratory illness associated with use of vaping products. However, this does not necessarily imply that nicotine is responsible, but rather, that other additives and the delivery technologies themselves may be contributing to such clinical effects.

When it comes to the brain, the potential dangerous effects e-cigarettes may have on the brain and their long term consequences stem from the well established effects nicotine in general has on the brain and brain development, the degree and concentration of nicotine e-cigarettes are capable of delivering, and the chemistry associated with how these devices deliver it. The microvascuature of the brain - the collection of specialized blood vessels that feed the brain and spinal cord and regulate their chemical environment - as well as the cells that make up the brain itself (neurons and other cells), are all vulnerable to damage.

The microvascuature of the brain and spinal cord consists of a vast collection of capillaries that provide brain cells with oxygen and nutrients. It also shuttles away cellular waste products. The brains microvascuature is unique compared to the rest of the body. The endothelial cells that make up these tiny blood vessels form a regulated barrier between the blood on one side (the lumen side of the blood vessels) and the chemical environment the brain and spinal cord float in on the other side. This barrier is called the blood brain barrier.

The normal compliment of molecules and immune cells capable of moving between the blood and the cellular spaces in the other tissues of the body cannot freely do so with the brain and spinal cord - which collectively form the central nervous system. The unique chemical environment of the central nervous system formed by the blood brain barrier is the cerebral spinal fluid.

There is a strong correlation between long term smoking, cognitive decline in the later decades of life, and disruption of the blood brain barrier and microvasculature of the brain. In fact, cognitive decline and microvascular dysfunction are essentially universal consequences of long term smoking for everyone. The exact pathophysiological mechanisms involved are still not completely clear though, warranting continued research. But a recently published paper suggests how the negative physiological effects nicotine has on brain cells when delivered via e-cigarettes mirrors the effects observed with combustible cigarettes.

The endothelial cells that make up the microvasculature are particularly vulnerable. This means that the normal regulatory mechanisms responsible for maintaining the unique chemical environment of the cerebral spinal fluid via the blood brain barrier may slowly break down, contributing to cognitive decline.

And in at least one mouse model study, the authors suggest that e-cigarettes may also have short term disruptive effects on cognitive and memory functions. So there may be more immediate and acute concerns with e-cigarette use, in particular in younger populations where the brain is still developing.

In another study, scientists found that e-cigarettes produce a stress response in neural stem cells, which are populations of cells that eventually become neurons and other important cell types in the brain. Again, potential effects on the still developing brain of adolescents is of immediate concern.

On a positive note, a clinically significant exception to the above effects is the use of nicotine to potentially treat Parkinsons disease. Nicotine and chemically related drugs have been shown to be effective in protecting the parts of the brain that are affected and degenerate in Parkinsons, as well as in treating the symptoms of the disease. Its use has also been indicated in reducing the significant side effects of other Parkinsons drugs.

At the moment there are more questions than answers when it comes to understanding the physiological and cellular effects e-cigarettes - and in particular high concentration nicotine delivery via these devices - has on the brain. The inclusion of additional additives may further exacerbate microvasculature and cellular damage to the brain. These risks should of course be balanced against e-cigarettes ability to help people quit combustible tobacco products, which for that population is judged to be significantly more dangerous than e-cigarettes. The long term epidemiological and public health consequences of e-cigarettes - both good and bad - will not be fully appreciated for years to come. But the data at the moment seems to suggest potential significant pathophysiological effects on brain function.

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Accumulating Evidence Suggests E-Cigarettes Are Likely As Harmful To The Brain As Regular Smoking - Forbes

Spinal Cord Stem Cells Comments Off on Accumulating Evidence Suggests E-Cigarettes Are Likely As Harmful To The Brain As Regular Smoking – Forbes | June 8th, 2021

Lymph nodes are small, bean-shaped glands that play a crucial role in the immune system. They filter lymphatic fluid, which helps rid the body of germs and remove waste products.

The body contains hundreds of lymph nodes. They form clusters around the body and are particularly prominent in areas such as the neck, armpit and groin and behind the ears.

The bodys cells and tissues dispose of waste products in lymphatic fluid, which lymph nodes then filter. During this process, they catch bacteria and viruses that could harm the rest of the body.

Lymph nodes are an essential part of the bodys immune system. Due to their function, they come into contact with toxins, which can cause them to swell. Although swollen lymph nodes are common, they may occasionally indicate lymph node cancer, or lymphoma.

Keep on reading to learn more about lymph nodes and their function within the immune system.

Lymph nodes are part of the lymphatic system, which is a complex network of nodes and vessels.

In certain areas of the body, such as the neck, armpit, and groin, lymph nodes sit close to the skin. This means a person may feel them swell when an infection develops.

Lymph nodes are also present in the stomach and between the lungs. However, there are no lymph nodes in the brain or spinal cord.

The name of a lymph node depends on its location in the body.

Lymph nodes form clusters throughout the body. Their main function is to filter out potentially harmful substances.

All tissues and cells in the body excrete lymphatic fluid, or lymph, in order to eliminate waste products. The lymph then travels through vessels in the lymphatic system and passes through lymph nodes for filtering.

Lymph nodes contain lymphocytes. These are a type of white blood cells that help destroy pathogens, such as bacteria, viruses, and fungi. When lymph nodes detect a pathogen in the lymph, they produce more lymphocytes, which causes them to swell.

Upon encountering bacteria or damaged cells, lymph nodes destroy them and turn them into a waste product.

When the lymph reenters the bloodstream, waste products travel to the kidneys and liver. The body then excretes waste products in the urine and feces.

Learn more about how the lymphatic system works here.

Swollen lymph nodes do not always indicate cancer. Below, we list some of many conditions that can cause lymph node swelling.

Lymphadenitis occurs when bacteria, viruses, or fungi in the lymph infect lymph nodes. When this happens, lymph nodes swell and are painful to the touch.

If multiple clusters of nodes become infected, a person may feel pain and swelling in both their neck and groin.

The most common type of lymphadenitis is localized lymphadenitis. This means the condition only affects a few nodes. If the infection occurs in several node clusters, a doctor will likely diagnose generalized lymphadenitis.

The condition usually results from an infection elsewhere in the body.

Symptoms of lymphadenitis include:

Lymphadenitis treatments include:

The type of treatment necessary will depend on a variety of factors, such as the severity of the disease and a persons underlying conditions and allergies. A doctor will help a person choose the most suitable treatment based on these factors.

Learn more about swollen lymph nodes in the neck here.

Swollen lymph nodes in the neck may be due to a viral or bacterial throat infection, such as strep throat.

Viral throat infections, such as colds, can present with swollen lymph nodes, a runny nose, and pinkeye.

These infections usually resolve on their own. However, a person can take over-the-counter pain relievers to alleviate pain they may experience when swallowing.

Strep throat is a bacterial infection that develops in the throat and tonsils due to group A streptococcus. People may contract strep throat if they come into contact with droplets containing the strep bacteria.

A person with strep throat may experience swollen lymph nodes on the neck, a sore throat, a fever, and red spots on the roof of the mouth.

Doctors treat strep throat with antibiotics.

Impetigo is an infection that develops due to group A streptococcus and may cause lymph nodes in the armpits and groin to swell.

A person can contract impetigo when the bacteria enter the body through a break in the skin. This can happen through sharing a towel, razor, or yoga mat.

Symptoms of impetigo include:

If a person has impetigo, they should seek medical attention to address their symptoms and prevent the condition from spreading to others.

Treatment will usually involve antibiotics.

Ringworm, or jock itch, is a fungal infection that can affect many areas of the body. If the fungus develops in the groin, a person may experience lymph node swelling in that area.

Typically, ringworm starts as a fungal lesion. The fungus often transmits when people share towels or razors.

Ringworm thrives in moist environments, and therefore a person should take care to dry thoroughly after a wash and try not to stay in damp clothes.

Common ringworm symptoms include:

A doctor will prescribe an antifungal treatment to address ringworm.

The best way to prevent ringworm is to wear breathable fabrics, avoid sharing towels and razors, and dry thoroughly after bathing.

Learn more about swollen lymph nodes in the groin here.

Lymphoma is a type of cancer that affects the lymphatic system. The two main types of lymphoma are Hodgkin lymphoma and non-Hodgkin lymphoma.

Hodgkin lymphoma occurs when cancer cells spread from one cluster of lymph nodes to another. By contrast, in non-Hodgkin lymphoma, there is no order in how cancer cells spread throughout the lymphatic system.

Typical symptoms of lymphoma include:

These are also common symptoms of viral infections, which can make lymphoma hard to diagnose. However, in people with lymphoma, symptoms tend to persist for longer periods of time.

It is of note that these symptoms do not clearly indicate cancer. If a person experiences any of these, they should contact a doctor to identify the cause of their symptoms.

Treatment options for lymphoma include:

A person should contact a healthcare professional if they are experiencing persistent swelling of lymph nodes.

Swelling usually indicates an infection, and therefore a person should not immediately worry about lymphoma.

After reaching a diagnosis, a doctor will recommend the appropriate course of treatment.

Lymph nodes are a part of the lymphatic system. They filter lymph, which contains pathogens and damaged cells, and send the dead cells to the kidneys and liver.

Lymph node swelling usually results from an infection. In rare cases, however, it may be due to lymphoma.

If a person is concerned about swelling and other symptoms they have, they should contact a doctor.

Read more:
Lymph nodes: Purpose, location, and disease warning signs - Medical News Today

Spinal Cord Stem Cells Comments Off on Lymph nodes: Purpose, location, and disease warning signs – Medical News Today | June 8th, 2021

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...

Cardiac Stem Cells Comments Off on Global Cell Therapy Markets, Technologies, and Competitive Landscape Report 2020-2030: Applications, Cardiovascular Disorders, Cancer, Neurological… | May 28th, 2021

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

Cardiac Stem Cells Comments Off on Global Cardiovascular Drug Delivery Markets Report 2021: Cell and Gene Therapies, Including Antisense and RNA Interference are Described in Detail -… | May 28th, 2021

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

Cardiac Stem Cells Comments Off on Obesity-Related Inflammation and Endothelial Dysfunction in COVID-19: | JIR – Dove Medical Press | May 28th, 2021

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

Cardiac Stem Cells Comments Off on Clinical Application of Cytokines in Cancer Immunotherapy | DDDT – Dove Medical Press | May 28th, 2021

World Blood Cancer Day is marked today. The celebration of this day was instituted in 2014 because, in 1991, Methchild Ehringer could not find a match by a German non-profit organisation DKMS founded by Methchilds family. She died because she was unable to find a match. The aim was and still remains to find a potential donor for every person needy of a bone marrow/stem cell transplant.

Today, there are 10 million potential donors registered when compared to the initial 3,000. Some might remember me as the person who needs Daratumumab included in the government formulary for free medicinals. I am interested in other matters too!

World Blood Cancer Day! This day highlights a more personal issue that concerns the realisation that I do not stand alone in my blood cancer journey. An issue that cuts across the three most common blood cancers: leukaemia, lymphoma and myeloma. Of these three, the lymphomas and the myelomas are the more common. For many people, such as myself, the diagnosis of a blood cancer is a shock. Like water, blood is meant to sustain a person.

Blood cancers bring along with them a lot of uncertainty and anxiety. For some blood cancers, such as multiple myeloma, a cure does not exist. To suddenly acknowledge that what is being generated in your bone marrow and what is circulating around your body and through your own blood is threatening you and your body, is psychologically invasive in a way like no other. I felt robbed. Robbed by my own body, my own immune system and, perhaps, by my own past lifestyle choices.

Stem cell transplantation offers hope of increased longevity, when appropriate, to a good proportion of blood cancer patients. A successful stem cell transplant means time out of hospital, visiting usually only every few months for monitoring. There are two types of stem cell transplantation: autologous and allogenic.

Autologous transplantation is when a person donates to oneself. As was the case with myself and my two attempts for autologous transplantation.

For many people, such as myself, the diagnosis of a blood cancer is a shock

Allogenic requires matching a donor to a patient. This is only suitable in specific cases. The government of Malta does pay for such transplantation. Charities such as Puttinu are incredibly supportive in supporting those undergoing stem cell transplantation by providing accommodation. On World Blood Cancer Day 2021, my wish is that the Maltese public understand what I consider to be three critical issues.

First, for many, stem cell/bone marrow transplants and, increasingly, cellular innovative therapies, potentially require a donor. Second, for a portion of those requiring such intervention/therapy they cannot donate to themselves and/or find a donor from their family. Third, millions of people are required to donate their stem cells.

As of today, I am under the impression that Malta and its generally very good healthcare system does not yet, have a register for stem cell donation. I hope I am wrong. Should I be right, I urge the powers that be to strongly consider this as part of their long-term strategic vision. What I do know, however, is that Malta has the local expertise and the equipment to collect stem cell transplants.

Perhaps because it is a small-island state, Malta does not have the facility for storage. The healthcare system is probably likely, especially at this point in time, not to possess as much capacity to assure consistent and sustained storage of stem cells according to European and international gold-standard criterion. This is likely to be primarily due to space issues given that the local expertise is available and excellent.

In more recent times especially, monoclonal agents, such as Daratumumab, are increasingly offered as more frontline treatment to those with an early diagnosis of multiple myeloma, at least in other EU countries and to those able to afford payment.

Chimeric Antigen therapy (CAR-T cell), which forms part of cellular therapeutic options, is also in the pipeline.

Monoclonal therapy and CAR-T cell are consequently likely to decrease the need for stem cell transplants. This is positive but, if anything, highlights even more the need for a stem cell database, register and repository. Newer therapies generally tend to be increasingly stem cell therapy dependent in some form or other.

What I would like the reader to appreciate is that I am not a medical professional. I have been at times accused of being a dreamer but life has taught me two things.

To turn lemons into lemonade and to give without the expectation of taking or receiving.

Today, I would like to go a bit beyond my myeloma, so to speak. I want to celebrate, as a blood cancer survivor, what works for me. I urge all of you to read about blood cancers and try to empathise with all blood cancer survivors.

Above all, let us not forget their carers: spouses, children, friends, doctors, nurses and everybody else whom I have inadvertently omitted.

I, for one, would not be here, especially, without the excellent care and patience of doctors, nurses, physiotherapists, etc. alongside the emotional care, motivation and interest offered by my sons, friends, work colleagues and those generally understanding and supportive of my condition, and, yes, my Lara still needs Dara quest! Thank you.

Lara Saids dream is to set up a non-profit organisation to advocate especially for the rights of patients with myeloma, leukaemia and lymphoma for Malta and Gozo. She is here appealing to survivors, their relatives and/or carers to help her set up a patient group.

Lara Said, Multiple myeloma survivor, member, Myeloma Patients Europe

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Living with blood cancer - Lara Said - Times of Malta

Bone Marrow Stem Cells Comments Off on Living with blood cancer – Lara Said – Times of Malta | May 28th, 2021

Ifeyinwa Osunkwo, MD, MPH: Pat, can you describe the steps people go through to do a bone marrow transplant to gene therapy? Set the stage to help people understand why busulfan and why were talking about mutations. Can you walk us through the whole gene therapy process?

Patrick McGann, MD, MS: With transplant or gene therapy, the term transplant could be autologous, meaning your own cell gene therapy transplant. Its not as if youre transplanting a solid organ or a kidney. Sometimes patients get confused about this. Its looks like a blood transfusion hanging when it eventually goes in. For a bone marrow transplant, the donorwhoever that iseither gets a bone marrow aspiration, where they get bone marrow cells taken from their bone marrow, or a medicine to rev up their blood cells and get blood taken just from their vein. The patient needs to get prepared because they need to get rid of all their sickle cells. They need to suppress their immune system, so they dont reject this foreign cell, which is someone elses.

We use strong chemotherapy. If you have leukemia, as a comparison, you need to kill every last leukemia cell, and you get blasted with really strong chemotherapy agents and strong immunosuppressive agents. This is usually a week or so before; the days are counted backward. The cells that go in your body are most vulnerable to infection to everything. Its a dangerous time. Thats when complications come in. If its a transplant, you get infused with that donors bone marrow cells and hope it takes. It takes some weeks time for your body to take those new cells, and youre often receiving antibiotics and getting transfused and sustaining it, because your bone marrow is still not working. Basically, your immune system is suppressed. Its a tough time.

Transplant conditioning, as this regimen is called, has gotten a little less toxicreduced conditioning is the term. But thats still serious conditioning. Even though its reduced from what it used to be, its a relative term. Gene therapy is a little different because youre giving back your own cell. The way gene therapy happens is its ex vivo, meaning they take it out of your body. There are different ways that this is being done. Many patients need to have a bone marrow aspiration or many procedures to take enough cells out of their bone marrow to take them to the lab to fix them. There have been new ways to do this with peripheral blood and a medication called plerixafor, which is much better than going to the operating room for these horrible procedures.

Those cells are then taken to the lab and edited, or whatever the mechanism of gene therapy is. You still need to ablate your bone marrow to get rid of all your sickle cells. Because if you have any or many sickle cells in there, when you give back your edited gene cells, those will just take over. You still need to suppress that bone marrow. Because its your own cells, the immune suppression isnt as much of a problem as it is with transplant. Still, its a week of pretty serious medicinebusulfan, traditionallyand youre in the hospital for less of a period of time than transplant. Its quite an ordeal. Similarly, it takes or doesnt, and you monitor over time if that gene therapy has worked and whether its sustainablecross your fingersin the long term.

Ifeyinwa Osunkwo, MD, MPH: Basically, you have 2 options. The first option, you have to kill off their own bone marrow cells using chemotherapy. Then you give them somebody elses bone marrow, like a blood transfusion. The stem cells from the other person finds its way into their bone marrow and then grows. Then you wait and see what happens. Do you fight it? Do you accept it? We know if it takes or not. For gene therapy, we take out the patient stem cells, take it to a laboratory. Its usually in New Jerseydont ask me why. They manipulate it to pick out the gene they dont want. Then they give that patient back their own modified stem cells and wait for it to grow. But you still have to wipe out that persons bone marrow, so you dont have this fight going on. Even though theyre your cells, theyre a little different with the new gene change that has been made. Its a complicated process, and its really the only way to cure your disease. Either stem cell or gene therapy. We have had some setbacks in the past and more recently, but I believe that science is going to prevail. Over time were going to get to the point where we figure out the way to do this in the safest way to make it available to the most people with sickle cell disease and other blood disorders.

Thank you so much for watching this HCPLive Peer Exchange. If you enjoyed the content, please subscribe to the e-newsletter to receive upcoming Peer Exchanges and other great content right in your in-box.

Transcript Edited for Clarity

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Stem Cell vs Gene Therapy Processes in SCD - MD Magazine

Bone Marrow Stem Cells Comments Off on Stem Cell vs Gene Therapy Processes in SCD – MD Magazine | May 28th, 2021

Fighter: A year long battle but Rory is still smiling

In January 10-year-old Aurora Pile-Grays family were told they may lose their little girl as a rare and aggressive cancer took its devastating toll.

The previous November Aurora affectionately known as Rory had been declared in remission from the disease that she had been fighting since May 2020 but a cruel blow saw a severe relapse with the cancer progressing to her skull, eyes, neck, spine, liver, kidneys, lungs, abdomen and pelvis.

A discussion with the consultant over going home from hospital and preparing for end of life care took place but lion-hearted Rory was not ready to give up her fight against Burkitts Lymphoma which affects blood and bone marrow and her family were not ready to let go.

After a year-long battle with the disease, seven rounds of intensive chemotherapy, stays in Royal Marsden and Great Ormond Street hospitals and targeted therapy with trial drug Inotuzumab, Aurora was declared cancer free again at the end of April.

The Inotuzumab drug also offered the family, who live in Garlinge, a chance for Aurora to be at home despite treatment.

Mum Keisha, 28, said: We chose Inotuzumab because it meant a better quality of life for Rory, she could come home to us and her brother and sister rather than having to be in hospital.

The trial drug, and cancer all clear, opened the way for the youngster to undergo a bone marrow transplant on May 13 all the more vital as chemo had wiped out her immune system.

First there were 10 radiotherapy sessions to get through involving cranial boost, where Auroras face was bolted to the bed so she couldnt move.

The transplant which is universally referred to as a new birthday to signal a second chance at life took place at the Marsden and involved replacing old bone marrow cells that are failing to produce healthy new cells, with cells from a donor. The aim is to create a new immune system and hopefully prevent cancer returning or mutations occurring.

The stem cells Aurora now carries should begin to reproduce in her own body and allow her bone marrow to work as normal in producing healthy red cells, white cells and platelets, since her body is no longer capable of doing so after the effects of both the cancer and the treatment.

Auroras donor cells were frozen in December which meant on the day they had to be defrosted and infused within 20 minutes. Auroras donor produced nine bags of stem cells. Only four were required, leaving 5 for future use if needed.

Each bag is defrosted in a water bath around 38 degrees so that by the time its infused its not too different to body temperature. They are then put into separate syringes and pushed through a central line into the body.

The family have been told there is just a 10-20% chance that the transplant will achieve long lasting remission, but the St Crispins school youngster has so far beaten the odds and mum Keisha says her little girl is a fighter.

In her blog Growing Pains and Paper Planes, Keisha says: Shes been amazing throughout this entire journey and Im unbelievably proud to call her my daughter. Im in awe of her strength, determination and resilience and shes shown us all that sometimes the smallest hearts overcome the biggest battles.

So far Aurora has responded well to the transplant and there is evidence of engraftment where the donor cells find their way to her bone marrow and begin to make new blood cells. White cells are the first to engraft which include neutrophils, then red cells and then platelets.

Keisha said: We were worried the effects would be awful as Rory was so sensitive to the chemo but it is going really well.

Rory is up every day, listening to audio books and doing lots of colouring, cracking jokes and just being Rory.

It is such a relief. In January when we realised the cancer had spread and she could lose the use of her legs, bladder and bowel we had to talk about making her comfortable at home. But we were not ready to give up. I said it wasnt that time yet, not all the time that Rory was laughing and joking and looking forward to seeing her brother and sister. She wasnt giving up and we werent.

Now there has been this complete 360 turnaround. Everything still depends on how she takes to the transplant. On day 28 a sample will hopefully show what percentage are donor (cells) and what percentage are her own. Hopefully it will show primarily donor.

Rory is on immunosuppressants for up to a year but potentially could be able to return to school in six months time.

Keisha said: Shell go back as a Year 6 and has missed two years but has been doing schoolwork for an hour each day.

She is looking forward to it, more the social side than the work!

Rory has won the hearts of the Thanet community during her battle with cancer with many following the familys progress through Keishas blog and also donating to a fundraiser for life-saving treatment. People also signed up on the bone marrow register following Keishas highlighting of the desperate need for donors, especially people of mixed ethnicity.

Keisha said: We are unbelievably grateful and humbled by the support people have shown us over the past year. The kind words, the gestures, the gifts, the donations, the shares, the marrow registrations and the sense of community has helped us more than you will ever know.

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'Lion-hearted' ten-year-old Aurora's 'second chance' at life after cancer remission and stem cell transplant - The Isle of Thanet News

Bone Marrow Stem Cells Comments Off on ‘Lion-hearted’ ten-year-old Aurora’s ‘second chance’ at life after cancer remission and stem cell transplant – The Isle of Thanet News | May 28th, 2021

Chronic lymphocytic leukemia (CLL) is a type of blood cancer. It affects lymphocytes, a type of immature white blood cell that the body produces in the bone marrow.

People with CLL may experience discomfort, swelling, and pain in the abdomen if their spleen enlarges. In rare cases, CLL can also affect mucosal tissues, such as those lining the gastrointestinal (GI) tract.

This article discusses the link between CLL and stomach pain in more detail. It also looks at ways to prevent and treat CLL, the other possible symptoms, and the outlook for people with this condition.

People with CLL may experience various symptoms involving the abdomen or stomach, such as:

In most cases, people with CLL do not experience abdominal symptoms due to the disease until it progresses and becomes more severe.

Research suggests that CLL infiltrates and affects the GI tract in about 5.713% of cases. When CLL affects the GI tract, doctors may call it Richters syndrome.

People with CLL may experience abdominal swelling, discomfort, and tenderness as a result of their spleen becoming larger. Less commonly, they may also feel full after eating small amounts of food, as the spleen can press on the stomach, making it smaller and able to hold less.

In rare cases, CLL infiltrates the lining of the GI tract, causing inflammation and ulcers or open wounds. People may experience symptoms similar to those of inflammatory bowel disease (IBD) and malabsorption disorders. These symptoms may include diarrhea, nausea, vomiting, abdominal pain and cramping, and unintentional weight loss.

CLL is cancer that develops in lymphocytes, which are white blood cells that form in the bone marrow and help fight infection.

Lymphocytes make up most of the lymph tissues in the lymph nodes, thymus gland, adenoids, tonsils, and spleen. They are also present in the GI tract, bone marrow, and respiratory system.

CLL is a type of leukemia that develops gradually over time.

About 5075% of people with CLL do not experience noticeable symptoms. Due to this, doctors diagnose most people with CLL during routine blood work.

The symptoms of CLL often begin when the cancerous cells crowd out healthy cells in the bone marrow or migrate to other organs or tissues. When symptoms first appear, they are typically mild, but they then become increasingly severe. CLL can cause many nonspecific symptoms, so a person may feel as though they have a cold or the flu.

Possible symptoms of CLL that do not relate to the abdominal area include:

Doctors do not yet have a way to prevent leukemia. However, some types of leukemia, including CLL, may have links to toxins, such as herbicides, pesticides, radon, and tobacco exposure. People can help reduce the risk of CLL by avoiding or practicing extreme caution around these toxins.

Many people do not experience symptoms of CLL for years and do not require treatment. However, as the disease progresses, these individuals may need treatment to extend their lives.

When and how a doctor treats someones CLL depends on a few factors, including:

Chemotherapy is typically the first-line therapy for CLL.

Doctors may use chemotherapy in conjunction with other treatment options, such as monoclonal antibody therapy. This therapy binds antibodies to cancer cells and destroys them. Treatment can also include medications to treat or prevent infections or improve low blood cell levels.

For instance, some people may take a combination of the monoclonal antibody rituximab and the chemotherapy drugs fludarabine and cyclophosphamide. Alongside the oral chemotherapy medication chlorambucil, doctors use obinutuzumab or ofatumumab, which have the same drug target as rituximab.

Small molecule inhibitors, such as bendamustine hydrochloride, idelalisib, and ibrutinib, can also sometimes form part of a CLL treatment regimen.

In 2017, the Food and Drug Administration (FDA) approved the combination medication Rituxan Hyecela (rituximab and hyaluronidase human) for CLL treatment.

In recurring or aggressive cases of CLL, an individual may have a blood or bone marrow stem cell transplant. This procedure replaces diseased cells with healthy blood cells that are able to mature into bone marrow cells.

Doctors may treat CLL until the symptoms lessen and then stop treatment until the symptoms worsen again.

Many people with CLL live for many years with a high quality of life.

There is no cure for CLL, so treatment aims to extend and improve someones life by reducing their symptoms. Doctors treat many people intermittently as their symptoms reoccur.

A persons outlook depends on their age, overall health, underlying conditions, and stage of CLL. Typically, people who are over the age of 65 years or have a more advanced stage of CLL have a less positive outlook.

Genetic changes in CLL cells and increased beta-2 microglobulin protein levels in the blood can make CLL more challenging to treat, potentially affecting a persons outlook.

Doctors classify people with CLL into different risk groups depending on certain factors. Based on these risk groups, the estimated percentages of people surviving 5 years or more after their diagnosis is:

People with more advanced or severe CLL may experience abdominal swelling, discomfort, tenderness, and pain. They may also feel full after eating small amounts. More rarely, someone with CLL may develop GI tract inflammation or ulcers, which can cause symptoms such as diarrhea, nausea, vomiting, cramping, and unexplained weight loss.

Anyone who thinks that they may have CLL should speak with a doctor. People with a confirmed diagnosis who experience symptoms of more advanced or severe CLL, such as abdominal pain, should also seek medical care.

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Chronic lymphocytic leukemia and stomach pain: What is the link? - Medical News Today

Bone Marrow Stem Cells Comments Off on Chronic lymphocytic leukemia and stomach pain: What is the link? – Medical News Today | May 28th, 2021

Magenta Therapeutics is losing its chief medical officer and head of R&D John Davis, M.D., with his last day coming July 30.

Davis helped steer the biotechs early path as well as deals with Avrobio and base editing biotech Beam Therapeutics around its lead stem cell conditioning program, MGTA-117, and MGTA-145 as a potentially new first-line standard of care for stem cell mobilization in a broad range of diseases. His departure comes three years after he joined the company from Pfizer, where he led its early R&D.

The biotech was keen to stress in an 8-K SEC filing (but not a press release) that his departure was not related to any disagreements with the Company on any matter relating to its operations, policies, practices or any issues regarding financial disclosures, accounting or legal matters.

It will now look for a new CMO, while Davis will become an adviser to the company.

RELATED: Magenta CSO Cooke jumps to IFM Therapeutics

This isnt the first move within the R&D ranks: Magenta started 2020 by losing its chief scientific officer when Michael Cooke, Ph.D., hopped over to IFM Therapeutics. In the fall, though, it nabbed Lisa Olson, Ph.D., who previously led immunology discovery at AbbVie, as his replacement.

Conditioning is a necessary step for some gene therapies, but one that can cause side effects like nausea, hair loss and mouth sores or make patients more vulnerable to infection. Magentas platform is based on looking to improve on current methods with an antibody-drug conjugate.

MGTA0117 is made up of an anti-CD117 antibody linked to amanitin, a cell-killing toxin. It is designed to target only hematopoietic, or blood-forming, stem cells and progenitor cells.

Animal studies suggest it could clear space in bone marrow for gene-modified stem cells to take root, Magenta said. The company plans to wrap IND-enabling studies for the antibody-drug conjugate this summer.

MGTA-145, meanwhile, just finished off a midstage test earlier this month, hitting its primary endpoint in a small multiple myeloma study.

Davis' replacement will already have a lot of clinical work on their hands.

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Magenta Therapeutics' research lead Davis hits the exit for family reasons - FierceBiotech

Bone Marrow Stem Cells Comments Off on Magenta Therapeutics’ research lead Davis hits the exit for family reasons – FierceBiotech | May 28th, 2021

KILLEEN, TX As blankets of snow covered the frigid ground and ice sheets laid atop of pavement, Michael or MJ Dixon was due for a sports physical at his normal pediatrician.

The doctor gave him a clearance, Chaundra Dixon, his mother explained. [The doctor] gave him a clearance and said, oh, he's healthy. He's fine.

MJ was used to getting hurt.

As a basketball, football, baseball and tennis star, its only a matter of time before one accumulates bruises and sore muscles.

However, MJ kept saying he was hurt.

His mom was convinced it was just growing pains, and so the duo worked on his stretching.

Until February 24, 2021.

She was at work on Fort Hood when MJs babysitter gave her a call.

She was just like, 'hey, I think you need to come home and get Michael, we need to get him to the hospital,' she recalled, as tears built up in her eyes thinking back to that day. He's crying non stop; he's saying he can't walk.

The panicked mother immediately picked up her son from their Killeen home and braved the icy road conditions, making it to Baylor Scott and White before EMS got to her house.

As they arrived at the hospital, they were met with COVID-19 tests. From there, physicians tested MJ for the basics, which all looked OK.

It wasnt until they ran blood work and found an abnormal amount of white blood cells in MJs body when they determined it was leukemia

About seven o'clock that night, I will never forget that, Dixon said. He was like, you're a great mom and I'm sorry, that I have to tell you, your son has leukemia, and I said leukemia? I lost it.

Living in this new reality brought on by his cancer, MJ and his family began looking for bone marrow donors.

The problem? He has less than a 25% chance of finding one due to his ethnicity.

http://www.bethematch.org

This is simply because we don't have the donor pool to pull from, Tressa Malone, a spokesperson with the Be The Match organization said. What that means is, we just need more people to join the registry. It's as simple as that.

Once you register to see if you can become a match, Malone explained that the following procedures are quite simple.

She said that if you match with a patient in need, 80% of their matches donate in a process similar to donating plasma.

You go and they take out the blood, separate the cells they need, then put it back in you.

She explained that this process is usually 3-4 hours.

The second procedure to donate is by retrieving the blood near your pelvic bone, the doctors separate the stem cells, all while youre under anesthesia.

Dr. Amy Mersiovsky, the director of nursing at Texas A&M Central Texas explained that she spent countless days caring for young kids with cancer as a pediatric nurse.

Realizing the low odds African Americans face when it comes to these donors, she explained that shes not necessarily surprised after the generations of mistrust that community typically has toward the medical field.

However, the Dixons dont go down without a fight because they know God is on their side.

Now, the family organizes bone marrow drives not only here in Central Texas, but across the county, hoping to find MJ a donor or anyone whos walking the same journey the 9-year-old boy is.

The family has set up a GoFundMe page to cover various expenses, and to see if you can be a match, you can text MJ to 61474 and a swab kit will be sent to your home.

To follow along with MJ's story, head over to his Facebook page.

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Killeen boy with leukemia needs a bone marrow donor, but the odds of finding one are slim - KXXV News Channel 25

Bone Marrow Stem Cells Comments Off on Killeen boy with leukemia needs a bone marrow donor, but the odds of finding one are slim – KXXV News Channel 25 | May 28th, 2021

REDWOOD CITY, Calif.--(BUSINESS WIRE)--Jasper Therapeutics, Inc., a biotechnology company focused on hematopoietic cell transplant therapies, today announced the initiation of a Phase 1/2 clinical trial to evaluate JSP191, the companys first-in-class anti-CD117 monoclonal antibody, as a targeted, non-toxic conditioning regimen prior to allogeneic transplant for chronic granulomatous disease (CGD). Jasper Therapeutics and the National Institute of Allergy and Infectious Diseases (NIAID) have entered into a clinical trial agreement in which NIAID will serve as the Investigational New Drug (IND) sponsor for this study.

CGD is a rare, inherited disease of the immune system that develops in infancy or early childhood and results in severe and sometimes life-threatening infections. Allogeneic hematopoietic stem cell transplant is a proven cure for CGD. However, its use is limited because current conditioning agents used to deplete stem cells in preparation for transplantation are genotoxic and associated with limited efficacy and serious adverse effects, including veno-occlusive disease, long-term infertility and secondary malignancies.

We look forward to collaborating with NIAID on this Phase 1/2 clinical trial, which should provide important information about the potential of JSP191 as a safer and more effective conditioning agent for patients with CGD undergoing hematopoietic stem cell transplant, said Kevin N. Heller, M.D., Executive Vice President, Research and Development, of Jasper Therapeutics. Through this clinical trial agreement with NIAID, as well as others with the National Institutes of Health and academic centers, we are continuing to develop JSP191 for additional pretransplant conditioning regimens beyond severe combined immunodeficiency and acute myeloid leukemia/myelodysplastic syndromes, which have demonstrated safety and efficacy in early-stage clinical trials to date.

About JSP191

JSP191 is a first-in-class humanized monoclonal antibody in clinical development as a conditioning agent that clears hematopoietic stem cells from bone marrow, creating an empty space for donor or gene-corrected transplanted stem cells to engraft. While hematopoietic cell transplantation can be curative for patients, its use is limited because standard high dose myeloablative conditioning is associated with severe toxicities and standard low dose conditioning has limited efficacy. To date, JSP191 has been evaluated in more than 90 healthy volunteers and patients. It is currently enrolling in two clinical trials for acute myeloid leukemia (AML)/myelodysplastic syndromes (MDS) and severe combined immunodeficiency (SCID) and is scheduled to begin enrollment in three additional studies in 2021 for severe autoimmune disease, sickle cell disease and Fanconi anemia patients undergoing hematopoietic cell transplantation.

About Jasper Therapeutics

Jasper Therapeutics is a biotechnology company focused on the development of novel curative therapies based on the biology of the hematopoietic stem cell. The company is advancing two potentially groundbreaking programs. JSP191, a first-in-class anti-CD117 monoclonal antibody, is in clinical development as a conditioning agent that clears hematopoietic stem cells from bone marrow in patients undergoing a hematopoietic cell transplantation. It is designed to enable safer and more effective curative allogeneic and autologous hematopoietic cell transplants and gene therapies. In parallel, Jasper Therapeutics is advancing its preclinical engineered hematopoietic stem cell (eHSC) platform, which is designed to overcome key limitations of allogeneic and autologous gene-edited stem cell grafts. Both innovative programs have the potential to transform the field and expand hematopoietic stem cell therapy cures to a greater number of patients with life-threatening cancers, genetic diseases and autoimmune diseases than is possible today. For more information, please visit us at jaspertherapeutics.com.

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Jasper Therapeutics Announces New Clinical Trial with the National Institute of Allergy and Infectious Diseases to Evaluate JSP191 in Chronic...

Bone Marrow Stem Cells Comments Off on Jasper Therapeutics Announces New Clinical Trial with the National Institute of Allergy and Infectious Diseases to Evaluate JSP191 in Chronic… | May 28th, 2021

CAMBRIDGE, Mass.--(BUSINESS WIRE)--Moderna, Inc., (Nasdaq: MRNA) a biotechnology company pioneering messenger RNA (mRNA) therapeutics and vaccines, today announced new research being highlighted as part of the Companys fourth annual Science Day. Modernas Science Day is designed to provide insight into the continued diverse efforts underway at Moderna to better understand how to use mRNA as medicines and vaccines and underscores the Companys continued commitment to basic science and innovation.

Science Day gives us an opportunity to provide insights into the advancements in our platform science and our further understanding of how to use mRNA as both a vaccine and a medicine. Our investments in basic science continue to result in major steps forward in our platforms capabilities, and these have allowed us to open new therapeutic areas and new scientific directions, said Stephen Hoge, M.D., President of Moderna. Our investments in platform research along with our digital backbone and manufacturing plants have enabled us to create first-in-class mRNA medicines and vaccines. Today, we're excited to highlight our work to identify and address SARS-CoV-2 variants of concern, optimize our proprietary lipid nanoparticle (LNP) technology, and deliver mRNA to hematopoietic stem cells.

Moderna currently has 24 mRNA development programs in its portfolio with 14 having entered clinical studies. The Companys updated pipeline can be found at http://www.modernatx.com/pipeline. Moderna and collaborators have published more than 65 peer-reviewed papers.

At this years Science Day, Moderna will present new platform science and preclinical research, including:

mRNA Delivery to Hematopoietic Stem and Progenitor Cells (HSPC)

Of the nearly 30 trillion cells in the body, approximately 90% are of hematopoietic origin. Hematopoietic lineages originating in the bone marrow are intimately involved in maintaining homeostasis and human health. Nonetheless, there are hundreds of hematologic or immune-related disorders caused or exacerbated by cells of the hematopoietic lineage. In a disease setting, cells of hematopoietic origin interact with host tissues to drive chronic inflammatory and immune disorders. Because some hematopoietic stem and progenitor cells (HSPC) have self-renewal and pluripotent properties, targeting HSPC has the potential to modulate underlying chronic inflammation and immune-related disorders.

Advances in lipid nanoparticle (LNP) technology has allowed for delivery to the bone marrow following systemic LNP administration in vivo. This achievement has enabled Moderna scientists to deliver mRNA directly to bone marrow HSPC in vivo, leading to HSPC transfection and long-term modulation of all hematopoietic lineages. This represents a major milestone in impacting chronic inflammatory and immune related disease.

mRNA Engineering: Optimizing Ribosome Load

The ribosome plays a central role in orchestrating the life of mRNA inside the cell. Understanding how to modulate translation by changing mRNA sequence could enable a powerful lever to control the performance of an mRNA drug. To develop such a lever, the Moderna's scientists strove to characterize mechanistically how differences in translation drive differences in protein expression. In this research, an unexpected relationship emerged where mRNAs with low translation initiation rates conferred the highest and most durable levels of protein expression. By understanding the mechanisms that drive this behavior, the Company is taking mRNA design from a guess-and-check discipline into an engineering discipline. With tools in place, and more under development, Moderna is improving its ability to make an mRNA that generates the right amount of the right protein, for the right amount of time, in the right cell type. As Moderna builds these levers into its mRNA drugs, the Company believes it will be able to target more indications with more precision.

Addressing SARS-CoV-2 Variants of Concern

One part of the Companys strategy to remain ahead of the SARS-CoV-2 virus is to closely monitor and address emerging variants of concern and waning immunity. Moderna is using artificial intelligence (AI) and machine learning to predict escape risk. This involves developing statistical models to understand and predict escape risk, including identifying breakthrough sequences from clinical trials and real-world data, examining spike protein biophysical and pseudovirus neutralization data, studying spike mutations and lineage info, and time tracking.

Science Day Webcast Information

Moderna will host its annual Science Day for analysts and investors at 8:00 a.m. ET on Thursday, May 27. A live webcast will be available under Events and Presentations in the Investors section of the Moderna website at investors.modernatx.com. A replay of the webcast will be archived on Modernas website for one year following the presentation.

About Moderna

In 10 years since its inception, Moderna has transformed from a science research-stage company advancing programs in the field of messenger RNA (mRNA), to an enterprise with a diverse clinical portfolio of vaccines and therapeutics across six modalities, a broad intellectual property portfolio in areas including mRNA and lipid nanoparticle formulation, and an integrated manufacturing plant that allows for both clinical and commercial production at scale and at unprecedented speed. Moderna maintains alliances with a broad range of domestic and overseas government and commercial collaborators, which has allowed for the pursuit of both groundbreaking science and rapid scaling of manufacturing. Most recently, Modernas capabilities have come together to allow the authorized use of one of the earliest and most-effective vaccines against the COVID-19 pandemic.

Modernas mRNA platform builds on continuous advances in basic and applied mRNA science, delivery technology and manufacturing, and has allowed the development of therapeutics and vaccines for infectious diseases, immuno-oncology, rare diseases, cardiovascular diseases and auto-immune diseases. Today, 24 development programs are underway across these therapeutic areas, with 14 programs having entered the clinic. Moderna has been named a top biopharmaceutical employer by Science for the past six years. To learn more, visit http://www.modernatx.com.

Forward-Looking Statements

This press release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including statements regarding: the potential for delivery of mRNA to hematopoietic stem and progenitor cells (HSPC) in vivo; methods of detecting and interrogating HSPC in vivo; the potential for delivering LNPs to hematopoietic stem cells in vivo; the ability to optimize codons and mRNA structures to increase total protein outputs; the potential for the Company to develop processes for controlling protein expression by modifying ribosomal loads; the Companys ability to engineer LNPs capable of accessing difficult-to-transfect primary cells with efficient endosomal escape and high functional mRNA delivery; the Companys strategy for combatting COVID-19, including processes for monitoring emerging variants and waning immunity; and strategies for modeling viral escape. In some cases, forward-looking statements can be identified by terminology such as will, may, should, could, expects, intends, plans, aims, anticipates, believes, estimates, predicts, potential, continue, or the negative of these terms or other comparable terminology, although not all forward-looking statements contain these words. The forward-looking statements in this press release are neither promises nor guarantees, and you should not place undue reliance on these forward-looking statements because they involve known and unknown risks, uncertainties, and other factors, many of which are beyond Modernas control and which could cause actual results to differ materially from those expressed or implied by these forward-looking statements. These risks, uncertainties, and other factors include, among others: the fact that there has never been a commercial product utilizing mRNA technology approved for use; the fact that the rapid response technology in use by Moderna is still being developed and implemented; the safety, tolerability and efficacy profile of the Moderna COVID-19 Vaccine observed to date may change adversely in ongoing analyses of trial data or subsequent to commercialization; the Moderna COVID-19 Vaccine may prove less effective against variants of the SARS-CoV-2 virus, or the Company may be unsuccessful in developing future versions of its vaccine against these variants; despite having ongoing interactions with the FDA or other regulatory agencies, the FDA or such other regulatory agencies may not agree with the Companys regulatory approval strategies, components of our filings, such as clinical trial designs, conduct and methodologies, or the sufficiency of data submitted; Moderna may encounter delays in meeting manufacturing or supply timelines or disruptions in its distribution plans for the Moderna COVID-19 Vaccine; whether and when any biologics license applications and/or additional emergency use authorization applications may be filed in various jurisdictions and ultimately approved by regulatory authorities; potential adverse impacts due to the global COVID-19 pandemic such as delays in regulatory review, manufacturing and clinical trials, supply chain interruptions, adverse effects on healthcare systems and disruption of the global economy; and those other risks and uncertainties described under the heading Risk Factors in Modernas most recent Annual Report on Form 10-K filed with the U.S. Securities and Exchange Commission (SEC) and in subsequent filings made by Moderna with the SEC, which are available on the SECs website at http://www.sec.gov. Except as required by law, Moderna disclaims any intention or responsibility for updating or revising any forward-looking statements contained in this press release in the event of new information, future developments or otherwise. These forward-looking statements are based on Modernas current expectations and speak only as of the date hereof.

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Moderna Highlights Advances in Platform Science and Innovative Vaccine Research at Fourth Annual Science Day - Business Wire

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For many years now, the topic of senescent cells has been the subject of plenty of research work. Back in the 1960s the Hayflick limit was noticed in cell culture: there was an apparent limit to the number of cell divisions that could take place before the cells just sort of stalled out. For human fibroblasts, that kicks in at around fifty divisions. Over time it was worked out that a primary mechanism involved is the shortening of telomeres with each cell division, specialized nucleotide sequences out at the ends of the chromosomes, and this cellular clock phenomenon has been making its way into the public consciousness ever since.

Its strange to think, but before these experiments human cells were considered to be more or less immortal and capable of unlimited numbers of divisions. Now, there are cells like that, but that (outside of some stem cell populations and a few other special cases) is a very short working definition of cancer. Those cells do indeed seem to be able to carry on for as long as conditions permit which in the artificial world of cell culture labs, means apparently forever. Henrietta Lacks died in 1951, but HeLa cells are still with us, and can be all too vigorous when they contaminate other lines. Tumor cells can pile up mutations that will make them die off, but short of that the jams have indeed been kicked out.

Its gradually become apparent that many aging or damaged tissues have a (sometimes substantial) population of cells that have reached their limit. Theyre alive and metabolically active but not really contributing much, in a stage of permanent growth arrest. Cellular senescence is a complex phenomenon, but its importance in aging, cancer, and tissue damaged by other factors (radiation, oxygen stress, etc.) is by now undeniable. Many of these non-aging states can be traced back to early telomere damage by other mechanisms, emphasizing that as a key countdown mechanism. But its clear that senescent have a different secretory profile (cytokines, growth factors and more) from the more vigorous cells around them and a number of other protein expression differences that can be used the characterize them.

Naturally enough, thoughts have turned to targeting such cells for therapy. There are a couple of very easy-to-picture hypotheses: first, could you keep telomeres from shortening (or shortening so much) and therefore keep cells in a non-senescent state for longer, potentially delaying biological aging? And second, could you somehow target cells that have already become senescent, and would doing so improve the health of the surrounding tissue? Though pretty obvious ideas, both of these are still very much in play. For now, Im going to talk about the second one, in light of a new paper.

That ones on the kidney. Younger people can regain some kidney function after an injury, but that ability goes down with aging, as youd imagine. It also goes down in states of chronic kidney disease, or after radiation damage. This new paper shows that targeting and removing senescent cells actually starts to reverse this phenotype once youve done that, the kidney tissue after injury shows increased function, increased regenerative ability, and less development of fibrosis. This is demonstrated both in aged tissue and in younger tissue exposed to radiation damage, in human cell culture and in mouse animal models.

You may well ask: how exactly does one target senescent cells? That takes us to ABT-263 (navitoclax), shown at right. This rather hefty molecule is part of a series of AbbVie protein-protein inhibitors for the Bcl-2 (B-cell-lymphoma) family. There are several of those, and navitoclax inhibits the function of Bcl-2, Bcl-xL, and Bcl-w. All of these proteins are intimately tied up in the pathways of apoptosis, programmed cell death, which is another monstrously huge pathway all its own. But one of the questions about senescent cells is why they dont go down some apoptotic pathway and just fall on their on cellular swords, instead of hanging around forever gumming up the works.

This one, like the others in its class, was developed to cause this to happen to tumor cells as an adjunct to other types of chemotherapy, but these have also turned out to be useful against senescent cells (although not all types of them). Similar to the kidney results reported in the new paper linked above, there have been reports in lung, CNS, muscle and other tissues of broadly similar enhancements (many of these summarized in this paper). So at this point you might be wondering why we dont just go ahead and put these things into the water supply already.

Theres a problem, unfortunately. It was clear from the clinical studies of the AbbVie compounds that platelet effects were dose-limiting. Cells in that pathway are sensitive to messing with these apoptosis pathways, and while you might be able to deal with that side effect in a chemotherapy situation, it doesnt exactly make for a good-for-what-ails-you drug. Navitoclax has alsorecently been shown to have profoundly bad effects on bone density and deposition, which is the exact opposite of what youd want for an aging population.

AbbVies next generation of such compounds, though, includes venetoclax, at right, also a lunker of a molecule and now approved for several types of leukemia. It still has platelet effects, but they arent nearly as disastrous as with navitoclax, thanks to deliberately lower binding to Bcl-xL. That also makes it a bit less of a mighty sword across senescent cell types for example, it appears that you need that pathway for activity against glioblastoma cells. But it has been reported to show strong protective effects against the development of Type I diabetes through the elimination of senescent cells in the islets of Langerhans. Meanwhile, other groups are looking at turning these ligands into targeted protein degraders, which (at least in some cases) seems to decrease the platelet problems and increase senolytic activity.

And before leaving the topic, it has to be noted that there are plenty of other ways to target these cells other than the Bcl pathway (although that one seems to be one of the most developed so far). What can I say? Im 59, and I doubtless have more senescent cells than I want or need, so I (and plenty of others) are interested in the idea. The whole cellular senescence pathway presumably developed as a way to avoid slipping into a tumor phenotype the more cellular divisions, the greater the chance of something going wrong along the way. Its a tradeoff, and evolution seems more than willing to shortchange older members of the species who have generally passed on their genes to all the offspring that theyre going to. But humans have other goals. We are looking at a rather rapidly aging planet, if current demographic trends hold up, and it would be extremely desirable to have that associated with less of a disease burden. Can we split the difference?

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Clearing Cellular Dead Wood | In the Pipeline - Science Magazine

Bone Marrow Stem Cells Comments Off on Clearing Cellular Dead Wood | In the Pipeline – Science Magazine | May 28th, 2021

REGULATED INFORMATION

Strong clinical progress especially in JTA-004 Phase III study thanks to high patient compliance and retention

Process development partnership and appointment of cell therapy expert Anthony Ting as CSO to further strengthen product pipeline

Gosselies, Belgium, 26May 2021, 7am CEST BONE THERAPEUTICS (Euronext Brussels and Paris: BOTHE), the cell therapy company addressing unmet medical needs in orthopedics and other diseases, today announces its business update for the first quarter, ended 31 March 2021.

Bone Therapeutics has continued the strong momentum into 2021, said Miguel Forte, MD, PhD, CEO of Bone Therapeutics. Bone Therapeutics mid-late stage clinical programs continue to advance largely on schedule, including the Phase IIb trial of the allogenic cell therapy platform ALLOB in difficult-to-heal tibial fractures and the Phase III trial of the enhanced viscosupplement JTA-004 in knee osteoarthritic pain. Alongside this, Bone Therapeutics has strengthened its manufacturing and R&D capabilities by signing a process development partnership with Rigenerand. It has also appointed the industry veteran Tony Ting as our new Chief Scientific Officer. Building on these achievements, Bone Therapeutics will be able to continue significant clinical and commercial advancements as we move towards the topline data of our JTA-004 Phase III study; a potential key inflection point for Bone Therapeutics.

Operational highlights

Financial highlights

Outlook for the remainder of 2021

(1) Unaudited number

About Bone Therapeutics

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

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

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

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

For further information, please contact:

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

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

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

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

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

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Bone Therapeutics Provides First Quarter 2021 Business Update - GlobeNewswire

Bone Marrow Stem Cells Comments Off on Bone Therapeutics Provides First Quarter 2021 Business Update – GlobeNewswire | May 28th, 2021

A five-year-old boy has returned from New York where he is taking part in a pioneering medical trial aimed at preventing his cancer from returning.

Oliver Stephenson from West Yorkshire was given the all-clear earlier this year after battling neuroblastoma a rare cancer originating from immature nerve cells throughout 2020.

His family crowdfunded 270,000 for treatment as the schoolboy underwent chemotherapy, surgery, stem cell treatment and a bone marrow transplant.

At one point, Oliver and his dad James had to isolate together in a 15sqft hospital room for seven weeks due to the coronavirus pandemic.

Oliver has spent the past month at New York's Memorial Sloan Kettering Cancer Center, where he is on a clinical trial vaccine called Bivalent.

It is hoped the treatment will train his immune system to identify and destroy neuroblastoma cells lurking in his body to prevent the disease from returning.

The vaccine, which costs 210,000, will require Oliver to make four more trips to the US this year.

He returned from the four-week trip to New York around a week ago with his mum Laura, dad James and little brother Alfie.

Mrs Stephenson said: "Everything went great. The doctors were all amazing and Oliver has responded well so far.

"It is a clinical trial so we don't know what will happen but we are hoping that this will prevent Oliver from suffering as he has done again.

"After everything he has been through it feels great to be on a positive track."

She added the treatment was "quick and easy", explaining Oliver had three vaccines and that, other than some discomfort, he had no obvious side effects.

Due to the length of their stay, Mrs Stephenson said the family were able to make a holiday out of the trip.

She added: "Oliver and Alfie had a brilliant time in New York, it was amazing.

"Because of the pandemic everywhere was really quiet, which meant we felt very safe all the time and didn't have to queue for anything."

The pioneering treatment comes almost 18 months after Oliver was diagnosed with neuroblastoma, which affects around 100 children annually.

The coronavirus pandemic complicated the family's ordeal as Oliver and his dad had to isolate together in hospital during chemotherapy.

Mr Stephenson was at his son's bedside throughout, but the rest of his family were unable to visit due to restrictions and had to make do with video calls.

Crowdfunding pages were set up to help the family pay for treatment once options on the NHS were exhausted, as neuroblastoma has a significant chance of returning.

Mrs Stephenson added: "The incredible support and donations not just from our friends and family but around the world has been overwhelming.

"We are so grateful to everyone who has helped.

"It's been a really difficult year for us and back in 2020 Oliver's prognosis wasn't good at all.

"But he has battled through everything is doing well now, he's fit and strong and even back at school.

Despite Oliver getting the all-clear, figures show 60% of people who recovered from neuroblastoma end up relapsing.

Mrs Stephenson said: "We want to everything we can to stop that happening."

Throughout Oliver's treatment, his family has been supported by the Solving Kids Cancer charity.

Original post:
Five-year-old boy returns home after first round of cancer treatment in New York - Surrey Live

Bone Marrow Stem Cells Comments Off on Five-year-old boy returns home after first round of cancer treatment in New York – Surrey Live | May 28th, 2021

bluebird bios cerebral adrenoleukodystrophy (CALD) gene therapy Skysona has moved closer towards EU approval after gaining a positive opinion from the European Medicines Agencys Committee for Medicinal Products for Human Use (CHMP).

The CHMP has recommended marketing authorisation for Skysona (elivaldogene autotemcel, Lenti-D) for the early treatment of CALD in patients under 18 years old with an ABCD1 genetic mutations, and who do not have a matched sibling haematopoietic stem cell (HSC) donor.

bluebird bio's Skysona is a potential one-time gene therapy designed to add functional copies of the ABCD1 gene into a patients hematopoietic stem cells.

Once this functional gene is added to a CALD patients stem cells, the patient's body can produce the adrenoleukodystrophy protein (ALDP), which is believed to allow for the breakdown of very-long-chain fatty acids that build up to toxic levels in the brain.

CALD is a progressive and fatal neurodegenerative disease that overwhelmingly affects males. It involves the breakdown of myelin the protective sheath of nerve cells in the brain that is responsible for muscle control and thinking.

The condition is caused by mutations in the ABCD1 gene that affect the production of ALDP which eventually causes damage to the adrenal cortex and white matter of the brain and spinal cord.

Currently, the only treatment for the disease is a stem cell transplant, although this carries a significant risk from the high-dose chemotherapy used to prepare patients for the procedure.

Other complications include graft-versus-host (GvHD) disease, which occurs when the transplanted cells recognise the recipients cells as foreign and attack them.

In the phase 2/3 Starbeam study evaluating Skysona, 90% of CALD patients met the month 24 major functional disability- (MFD) free survival endpoint as of the last data cutoff date.

MFDs are the six severe disabilities commonly attributed to CALD, which have the most severe effect on a patients ability to function independently.

In addition, 26 out of 28 evaluable patients maintained a neurologic function score (NFS) less than or equal to one until month 24, with 24 of those patients having no change in their NFS.

The CHMPs positive opinion is now due to be reviewed by the European Commission, with a final decision for Skysona expected in mid-2021.

Read the rest here:
bluebird bio's CALD gene therapy Skysona gains positive opinion from CHMP - PMLiVE

Spinal Cord Stem Cells Comments Off on bluebird bio’s CALD gene therapy Skysona gains positive opinion from CHMP – PMLiVE | May 28th, 2021

NEARLY 10,000 has been raised in less than a week to help pay for a Dunfermline mum to have life-changing treatment in Russia.

Lynda Hogg was first diagnosed with Primary Progressive Multiple Sclerosis (PPMS) seven years ago and since then, she has seen her health deteriorate.

She has been forced to give up her job as a theatre nurse at Queen Margaret Hospital and faces a daily battle with pain from a condition for which there is no cure and no treatment on the NHS.

Lynda, 58, and her family are now pinning their hopes on travelling to Russia for Hematopoietic Stem Cell Transplantation which, it is hoped, will stop the progression of her MS.

Within a week of being set up, a GoFundMe page has already raised nearly a quarter of the required 45,000, something which Lynda said she was overwhelmed and humbled by.

It was my son who said how do you feel in 10 years time if you are wheelchair-bound knowing you had this opportunity and didnt take it? she explained.

We have considered selling the house if we have to.

Daughter Popsi is hopeful that the GoFundMe page will mean such a drastic step is not necessary.

Before the GoFundMe, these were all considerations, she said. Shall we sell the house, will my dad have to give up his job and become a carer?

After we found out about this treatment years ago, I dont think we ever really considered it a possibility. It has been quite tough. I have three brothers. Speaking on behalf of the younger ones, I want them to remember them as I remember her, full of life.

This treatment is kind of a possibility that it would not get any worse. It is a pipe dream. We never thought it could happen. The support and kindness of others has been so touching.

Multiple Sclerosis (MS) is a disabling disease of the brain and spinal cord which results in muscle and nerve damage, ongoing pain and fatigue.

Since her diagnosis, Lynda, who is married to Murray and has four children, Adam, 29, Popsi, 23, Mitchell, 18, and Charlie, 15, has seen her health go downhill with her mobility decreasing.

She has been shocked at the lack of available medication.

Basically, I get co-codamol, she said. Progressive Primary MS is the worst form of it. There is no treatment and there is really little in the way of emotional support. It is not a fault of the NHS. There is nothing and there needs to be something.

PPMS is 15 per cent of people diagnosed. It is horrible to think there is nothing out there for them.

Trials for the treatment which Lynda hopes to have in Russia have taken place in the UK but, to date, are not available on the NHS for patients like herself.

They give you medication to increase the quality of stem cells. They are harvested and then you are given chemo then the stem cells are reintroduced to you. They can stop MS in its tracks. It will stop the progression. It wont give you functions back.

Even just to stop it progressing; I can cope with the disabilities I have but I dont know if I can cope with them moving forward and getting worse.

Popsi and her siblings are planning to hold further fundraisers when coronavirus restrictions allow while also raising awareness of MS.

We have had to watch our mum struggle with the disease and battle with pain every day, she added. We feel that its time to give back to our mum, and everything that she has done for us over the years.

Anyone wanting to help can do so by visiting https://gofund.me/61401f6e.

Read more:
Dunfermline mum "overwhelmed" by fundraiser aiming to fund vital treatment in Russia - Dunfermline Press

Spinal Cord Stem Cells Comments Off on Dunfermline mum "overwhelmed" by fundraiser aiming to fund vital treatment in Russia – Dunfermline Press | May 28th, 2021